%0 Journal Article %J Nature Communications %D 2023 %T Author Correction: The evolution and international spread of extensively drug resistant Shigella sonnei %A Lewis C. E. Mason %A David R. Greig %A Lauren A. Cowley %A Sally R. Partridge %A Elena Martinez %A Grace A. Blackwell %A Charlotte E. Chong %A P. Malaka De Silva %A Rebecca J. Bengtsson %A Jenny L. Draper %A Andrew N. Ginn %A Indy Sandaradura %A Eby M. Sim %A Jonathan R. Iredell %A Vitali Sintchenko %A Danielle J. Ingle %A Benjamin P. Howden %A Sophie Lefèvre %A Elisabeth Njamkepo %A Weill, François-Xavier %A Pieter-Jan Ceyssens %A Claire Jenkins %A Kate S. Baker %B Nature Communications %V 14 %8 Jan-12-2023 %G eng %N 1 %R 10.1038/s41467-023-38041-3 %0 Journal Article %J Viruses %D 2023 %T Characterization of a Bacteriophage GEC_vB_Bfr_UZM3 Active against Bacteroides fragilis %A Nata Bakuradze %A Merabishvili, Maia %A Ia Kusradze %A Pieter-Jan Ceyssens %A Jolien Onsea %A Willem-Jan Metsemakers %A Nino Grdzelishvili %A Natroshvili, Guliko %A Tamar Tatrishvili %A Davit Lazvliashvili %A Nunu Mitskevich %A Pirnay, Jean-Paul %A Chanishvili, Nina %B Viruses %V 15 %8 Jan-05-2023 %G eng %N 5 %R 10.3390/v15051042 %0 Journal Article %J Viruses %D 2023 %T Characterization of a Bacteriophage GEC_vB_Bfr_UZM3 Active against . %A Nata Bakuradze %A Merabishvili, Maia %A Ia Kusradze %A Pieter-Jan Ceyssens %A Jolien Onsea %A Willem-Jan Metsemakers %A Nino Grdzelishvili %A Natroshvili, Guliko %A Tamar Tatrishvili %A Davit Lazvliashvili %A Nunu Mitskevich %A Pirnay, Jean-Paul %A Chanishvili, Nina %K Anti-Bacterial Agents %K Bacterial Infections %K Bacteriophages %K Bacteroides fragilis %K Humans %X

is a commensal gut bacterium that is associated with a number of blood and tissue infections. It has not yet been recognized as one of the drug-resistant human pathogens, but cases of the refractory infections, caused by strains that are not susceptible to the common antibiotic regimes established for have been more frequently reported. Bacteriophages (phages) were found to be a successful antibacterial alternative to antibiotic therapy in many cases of multidrug-resistant (MDR) bacterial infections. We have characterized the bacteriophage GEC_vB_Bfr_UZM3 (UZM3), which was used for the treatment of a patient with a chronic osteomyelitis caused by a mixed infection. Studied biological and morphological properties of UZM3 showed that it seems to represent a strictly lytic phage belonging to a siphovirus morphotype. It is characterized by high stability at body temperature and in pH environments for about 6 h. Whole genome sequencing analysis of the phage UZM3 showed that it does not harbor any known virulence genes and can be considered as a potential therapeutic phage to be used against infections.

%B Viruses %V 15 %8 2023 Apr 25 %G eng %N 5 %R 10.3390/v15051042 %0 Journal Article %J Viruses %D 2023 %T Foundation of the Belgian Society for Viruses of Microbes and Meeting Report of Its Inaugural Symposium %A Agnieszka Latka %A Aertsen, Abram %A Dimitri Boeckaerts %A Blasdel, Bob %A Pieter-Jan Ceyssens %A Abel Garcia-Pino %A Annika Gillis %A Lavigne, Rob %A Gipsi Lima-Mendez %A Jelle Matthijnssens %A Jolien Onsea %A Eveline Peeters %A Pirnay, Jean-Paul %A Thiry, Damien %A Vandenheuvel, Dieter %A Els Van Mechelen %A Jolien Venneman %A Verbeken, Gilbert %A Jeroen Wagemans %A Yves Briers %B Viruses %V 15 %8 Jan-05-2023 %G eng %N 5 %R 10.3390/v15051213 %0 Journal Article %J Viruses %D 2023 %T Foundation of the Belgian Society for Viruses of Microbes and Meeting Report of Its Inaugural Symposium. %A Agnieszka Latka %A Aertsen, Abram %A Dimitri Boeckaerts %A Blasdel, Bob %A Pieter-Jan Ceyssens %A Abel Garcia-Pino %A Annika Gillis %A Lavigne, Rob %A Gipsi Lima-Mendez %A Jelle Matthijnssens %A Jolien Onsea %A Eveline Peeters %A Pirnay, Jean-Paul %A Thiry, Damien %A Vandenheuvel, Dieter %A Els Van Mechelen %A Jolien Venneman %A Verbeken, Gilbert %A Jeroen Wagemans %A Yves Briers %K Belgium %K Host Microbial Interactions %K Humans %K Viruses %X

The Belgian Society for Viruses of Microbes (BSVoM) was founded on 9 June 2022 to capture and enhance the collaborative spirit among the expanding community of microbial virus researchers in Belgium. The sixteen founders are affiliated to fourteen different research entities across academia, industry and government. Its inaugural symposium was held on 23 September 2022 in the Thermotechnical Institute at KU Leuven. The meeting program covered three thematic sessions launched by international keynote speakers: (1) virus-host interactions, (2) viral ecology, evolution and diversity and (3) present and future applications. During the one-day symposium, four invited keynote lectures, ten selected talks and eight student pitches were given along with 41 presented posters. The meeting hosted 155 participants from twelve countries.

%B Viruses %V 15 %8 2023 May 22 %G eng %N 5 %R 10.3390/v15051213 %0 Journal Article %J Nat Commun %D 2023 %T A genomic appraisal of invasive Salmonella Typhimurium and associated antibiotic resistance in sub-Saharan Africa. %A Sandra Van Puyvelde %A Tessa de Block %A Sushmita Sridhar %A Matt Bawn %A Robert A Kingsley %A Brecht Ingelbeen %A Mathew A Beale %A Barbé, Barbara %A Jeon, Hyon Jin %A Lisette Mbuyi-Kalonji %A Phoba, Marie-France %A Falay, Dadi %A Martiny, Delphine %A Vandenberg, Olivier %A Affolabi, Dissou %A Jean Pierre Rutanga %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Wim L Cuypers %A Marianne A B van der Sande %A Park Se Eun %A Simon Kariuki %A Kephas Otieno %A John P A Lusingu %A Joyce R Mbwana %A Samuel Adjei %A Anima Sarfo %A Seth O Agyei %A Kwaku P Asante %A Walter Otieno %A Lucas Otieno %A Marc C Tahita %A Lompo, Palpouguini %A Irving F Hoffman %A Mvalo, Tisungane %A Msefula, Chisomo %A Hassan-Hanga, Fatimah %A Stephen Obaro %A Mackenzie, Grant %A Stijn Deborggraeve %A Nicholas Feasey %A Florian Marks %A Calman A MacLennan %A Nicholas R Thomson %A Jacobs, Jan %A Gordon Dougan %A Samuel Kariuki %A Lunguya, Octavie %K Africa South of the Sahara %K Drug Resistance, Microbial %K Genomics %K Humans %K Salmonella Infections %K Salmonella typhimurium %X

Invasive non-typhoidal Salmonella (iNTS) disease manifesting as bloodstream infection with high mortality is responsible for a huge public health burden in sub-Saharan Africa. Salmonella enterica serovar Typhimurium (S. Typhimurium) is the main cause of iNTS disease in Africa. By analysing whole genome sequence data from 1303 S. Typhimurium isolates originating from 19 African countries and isolated between 1979 and 2017, here we show a thorough scaled appraisal of the population structure of iNTS disease caused by S. Typhimurium across many of Africa's most impacted countries. At least six invasive S. Typhimurium clades have already emerged, with ST313 lineage 2 or ST313-L2 driving the current pandemic. ST313-L2 likely emerged in the Democratic Republic of Congo around 1980 and further spread in the mid 1990s. We observed plasmid-borne as well as chromosomally encoded fluoroquinolone resistance underlying emergences of extensive-drug and pan-drug resistance. Our work provides an overview of the evolution of invasive S. Typhimurium disease, and can be exploited to target control measures.

%B Nat Commun %V 14 %8 2023 Oct 23 %G eng %N 1 %R 10.1038/s41467-023-41152-6 %0 Journal Article %J Nature Communications %D 2023 %T A genomic appraisal of invasive Salmonella Typhimurium and associated antibiotic resistance in sub-Saharan AfricaAbstract %A Sandra Van Puyvelde %A Tessa de Block %A Sushmita Sridhar %A Matt Bawn %A Robert A. Kingsley %A Brecht Ingelbeen %A Mathew A. Beale %A Barbé, Barbara %A Jeon, Hyon Jin %A Lisette Mbuyi-Kalonji %A Phoba, Marie-France %A Falay, Dadi %A Martiny, Delphine %A Vandenberg, Olivier %A Affolabi, Dissou %A Jean Pierre Rutanga %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Wim L. Cuypers %A Marianne A. B. van der Sande %A Park Se Eun %A Simon Kariuki %A Kephas Otieno %A John P.A. Lusingu %A Joyce R. Mbwana %A Samuel Adjei %A Anima Sarfo %A Seth O. Agyei %A Kwaku P. Asante %A Walter Otieno %A Lucas Otieno %A Marc C. Tahita %A Lompo, Palpouguini %A Irving F. Hoffman %A Mvalo, Tisungane %A Msefula, Chisomo %A Hassan-Hanga, Fatimah %A Stephen Obaro %A Mackenzie, Grant %A Stijn Deborggraeve %A Nicholas Feasey %A Florian Marks %A Calman A. MacLennan %A Nicholas R. Thomson %A Jacobs, Jan %A Gordon Dougan %A Samuel Kariuki %A Lunguya, Octavie %B Nature Communications %V 14 %8 Jan-12-2023 %G eng %N 1 %R 10.1038/s41467-023-41152-6 %0 Journal Article %J Nat Commun %D 2023 %T A global genomic analysis of Salmonella Concord reveals lineages with high antimicrobial resistance in Ethiopia. %A Wim L Cuypers %A Pieter Meysman %A Weill, François-Xavier %A Rene S Hendriksen %A Beyene, Getenet %A John Wain %A Nair, Satheesh %A Marie A Chattaway %A Blanca M Perez-Sepulveda %A Pieter-Jan Ceyssens %A Tessa de Block %A Winnie W Y Lee %A Maria Pardos de la Gandara %A Kornschober, Christian %A Jacob Moran-Gilad %A Kees T Veldman %A Martin Cormican %A Torpdahl, Mia %A Patricia I Fields %A Tomáš Černý %A Liselotte Hardy %A Bieke Tack %A Kate C Mellor %A Nicholas Thomson %A Gordon Dougan %A Stijn Deborggraeve %A Jacobs, Jan %A Kris Laukens %A Sandra Van Puyvelde %K Anti-Bacterial Agents %K Drug Resistance, Bacterial %K Ethiopia %K Genomics %K Humans %K Salmonella %X

Antimicrobial resistant Salmonella enterica serovar Concord (S. Concord) is known to cause severe gastrointestinal and bloodstream infections in patients from Ethiopia and Ethiopian adoptees, and occasional records exist of S. Concord linked to other countries. The evolution and geographical distribution of S. Concord remained unclear. Here, we provide a genomic overview of the population structure and antimicrobial resistance (AMR) of S. Concord by analysing genomes from 284 historical and contemporary isolates obtained between 1944 and 2022 across the globe. We demonstrate that S. Concord is a polyphyletic serovar distributed among three Salmonella super-lineages. Super-lineage A is composed of eight S. Concord lineages, of which four are associated with multiple countries and low levels of AMR. Other lineages are restricted to Ethiopia and horizontally acquired resistance to most antimicrobials used for treating invasive Salmonella infections in low- and middle-income countries. By reconstructing complete genomes for 10 representative strains, we demonstrate the presence of AMR markers integrated in structurally diverse IncHI2 and IncA/C2 plasmids, and/or the chromosome. Molecular surveillance of pathogens such as S. Concord supports the understanding of AMR and the multi-sector response to the global AMR threat. This study provides a comprehensive baseline data set essential for future molecular surveillance.

%B Nat Commun %V 14 %8 2023 Jun 14 %G eng %N 1 %R 10.1038/s41467-023-38902-x %0 Journal Article %J European Journal of Clinical Microbiology & Infectious Diseases %D 2023 %T Multicenter interlaboratory study of routine systems for the susceptibility testing of temocillin using a challenge panel of multidrug-resistant strains %A Corentin Deckers %A Florian Bélik %A Denis, Olivier %A Isabel Montesinos %A Bogaerts, Pierre %A Anne Marie Van den Abeele %A Pieter-Jan Ceyssens %A Huang, Te-Din %A Jerina Boelens %A Laetitia Brassinne %A Lucy Catteau %A Pieter-Jan Ceyssens %A Julie Descy %A Stefanie Desmet %A Sarah Gils %A Katrien Latour %A Bénédicte Lissoir %A Koen Magerman %A Veerle Matheeussen %A Cécile Meex %A Hector Rodriguez Villalobos %A Sarah Vandamme %A Van den Abeele, Anne-Marie %A Aline Vilain %A Kris Vernelen %A Ingrid Wybo %A Harun Yaras %A Nicolas Yin %B European Journal of Clinical Microbiology & Infectious Diseases %V 42 %8 Jan-12-2023 %G eng %N 12 %R 10.1007/s10096-023-04681-y %0 Journal Article %J Eur J Clin Microbiol Infect Dis %D 2023 %T Multicenter interlaboratory study of routine systems for the susceptibility testing of temocillin using a challenge panel of multidrug-resistant strains. %A Corentin Deckers %A Florian Bélik %A Denis, Olivier %A Isabel Montesinos %A Bogaerts, Pierre %A Jerina Boelens %A Laetitia Brassinne %A Julie Descy %A Stefanie Desmet %A Sarah Gils %A Bénédicte Lissoir %A Koen Magerman %A Veerle Matheeussen %A Cécile Meex %A Hector Rodriguez Villalobos %A Anne Marie Van den Abeele %A Kris Vernelen %A Pieter-Jan Ceyssens %A Huang, Te-Din %X

Accurate susceptibility result of temocillin (TMO) is important for treating infections caused by multidrug-resistant Enterobacterales. This multicenter study aimed to investigate the performance of routine temocillin testing assays against Enterobacterales challenging strains. Forty-seven selected clinical isolates were blindly analyzed by 12 Belgian laboratories using VITEK® 2 (n = 5) and BD Phoenix™ (n = 3) automated systems, ETEST® gradient strip (n = 3), and disk (3 brands) diffusion method (DD; n = 6) for temocillin susceptibility using standardized methodology. Results were interpreted using EUCAST 2023 criteria and compared to the broth microdilution (BMD; Sensititre™ panel) method used as gold standard. Methods' reproducibility was assessed by testing 3 reference strains in triplicate. A total of 702 organism-drug results were obtained against 33 TMO-susceptible and 14 TMO-resistant isolates. Excluding Proteae species (P. mirabilis and M. morganii), the essential agreement rates were excellent (91.5-100%) for all MIC-based methods. The highest category agreement was achieved by ETEST® (97.5%) followed by VITEK® 2 (93.2%), disk diffusion (91.6%), and BD Phoenix™ (88.5%). BD Phoenix™ and paper disk diffusion overcalled resistance (11.5% and 6.8% of major discrepancies, respectively), while ROSCO tablets diffusion and VITEK® 2 generated higher very major discrepancies (7.1% and 4.2% respectively). Inter-assay reproducibility was unsatisfactory using recommended E. coli ATCC 25922 strain but was excellent with E. coli ATCC 35218 and K. pneumoniae ATCC 700603 strains. This interlaboratory study suggests that routine testing methods provide accurate and reproducible TMO categorization results except for Proteae species.

%B Eur J Clin Microbiol Infect Dis %V 42 %8 2023 Dec %G eng %N 12 %R 10.1007/s10096-023-04681-y %0 Journal Article %J Microbiol Spectr %D 2023 %T The phage-encoded PIT4 protein affects multiple two-component systems of Pseudomonas aeruginosa %A Kaat Schroven %A Leena Putzeys %A Alison Kerremans %A Pieter-Jan Ceyssens %A Marta Vallino %A Paeshuyse, Jan %A Farhana Haque %A Yusuf, Ahmed %A Matthias D Koch %A Lavigne, Rob %X

More and more isolates have become resistant to antibiotics like carbapenem. As a consequence, ranks in the top three of pathogens for which the development of novel antibiotics is the most crucial. The pathogen causes both acute and chronic infections, especially in patients who are the most vulnerable. Therefore, efforts are urgently needed to develop alternative therapies. One path explored in this article is the use of bacteriophages and, more specifically, phage-derived proteins. In this study, a phage-derived protein was studied that impacts key virulence factors of the pathogen via interaction with multiple histidine kinases of TCSs. The fundamental insights gained for this protein can therefore serve as inspiration for the development of an anti-virulence compound that targets the bacterial TCS.

%B Microbiol Spectr %8 2023 Nov 14 %G eng %R 10.1128/spectrum.02372-23 %0 Generic %D 2023 %T Pyrogen testing of phage therapeutic products %A Marie Willocx %A Mathieu de Jode %A Flore Laurent %A Merabishvili, Maya %A Lavigne, Rob %A Pieter-Jan Ceyssens %A Celine Vanhee %K Endotoxins %K monocyte activation test %K phage therapy %K pyrogens %X

The use of phage therapeutic products has gained popularity in Belgium these last years, with treatments provided to more than 100 patients, against more than 10 different bacterial species. Therapeutic phage products are produced  in a bacterial host strain, hence pyrogen contamination (e.g. cell wall debris) is a major safety concern as these pro-inflammatory agents can cause unwanted clinical reactions with potential detrimental outcome. The most common and potent pyrogens are endotoxins, components of the cell wall of gram-negative bacteria which are released upon bacterial lysis.

 

In our routine phage quality control (QC) workflow, endotoxins are quantified using the Limulus amoebocyte lysate (LAL) assay and the animal-friendly alternative, the recombinant factor C (rFC) test. However, other (non-endotoxin) pyrogenic substances including compounds of the gram-positive bacterial cell wall, flagellin, etc. are not detected by those tests. As phages are being produced in a growing variety of bacterial host strains, including gram-positive bacteria, an expansion of the QC workflow is pivotal to limit the risk of non-endotoxin contamination. These pyrogens can be detected by the monocyte activation test (MAT), which employs human blood cells to quantify pyrogenicity in vitro. In this work, we discuss and compare the applicability of the LAL, rFC and MAT tests for pyrogen testing of clinical grade phage products based on the analysis of various productions manufactured by the Queen Astrid Military Hospital in Brussels. With a few exceptions, the results on different phages demonstrate that in general pyrogenic contamination is low for most samples.

%B BSVoM Symposium %I BSVoM %C Liège, Belgium %8 08/09/2023 %G eng %0 Journal Article %J BMC Infect Dis %D 2023 %T Salmonella Durban meningitis: case report and genomics study. %A Christelle Nanga Diasi %A Pieter-Jan Ceyssens %A Alexandra Vodolazkaia %A Marina Mukovnikova %A Sarah Dorval %A Olivia Bauraind %A Wesley Mattheus %K Genomics %K Humans %K Infant %K Male %K Meningitis, Bacterial %K Salmonella %K Salmonella Infections %K South Africa %X

BACKGROUND: Bacterial meningitis caused by non-typhoid Salmonella can be a fatal condition which is more common in low and middle-income countries.

CASE PRESENTATION: We report the case of a Salmonella meningitis in a Belgian six-month old male infant. The first clinical examination was reassuring, but after a few hours, his general state deteriorated. A blood test and a lumbar puncture were therefore performed. The cerebrospinal fluid analysis was compatible with a bacterial meningitis which was later identified by the NRC (National Reference Center) as Salmonella enterica serovar Durban.

CONCLUSIONS: In this paper, we present the clinical presentation, genomic typing, and probable sources of infection for an unusually rare serovar of Salmonella. Through an extended genomic analysis, we established its relationship to historical cases with links to Guinea.

%B BMC Infect Dis %V 23 %8 2023 May 20 %G eng %N 1 %R 10.1186/s12879-023-08308-7 %0 Journal Article %J Microbiol Spectr %D 2023 %T Targeted Chromosomal Barcoding Establishes Direct Genotype-Phenotype Associations for Antibiotic Resistance in Mycobacterium abscessus. %A Juan Calvet-Seral %A Estefanía Crespo-Yuste %A Vanessa Mathys %A Rodriguez-Villalobos, Hector %A Pieter-Jan Ceyssens %A Martin, Anandi %A Jesús Gonzalo-Asensio %X

A bedaquiline-resistant Mycobacterium abscessus isolate was sequenced, and a candidate mutation in the gene was identified as responsible for the antibiotic resistance phenotype. To establish a direct genotype-phenotype relationship of this mutation which results in a Asp-to-Ala change at position 29 (D29A), we developed a recombineering-based method consisting of the specific replacement of the desired mutation in the bacterial chromosome. As surrogate bacteria, we used two M. abscessus bedaquiline-susceptible strains: ATCC 19977 and the SL541 clinical isolate. The allelic exchange substrates used in recombineering carried either the sole D29A mutation or a genetic barcode of silent mutations in codons flanking the D29A mutation. After selection of bedaquiline-resistant M. abscessus colonies transformed with both substrates, we obtained equivalent numbers of recombinants. These resistant colonies were analyzed by allele-specific PCR and Sanger sequencing, and we demonstrated that the presence of the genetic barcode was linked to the targeted incorporation of the desired mutation in its chromosomal location. All recombinants displayed the same MIC to bedaquiline as the original isolate, from which the D29A mutation was identified. Finally, to demonstrate the broad applicability of this method, we confirmed the association of bedaquiline resistance with the A64P mutation in analysis performed in independent M. abscessus strains and by independent researchers. Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections caused by microorganisms. On the other hand, infections caused by affect people with chronic lung diseases, and their incidence has grown alarmingly in recent years. Further, these bacteria are known to easily develop AMR to the few therapeutic options available, making their treatment long-lasting and challenging. The recent introduction of new antibiotics against , such as bedaquiline, makes us anticipate a future when a plethora of antibiotic-resistant strains will be isolated and sequenced. However, in the era of whole-genome sequencing, one of the challenges is to unequivocally assign a biological function to each identified polymorphism. Thus, in this study, we developed a fast, robust, and reliable method to assign genotype-phenotype associations for putative antibiotic-resistant polymorphisms in .

%B Microbiol Spectr %8 2023 Mar 29 %G eng %R 10.1128/spectrum.05344-22 %0 Journal Article %J Microbial Genomics %D 2023 %T Using a combination of short and long read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum-beta-lactamases (ESBL) in clinical Shigella and Salmonella isolates in Belgium %A Bas Berbers %A Kevin Vanneste %A Nancy Roosens %A Marchal,K. %A Pieter-Jan Ceyssens %A Sigrid C.J. De Keersmaecker %K Antimicrobial resistance %K extended spectrum beta-lactamase %K Plasmids %K Salmonella %K Shigella %K whole genome sequencing. %B Microbial Genomics %V 9:000925 %8 23/01/2023 %G eng %& 1 %R DOI 10.1099/mgen.0.000925 %0 Journal Article %J Microbial Genomics %D 2023 %T Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical Shigella and Salmonella isolates in Belgium %A Bas Berbers %A Kevin Vanneste %A Nancy Roosens %A Marchal, Kathleen %A Pieter-Jan Ceyssens %A Sigrid C.J. De Keersmaecker %B Microbial Genomics %V 9 %8 Nov-01-2024 %G eng %N 1 %R 10.1099/mgen.0.000925 %0 Journal Article %J J Microbiol Methods %D 2022 %T The AMR-ARRAY: A modular bead array detecting β-lactam, (fluoro) quinolone, colistin, aminoglycoside and macrolide resistance determinants in Gram-negative bacteria. %A Michaël Timmermans %A Samuel Latour %A Pieter-Jan Ceyssens %A Cristina Garcia-Graells %A Carole Kowalewicz %A David Fretin %A Denis, Olivier %A P Wattiau %A Cécile Boland %K Aminoglycosides %K Anti-Bacterial Agents %K beta-Lactams %K Colistin %K Drug Resistance, Bacterial %K Escherichia coli %K Gram-Negative Bacteria %K Macrolides %K Quinolones %X

The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the β-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.

%B J Microbiol Methods %V 196 %8 2022 05 %G eng %R 10.1016/j.mimet.2022.106472 %0 Journal Article %J Cell Rep %D 2022 %T Metabolic reprogramming of Pseudomonas aeruginosa by phage-based quorum sensing modulation. %A Hendrix, Hanne %A Maria Zimmermann-Kogadeeva %A Zimmermann, Michael %A Sauer, Uwe %A De Smet, Jeroen %A Laurens Muchez %A Maries Lissens %A Ines Staes %A Voet, Marleen %A Jeroen Wagemans %A Pieter-Jan Ceyssens %A Noben, Jean-Paul %A Aertsen, Abram %A Lavigne, Rob %K Acetyltransferases %K Anti-Bacterial Agents %K Bacterial Proteins %K Bacteriophages %K Carbon %K Metabolic Networks and Pathways %K Metabolome %K Metabolomics %K Models, Biological %K Pseudomonas aeruginosa %K Quinolones %K Quorum Sensing %K Secondary Metabolism %K Viral Proteins %X

The Pseudomonas quinolone signal (PQS) is a multifunctional quorum sensing molecule of key importance to P. aeruginosa. Here, we report that the lytic Pseudomonas bacterial virus LUZ19 targets this population density-dependent signaling system by expressing quorum sensing targeting protein (Qst) early during infection. We demonstrate that Qst interacts with PqsD, a key host quinolone signal biosynthesis pathway enzyme, resulting in decreased levels of PQS and its precursor 2-heptyl-4(1H)-quinolone. The lack of a functional PqsD enzyme impairs LUZ19 infection but is restored by external supplementation of 2-heptyl-4(1H)-quinolone, suggesting that LUZ19 exploits the PQS system for successful infection. We establish a broad functional interaction network of Qst, which includes enzymes of cofactor biosynthesis pathways (CoaC/ThiD) and a non-ribosomal peptide synthetase pathway (PA1217). Qst therefore represents an exquisite example of intricate reprogramming of the bacterium by a phage, which may be further exploited as tool to combat antibiotic resistant bacterial pathogens.

%B Cell Rep %V 38 %8 2022 02 15 %G eng %N 7 %R 10.1016/j.celrep.2022.110372 %0 Journal Article %J Sci Rep %D 2022 %T Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification. %A David Rodriguez-Temporal %A Fernando Alcaide %A Ivana Mareković %A James Anthony O'Connor %A Rebecca Gorton %A Jakko van Ingen %A An Van den Bossche %A Genevieve Héry-Arnaud %A Clémence Beauruelle %A Dorothea Orth-Höller %A Juan-José Palacios-Gutiérrez %A Griselda Tudó %A Germán Bou %A Pieter-Jan Ceyssens %A Montserrat Garrigó %A Julià González-Martin %A Gilbert Greub %A Jaroslav Hrabak %A André Ingebretsen %A Maria Concepción Mediavilla-Gradolph %A Marina Oviaño %A Begoña Palop %A Arthur B Pranada %A Lidia Quiroga %A Maria Jesús Ruiz-Serrano %A Belén Rodriguez-Sanchez %X

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

%B Sci Rep %V 12 %8 2022 Jan 24 %G eng %N 1 %R 10.1038/s41598-022-05315-7 %0 Journal Article %J J Clin Microbiol %D 2022 %T Validation of Fourier Transform Infrared Spectroscopy for Serotyping of Streptococcus pneumoniae. %A I Passaris %A N Mauder %A M Kostrzewa %A I Burckhardt %A S Zimmermann %A N M van Sorge %A H-C Slotved %A S Desmet %A Pieter-Jan Ceyssens %K bacteria %K Humans %K Pneumococcal Infections %K Serogroup %K Serotyping %K Spectroscopy, Fourier Transform Infrared %K Streptococcus pneumoniae %X

Fourier transform infrared (FT-IR) spectroscopy (IR Biotyper; Bruker) allows highly discriminatory fingerprinting of closely related bacterial strains. In this study, FT-IR spectroscopy-based capsular typing of Streptococcus pneumoniae was validated as a rapid, cost-effective, and medium-throughput alternative to the classical phenotypic techniques. A training set of 233 strains was defined, comprising 34 different serotypes and including all 24 vaccine types (VTs) and 10 non-vaccine types (NVTs). The acquired spectra were used to (i) create a dendrogram where strains clustered together according to their serotypes and (ii) train an artificial neural network (ANN) model to predict unknown pneumococcal serotypes. During validation using 153 additional strains, we reached 98.0% accuracy for determining serotypes represented in the training set. Next, the performance of the IR Biotyper was assessed using 124 strains representing 59 non-training set serotypes. In this setting, 42 of 59 serotypes (71.1%) could be accurately categorized as being non-training set serotypes. Furthermore, it was observed that comparability of spectra was affected by the source of the Columbia medium used to grow the pneumococci and that this complicated the robustness and standardization potential of FT-IR spectroscopy. A rigorous laboratory workflow in combination with specific ANN models that account for environmental noise parameters can be applied to overcome this issue in the near future. The IR Biotyper has the potential to be used as a fast, cost-effective, and accurate phenotypic serotyping tool for S. pneumoniae.

%B J Clin Microbiol %V 60 %8 2022 Jul 20 %G eng %N 7 %R 10.1128/jcm.00325-22 %0 Journal Article %J Cell Rep %D 2021 %T The bacteriophage LUZ24 "Igy" peptide inhibits the Pseudomonas DNA gyrase. %A De Smet, Jeroen %A Jeroen Wagemans %A Maarten Boon %A Pieter-Jan Ceyssens %A Voet, Marleen %A Noben, Jean-Paul %A Julia Andreeva %A Dmitry Ghilarov %A Konstantin Severinov %A Lavigne, Rob %K Bacteriophage %K DNA Gyrase %X

The bacterial DNA gyrase complex (GyrA/GyrB) plays a crucial role during DNA replication and serves as a target for multiple antibiotics, including the fluoroquinolones. Despite it being a valuable antibiotics target, resistance emergence by pathogens including Pseudomonas aeruginosa are proving problematic. Here, we describe Igy, a peptide inhibitor of gyrase, encoded by Pseudomonas bacteriophage LUZ24 and other members of the Bruynoghevirus genus. Igy (5.6 kDa) inhibits in vitro gyrase activity and interacts with the P. aeruginosa GyrB subunit, possibly by DNA mimicry, as indicated by a de novo model of the peptide and mutagenesis. In vivo, overproduction of Igy blocks DNA replication and leads to cell death also in fluoroquinolone-resistant bacterial isolates. These data highlight the potential of discovering phage-inspired leads for antibiotics development, supported by co-evolution, as Igy may serve as a scaffold for small molecule mimicry to target the DNA gyrase complex, without cross-resistance to existing molecules.

%B Cell Rep %V 36 %8 2021 Aug 24 %G eng %N 8 %R 10.1016/j.celrep.2021.109567 %0 Journal Article %J J Clin Microbiol %D 2021 %T A Bioinformatics Whole-Genome Sequencing Workflow for Clinical Mycobacterium tuberculosis Complex Isolate Analysis, Validated Using a Reference Collection Extensively Characterized with Conventional Methods and In Silico Approaches %A Bert Bogaerts %A Thomas Delcourt %A Karine Soetaert %A Samira Boarbi %A Pieter-Jan Ceyssens %A Raf Winand %A Julien Van Braekel %A Sigrid C.J. De Keersmaecker %A Nancy Roosens %A Marchal, Kathleen %A Vanessa Mathys %A Kevin Vanneste %K Antimicrobial resistance %K Mycobacterium tuberculosis %K national reference center %K public health %K single nucleotide polymorphism %K VALIDATION %K whole genome sequencing %X

The use of whole genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For (MTB), in particular, WGS has the benefit of drastically reducing the time to generate results compared to most conventional phenotypic methods. Consequently, a multitude of solutions for analyzing WGS MTB data have been developed, but their successful integration in clinical and national reference laboratories is hindered by the requirement for their validation, for which a consensus framework is still largely absent. We developed a bioinformatics workflow for (Illumina) WGS-based routine typing of MTB Complex (MTBC) member isolates allowing complete characterization including (sub)species confirmation and identification (16S, /RD, , Single Nucleotide Polymorphism (SNP)-based antimicrobial resistance (AMR) prediction, and pathogen typing (spoligotyping, SNP barcoding, and core genome MultiLocus Sequence Typing). Workflow performance was validated on a per-assay basis using a collection of 238 in-house sequenced MTBC isolates, extensively characterized with conventional molecular biology-based approaches supplemented with public data. For SNP-based AMR prediction, results from molecular genotyping methods were supplemented with modified datasets allowing to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics >99% for all assays, except for spoligotyping where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid standardization of bioinformatics tools by the MTB community and other SNP-based applications regardless of the targeted pathogen(s). The bioinformatics workflow is available for academic and non-profit usage through the Galaxy instance of our institute at https://galaxy.sciensano.be.

%B J Clin Microbiol %8 2021 Mar 31 %G eng %R 10.1128/JCM.00202-21 %0 Journal Article %J PLoS One %D 2021 %T Coverage of the national surveillance system for human Salmonella infections, Belgium, 2016-2020. %A Nina Van Goethem %A An Van den Bossche %A Pieter-Jan Ceyssens %A Adrien Lajot %A Wim Coucke %A Kris Vernelen %A Nancy Roosens %A Sigrid C.J. De Keersmaecker %A Dieter Van Cauteren %A Wesley Mattheus %K Belgium %K Diagnostic Tests, Routine %K Disease Outbreaks %K Humans %K Public Health Surveillance %K Salmonella %K Salmonella Food Poisoning %K Salmonella Infections %K whole genome sequencing %X

INTRODUCTION: The surveillance of human salmonellosis in Belgium is dependent on the referral of human Salmonella isolates to the National Reference Center (NRC). Knowledge of current diagnostic practices and the coverage of the national Salmonella surveillance system are important to correctly interpret surveillance data and trends over time, to estimate the true burden of salmonellosis in Belgium, and to evaluate the appropriateness of implementing whole-genome sequencing (WGS) at this central level.

METHODS: The coverage of the NRC was defined as the proportion of all diagnosed human Salmonella cases in Belgium reported to the NRC and was assessed for 2019 via a survey among all licensed Belgian medical laboratories in 2019, and for 2016-2020 via a capture-recapture study using the Sentinel Network of Laboratories (SNL) as the external source. In addition, the survey was used to assess the impact of the implementation of culture-independent diagnostic tests (CIDTs) at the level of peripheral laboratory sites, as a potential threat to national public health surveillance programs.

RESULTS: The coverage of the NRC surveillance system was estimated to be 83% and 85%, based on the results of the survey and on the two-source capture-recapture study, respectively. Further, the results of the survey indicated a limited use of CIDTs by peripheral laboratories in 2019.

CONCLUSION: Given the high coverage and the limited impact of CIDTs on the referral of isolates, we may conclude that the NRC can confidently monitor the epidemiological situation and identify outbreaks throughout the country. These findings may guide the decision to implement WGS at the level of the NRC and may improve estimates of the true burden of salmonellosis in Belgium.

%B PLoS One %V 16 %8 2021 %G eng %N 8 %R 10.1371/journal.pone.0256820 %0 Journal Article %J Journal of Antimicrobial Chemotherapy %D 2021 %T Genomic epidemiology of persistently circulating MDR Shigella sonnei strains associated with men who have sex with men (MSM) in Belgium (2013–19) %A Nathalie Fischer %A Margo Maex %A Wesley Mattheus %A An Van den Bossche %A Dieter Van Cauteren %A Valeska Laisnez %A Hammami, Naïma %A Pieter-Jan Ceyssens %X

OBJECTIVESShigella sonnei resistant to first-line antibiotics azithromycin and ciprofloxacin are on the rise globally. The aim of this study was to describe the epidemiology of MDR S. sonnei in Belgium and to identify origins and circulating clusters through WGS.

METHODS: We undertook demographic, temporal and geographical analysis of 930 S. sonnei isolates submitted to the Belgian National Reference Centre for Salmonella and Shigella between 2017 and 2019. Phylogenetic analysis of WGS data, genotyping and identification of genetic markers of antimicrobial resistance was performed on 372 Belgian isolates submitted between 2013 and 2019.

RESULTSS. sonnei was identified in 75% (930/1253) of Belgian Shigella isolates submitted between 2017 and 2019. Overall, 7% (69/930) of isolates were resistant to ciprofloxacin alone, 6% (57/930) showed reduced susceptibility to azithromycin alone, and 24% (223/930) exhibited both. Men were at higher risk of carrying a double resistant S. sonnei strain, compared with women (risk ratio = 8.6, 95% CI = 5.4-13.9). Phylogenetic analysis revealed four independent Belgian clusters of persistently circulating MDR strains, associated with men who have sex with men (MSM) and of the same genotypes as previously described international MSM-related clades. Belgian isolates carried various incompatibility (Inc)-type plasmids, the SpA plasmid and ESBL genes.

CONCLUSIONS: In Belgium, S. sonnei isolates from men are much more likely to be resistant to important first-line antibiotics than isolates from women. Multiple co-circulating MDR S. sonnei clusters of different genotypes were identified in the MSM community. Further studies on risk groups are needed for targeted prevention, improved clinical and public health management and antimicrobial stewardship in Belgium.

%B Journal of Antimicrobial Chemotherapy %V 77 %8 Sep-10-2022 %G eng %N 1 %R 10.1093/jac/dkab377 %0 Journal Article %J Journal of Microbiological Methods %D 2021 %T A molecular assay for rapidly distinguishing the AviPro SALMONELLA VAC T vaccine strain from wild-type field isolates %A Pieter-Jan Ceyssens %A An Van den Bossche %A Lac Kim Phan %A Koenraad Van Hoorde %A Wesley Mattheus %K Luminex-based multiplex test %K Molecular differentiation %K Salmonella typhimurium %K Vac T %K vaccin %X

Rapid differentiation of the AviPro Salmonella VAC T strain from wild-type Salmonella ser. Typhimurium isolates is essential for the monitoring of veterinary isolates and targeted control actions. The distinction between the two strain types is routinely made by phenotypic antimicrobial resistance testing, but this sometime leads to ambiguous results with major economic implications. In this study, we used whole-genome sequencing to identify conserved and specific mutations in resistance and virulence genes which enable to distinguish field and vaccine strains. Based on this information, we developed and validated (n = 199) a Luminex-based assay targeting seven specific single-nucleotide polymorphisms. This molecular test is able to distinguish both Salmonella ser. Typhimurium types with 100% sensitivity and specificity within one working day.

%B Journal of Microbiological Methods %V 184 %8 May-2021 %G eng %R 10.1016/j.mimet.2021.106190 %0 Journal Article %J European Journal of Clinical Microbiology & Infectious Diseases %D 2021 %T Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019 %A An Van den Bossche %A Pieter-Jan Ceyssens %A Sarah Denayer %A Naïma Hammami %A Maaike van den Beld %A Timothy J Dallman %A Wesley Mattheus %K Cluster analyses %K Next-generation sequencing %K outbreak %K Shigella sonnei %X

In 2019, an outbreak of Shigella sonnei occurred during two youth camps in Belgium. The clustering of isolates from both camps was confirmed by next-generation sequencing, as well as a secondary infection of a technician. The outbreak strain clustered with internationally isolated strains from patients with recent travel history to Central America. This report exemplifies enhanced surveillance and international collaboration between public health institutes by enabling to link local outbreaks to region-specific sublineages circulating abroad. 

%B European Journal of Clinical Microbiology & Infectious Diseases %8 2021 Feb 11 %G eng %R https://doi.org/10.1007/s10096-021-04164-y %0 Journal Article %J Curr Opin Biotechnol %D 2021 %T Phage Biobank: Present Challenges and Future Perspectives. %A Ruby Cy Lin %A Jessica C Sacher %A Pieter-Jan Ceyssens %A Jan Zheng %A Ali Khalid %A Jonathan R Iredell %K Australia %K Bacteriophages %K Biological Specimen Banks %K Humans %K phage therapy %K Reproducibility of Results %X

After a century of use in human infection, the preparation and administration of therapeutic bacteriophages (phages) still relies on ad hoc partnerships of researchers, biotech companies, clinicians and regulators. There is a clear need to improve the reproducibility, safety and speed of the provision of suitable phages. Here we discuss the specific characteristics and challenges of a sustainable phage biobank and, as we build a national consortium aimed at delivering phage therapeutics, suggest a roadmap toward national biobanking and phage therapy initiatives using the Australian context as a model.

%B Curr Opin Biotechnol %V 68 %8 2021 04 %G eng %R 10.1016/j.copbio.2020.12.018 %0 Journal Article %J Microorganisms %D 2021 %T Phylogenomic Investigation of Increasing Fluoroquinolone Resistance among Belgian Cases of Shigellosis between 2013 and 2018 Indicates Both Travel-Related Imports and Domestic Circulation. %A Bert Bogaerts %A Raf Winand %A Julien Van Braekel %A Wesley Mattheus %A Sigrid C.J. De Keersmaecker %A Nancy Roosens %A Marchal, Kathleen %A Kevin Vanneste %A Pieter-Jan Ceyssens %X

Shigellosis is an acute enteric infection caused mainly by the species and . Since surveillance of these pathogens indicated an increase in ciprofloxacin-resistant samples collected in Belgium between 2013 and 2018, a subset of 148 samples was analyzed with whole genome sequencing (WGS) to investigate their dispersion and underlying genomic features associated with ciprofloxacin resistance. A comparison between observed phenotypes and WGS-based resistance prediction to ciprofloxacin revealed perfect correspondence for all samples. Core genome multi-locus sequence typing and single nucleotide polymorphism-typing were used for phylogenomic investigation to characterize the spread of these infections within Belgium, supplemented with data from international reference collections to place the Belgian isolates within their global context. For , substantial diversity was observed with ciprofloxacin-resistant isolates assigned to several phylogenetic groups. Besides travel-related imports, several clusters of highly similar Belgian isolates could not be linked directly to international travel suggesting the presence of domestically circulating strains. For , Belgian isolates were all limited to lineage III, and could often be traced back to travel to countries in Asia and Africa, sometimes followed by domestic circulation. For both species, several clusters of isolates obtained exclusively from male patients were observed. Additionally, we illustrated the limitations of conventional serotyping of , which was impacted by serotype switching. This study contributes to a better understanding of the spread of shigellosis within Belgium and internationally, and highlights the added value of WGS for the surveillance of this pathogen.

%B Microorganisms %V 9 %8 2021 Apr 06 %G eng %N 4 %R 10.3390/microorganisms9040767 %0 Journal Article %J Acta Clin Belg %D 2021 %T Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019. %A Karine Soetaert %A Pieter-Jan Ceyssens %A Samira Boarbi %A Bert Bogaerts %A Thomas Delcourt %A Kevin Vanneste %A Sigrid C.J. De Keersmaecker %A Nancy Roosens %A Alexandra Vodolazkaia %A Marina Mukovnikova %A Vanessa Mathys %X

OBJECTIVES: Since January 2019, the Belgian National Reference Center for Mycobacteria (NRC) has switched from conventional typing to prospective whole-genome sequencing (WGS) of all submitted complex (MTB) isolates. The ISO17025 validated procedure starts with semi-automated extraction and purification of gDNA directly from the submitted MGIT tubes, without preceding subculturing. All samples are then sequenced on an Illumina MiSeq sequencer and analyzed using an in-house developed and validated bioinformatics workflow to determine the species and antimicrobial resistance. In this study, we retrospectively compare results obtained via WGS to conventional phenotypic and genotypic testing, for all Belgian MTB strains analyzed in 2019 (n = 306).

RESULTS: In all cases, the WGS-based procedure was able to identify correctly the MTB species. Compared to MGIT drug susceptibility testing (DST), the sensitivity and specificity of genetic prediction of resistance to first-line antibiotics were respectively 100 and 99% (rifampicin, RIF), 90.5 and 100% (isoniazid, INH), 100 and 98% (ethambutol, EMB) and 61.1 and 100% (pyrazinamide, PZA). The negative predictive value was above 95% for these four first-line drugs. A positive predictive value of 100% was calculated for INH and PZA, 80% for RIF and 45% for EMB.

CONCLUSIONS: Our study confirms the effectiveness of WGS for the rapid detection of complex and its drug resistance profiles for first-line drugs even when working directly on MGIT tubes, and supports the introduction of this test into the routine workflow of laboratories performing tuberculosis diagnosis.

%B Acta Clin Belg %8 2021 Nov 09 %G eng %R 10.1080/17843286.2021.1999588 %0 Journal Article %J Int J Tuberc Lung Dis %D 2021 %T Whole-genome sequencing for TB source investigations: principles of ethical precision public health. %A A Van Rie %A D G de Viedma %A C Meehan %A I Comas %A T H Heupink %A E De Vos %A W A de Oñate %A Vanessa Mathys %A Pieter-Jan Ceyssens %A Groenen, G %A F González-Candelas %A A Forier %A E Juengst %X

Whole-genome sequencing (WGS) of allows rapid, accurate inferences about the sources, location and timing of transmission. However, in an era of heightened concern for personal privacy and science distrust, such inferences could result in unintended harm and undermine the public´s trust. We held interdisciplinary stakeholder discussions and performed ethical analyses of real-world illustrative cases to identify principles that optimise benefit and mitigate harm of WGS-driven TB source investigations. The speed and precision with which real-time WGS can be used to associate strains with sensitive information has raised important concerns. While detailed understanding of transmission events could mitigate harm to vulnerable patients and communities when otherwise unfairly blamed for TB outbreaks, the precision of WGS can also identify transmission events resulting in social blame, fear, discrimination, individual or location stigma, and the use of defaming language by the public, politicians and scientists. Public health programmes should balance the need to safeguard privacy with public health goals, transparency and individual rights, including the right to know who infects whom or where. Ethical challenges raised by real-time WGS-driven TB source investigation requires public health authorities to move beyond their current legal mandate and embrace transparency, privacy and community engagement.

%B Int J Tuberc Lung Dis %V 25 %8 2021 Mar 01 %G eng %N 3 %R 10.5588/ijtld.20.0886 %0 Journal Article %J Antibiotics (Basel) %D 2020 %T Development of an NGS-Based Workflow for Improved Monitoring of Circulating Plasmids in Support of Risk Assessment of Antimicrobial Resistance Gene Dissemination. %A Bas Berbers %A Pieter-Jan Ceyssens %A Bogaerts, Pierre %A Kevin Vanneste %A Nancy Roosens %A Marchal, Kathleen %A Sigrid C.J. De Keersmaecker %K Antimicrobial resistance %K MinION %K Plasmid sequencing %X

Antimicrobial resistance (AMR) is one of the most prominent public health threats. AMR genes localized on plasmids can be easily transferred between bacterial isolates by horizontal gene transfer, thereby contributing to the spread of AMR. Next-generation sequencing (NGS) technologies are ideal for the detection of AMR genes; however, reliable reconstruction of plasmids is still a challenge due to large repetitive regions. This study proposes a workflow to reconstruct plasmids with NGS data in view of AMR gene localization, i.e., chromosomal or on a plasmid. Whole-genome and plasmid DNA extraction methods were compared, as were assemblies consisting of short reads (Illumina MiSeq), long reads (Oxford Nanopore Technologies) and a combination of both (hybrid). Furthermore, the added value of conjugation of a plasmid to a known host was evaluated. As a case study, an isolate harboring a large, low-copy -carrying plasmid (>200 kb) was used. Hybrid assemblies of NGS data obtained from whole-genome DNA extractions of the original isolates resulted in the most complete reconstruction of plasmids. The optimal workflow was successfully applied to multidrug-resistant Kentucky isolates, where the transfer of an ESBL-gene-containing fragment from a plasmid to the chromosome was detected. This study highlights a strategy including wet and dry lab parameters that allows accurate plasmid reconstruction, which will contribute to an improved monitoring of circulating plasmids and the assessment of their risk of transfer.

%B Antibiotics (Basel) %V 9 %8 2020 Aug 11 %G eng %N 8 %R 10.3390/antibiotics9080503 %0 Journal Article %J Emerg Microbes Infect %D 2020 %T Genomic epidemiology of emerging ESBL-producing Kentucky in Europe. %A Claudia E Coipan %A Therese Westrell %A Angela H A M van Hoeck %A Erik Alm %A Saara Kotila %A Bas Berbers %A Sigrid C.J. De Keersmaecker %A Pieter-Jan Ceyssens %A Maria Louise Borg %A Marie Chattaway %A Jacquelyn McCormick %A Timothy J Dallman %A Eelco Franz %K ESBL %K Genomics %K Salmonella Kentucky %X

Global dissemination of ciprofloxacin-resistant Kentucky has been observed over the past decades. In recent years, there have been reports of extended-spectrum β-lactamase (ESBL) producing . Kentucky. Routine surveillance at the European Centre for Disease Prevention and Control (ECDC) detected cases with a ciprofloxacin-resistant . Kentucky with the ESBL-gene . Ensuing research identified 78 cases in 2013-2018 in eight European countries. Compared to other . Kentucky and non-typhoidal infections, reported to the European Surveillance System, these cases were more likely to be elderly and to present urinary-tract infections. Bayesian time-scaled phylogeny on whole genome sequences of isolates from these cases and supplementary isolates from public sequence databases was used to infer the origin and spread of this clone. We dated the origin of the clone to approximately 2005 in Northern Africa, most likely in Egypt. The geographic origin predicted by the phylogenetic analysis is consistent with the patients' travel history. Next to multiple introductions of the clone to Europe from Egypt, our analysis suggests that in some parts of Europe the clone might have formed a stable population, from which further spread has occurred. Comparative genomics indicated that the gene is present on the bacterial chromosome, within the type VI secretion system region. The gene is integrated downstream of the gene, on a 2854 bp plasmid fragment containing also IS. This is the first report of a chromosomally integrated CTX-M gene in spp. in Europe, previous studies having identified similar genes only on plasmids.

%B Emerg Microbes Infect %V 9 %8 2020 Dec %G eng %N 1 %R 10.1080/22221751.2020.1821582 %0 Journal Article %J Viruses %D 2020 %T The Phage-Encoded -Acetyltransferase Rac Mediates Inactivation of Transcription by Cleavage of the RNA Polymerase Alpha Subunit. %A Pieter-Jan Ceyssens %A De Smet, Jeroen %A Jeroen Wagemans %A Natalia Akulenko %A Evgeny Klimuk %A Subray Hedge %A Voet, Marleen %A Hendrix, Hanne %A Paeshuyse, Jan %A Bart Landuyt %A Xu, Hua %A John Blanchard %A Konstantin Severinov %A Lavigne, Rob %K acetylomics %K bacterial shutdown %K Bacteriophage %X

In this study, we describe the biological function of the phage-encoded protein RNA polymerase alpha subunit cleavage protein (Rac), a predicted Gcn5-related acetyltransferase encoded by phiKMV-like viruses. These phages encode a single-subunit RNA polymerase for transcription of their late (structure- and lysis-associated) genes, whereas the bacterial RNA polymerase is used at the earlier stages of infection. Rac mediates the inactivation of bacterial transcription by introducing a specific cleavage in the α subunit of the bacterial RNA polymerase. This cleavage occurs within the flexible linker sequence and disconnects the C-terminal domain, required for transcription initiation from most highly active cellular promoters. To achieve this, Rac likely taps into a novel post-translational modification (PTM) mechanism within the host . From an evolutionary perspective, this novel phage-encoded regulation mechanism confirms the importance of PTMs in the prokaryotic metabolism and represents a new way by which phages can hijack the bacterial host metabolism.

%B Viruses %V 12 %8 2020 09 02 %G eng %N 9 %R 10.3390/v12090976 %0 Journal Article %J Nat Commun %D 2019 %T Dissecting the molecular evolution of fluoroquinolone-resistant Shigella sonnei. %A Chung The, Hao %A Christine Boinett %A Pham Thanh, Duy %A Claire Jenkins %A Weill, François-Xavier %A Benjamin P Howden %A Mary Valcanis %A De Lappe, Niall %A Martin Cormican %A Sonam Wangchuk %A Ladaporn Bodhidatta %A Carl J Mason %A Nguyen, To Nguyen Thi %A Ha Thanh, Tuyen %A Phat Vinh Voong %A Duong, Vu Thuy %A Phu Huong Lan Nguyen %A Paul Turner %A Ryan Wick %A Pieter-Jan Ceyssens %A Guy Thwaites %A Kathryn E Holt %A Nicholas R Thomson %A Maia A Rabaa %A Stephen Baker %K Antimicrobial resistance %K Evolution %K fluoroquinolone %K Genomics %K Shigella sonnei %X

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.

%B Nat Commun %V 10 %8 2019 10 23 %G eng %N 1 %R 10.1038/s41467-019-12823-0 %0 Journal Article %J Microb Genom %D 2019 %T Global phylogenomics of multidrug-resistant Salmonella enterica serotype Kentucky ST198. %A Jane Hawkey %A Le Hello, Simon %A Benoît Doublet %A Sophie A Granier %A Rene S Hendriksen %A Florian W Fricke %A Pieter-Jan Ceyssens %A Camille Gomart %A Helen Billman-Jacobe %A Kathryn E Holt %A Weill, François-Xavier %K Antimicrobial resistance %K Salmonella enterica %K sequencing %X

Salmonella enterica serotype Kentucky can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S.enterica serotype Kentucky sequence type 198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug-resistant (MDR) S.enterica serotype Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island (SGI) 1 (variant SGI1-K) conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole and tetracycline. Phylogeographical analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone-resistance-determining regions (QRDRs) of DNA gyrase (gyrA) and DNA topoisomerase IV (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. enterica serotype Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome of the MDR clone carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems and/or azithromycin. These results indicate that most MDR S. enterica serotype Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials. This article contains data hosted by Microreact.

%B Microb Genom %V 5 %8 2019 07 %G eng %N 7 %R 10.1099/mgen.0.000269 %0 Generic %D 2019 %T RNA-Based susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Roby Bhattacharyya %A Jean-Yves Coppee %A Alain Baulard %A Deborah Hung %A Vanessa Mathys %A Pieter-Jan Ceyssens %B 29th ECCMID %C Amsterdam, The Netherlands %8 1316 April, 2019 %G eng %N ESCMID %0 Journal Article %J Microbiologyopen %D 2019 %T Shifting national surveillance of Shigella infections toward geno-serotyping by the development of a tailored Luminex assay and NGS workflow. %A Eleonora Ventola %A Bert Bogaerts %A Sigrid C.J. De Keersmaecker %A Kevin Vanneste %A Nancy Roosens %A Wesley Mattheus %A Pieter-Jan Ceyssens %K Luminex %K multiplex %K Public Health Surveillance %K sequencing %K Shigella %X

The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow-up by public health authorities. However, many clinical laboratories struggle to differentiate non-EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E. coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low-cost Luminex-based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S. sonnei and S. flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non-EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella-specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole-genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next-Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping-based surveillance of shigellosis in Belgium.

%B Microbiologyopen %8 2019 Mar 28 %G eng %R 10.1002/mbo3.807 %0 Journal Article %J Euro Surveill %D 2019 %T Strong increase of true and false positive mycobacterial cultures sent to the National Reference Centre in Belgium, 2007 to 2016. %A Karine Soetaert %A Lorenzo Subissi %A Pieter-Jan Ceyssens %A Vanfleteren, Brigitte %A Marianne Chantrenne %A Tommi Asikainen %A Els Duysburgh %A Vanessa Mathys %X

IntroductionIn 2007, a new federal legislation in Belgium prohibited non-biosafety level 3 laboratories to process culture tubes suspected of containing mycobacteria.AimTo present mycobacterial surveillance/diagnosis data from the Belgian National Reference Centre for mycobacteria (NRC) from 2007 to 2016.MethodsThis retrospective observational study investigated the numbers of analyses at the NRC and false positive cultures (interpreted as containing mycobacteria at referring clinical laboratories, but with no mycobacterial DNA detected by PCR in the NRC). We reviewed mycobacterial species identified and assessed trends over time of proportions of nontuberculous mycobacteria (NTM) vs complex (MTBc), and false positive cultures vs NTM.ResultsFrom 2007 to 2016, analyses requests to the NRC doubled from 12.6 to 25.3 per 100,000 inhabitants. A small but significant increase occurred in NTM vs MTBc proportions, from 57.9% (587/1,014) to 60.3% (867/1,437) (p < 0.001). Although NTM infection notification is not mandatory in Belgium, we annually received up to 8.6 NTM per 100,000 inhabitants. predominated (ca 20% of NTM cultures), but culture numbers rose significantly, from 13.0% (74/587) of NTM cultures in 2007 to 21.0% (178/867) in 2016 (RR: 1.05; 95% CI: 1.03-1.07). The number of false positive cultures also increased, reaching 43.3% (1,097/2,534) of all samples in 2016.ConclusionWe recommend inclusion of NTM in sentinel programmes. The large increase of false positive cultures is hypothesised to result from processing issues prior to arrival at the NRC, highlighting the importance of sample decontamination/transport and equipment calibration in peripheral laboratories.

%B Euro Surveill %V 24 %8 2019 Mar %G eng %N 11 %R 10.2807/1560-7917.ES.2019.24.11.1800205 %0 Journal Article %J Tuberculosis (Edinb) %D 2019 %T Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid. %A An Van den Bossche %A Hugo Varet %A Amandine Sury %A Odile Sismeiro %A Rachel Legendre %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %K Mycobacterium tuberculosis %K Nitroimidazoles %K Transcriptome %X

Tuberculosis (TB) is the most deadly infectious disease worldwide. To reduce TB incidence and counter the spread of multidrug resistant TB, the discovery and characterization of new drugs is essential. In this study, the transcriptional response of two Mycobacterium tuberculosis strains to a pressure of the recently approved delamanid is investigated. Total RNA sequencing revealed that the response to this bicyclic nitroimidazole shows many similarities with pretomanid, an anti-tuberculous drug from the same class. Although delamanid is found to inhibit cell wall synthesis, the expression of genes involved in this process were only mildly affected. In contrast, a clear parallel was found with components that affect aerobic respiration. This demonstrates that, besides the inhibition of cell wall synthesis, respiratory poisoning plays a fundamental role in the bactericidal effect of delamanid. Remarkably, the most highly induced genes comprise poorly characterized genes for which functional characterization might hint to the target molecule(s) of delamanid and its exact mode(s) of action.

%B Tuberculosis (Edinb) %V 117 %8 2019 07 %G eng %R 10.1016/j.tube.2019.05.002 %0 Journal Article %J Tuberculosis %D 2019 %T Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid %A An Van den Bossche %A Hugo Varet %A Amandine Sury %A Odile Sismeiro %A Rachel Legendre %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %K Delamanid %K Mycobacterium tuberculosis %K RNA sequencing %K Transciptomics %X

Tuberculosis (TB) is the most deadly infectious disease worldwide. To reduce TB incidence and counter the

spread of multidrug resistant TB, the discovery and characterization of new drugs is essential. In this study, the

transcriptional response of two Mycobacterium tuberculosis strains to a pressure of the recently approved delamanid

is investigated. Total RNA sequencing revealed that the response to this bicyclic nitroimidazole shows

many similarities with pretomanid, an anti-tuberculous drug from the same class. Although delamanid is found

to inhibit cell wall synthesis, the expression of genes involved in this process were only mildly affected. In

contrast, a clear parallel was found with components that affect aerobic respiration. This demonstrates that,

besides the inhibition of cell wall synthesis, respiratory poisoning plays a fundamental role in the bactericidal

effect of delamanid. Remarkably, the most highly induced genes comprise poorly characterized genes for which

functional characterization might hint to the target molecule(s) of delamanid and its exact mode(s) of action.

%B Tuberculosis %V 117 %8 Jan-07-2019 %G eng %R 10.1016/j.tube.2019.05.002 %0 Generic %D 2019 %T Transcriptional profiling of Mycobacterium tuberculosis suggests respiratory poisoning upon exposure to delamanid %A An Van den Bossche %A Hugo Varet %A Amandine Sury %A Odile Sismeiro %A Rachel Legendre %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %X

Tuberculosis (TB) is the most deadly infectious disease worldwide. To reduce TB incidence and counter the spread of multidrug resistant TB, the discovery and characterization of new drugs is essential. In this study, the transcriptional response of two Mycobacterium tuberculosis strains to a pressure of the recently approved delamanid is investigated. Total RNA sequencing revealed that the response to this bicyclic nitroimidazole shows many similarities with pretomanid, an anti-tuberculous drug from the same class. Although delamanid is found to inhibit cell wall synthesis, the expression of genes involved in this process were only mildly affected. In contrast, a clear parallel was found with components that affect aerobic respiration. This demonstrates that, besides the inhibition of cell wall synthesis, respiratory poisoning plays a fundamental role in the bactericidal effect of delamanid. Remarkably, the most highly induced genes comprise poorly characterized genes for which functional characterization might hint to the target molecule(s) of delamanid and its exact mode(s) of action.

%B 40th ESM conference %C Valencia, Spain %8 30/6-3/7/2019 %G eng %N European Society of Mycobacteria %0 Journal Article %J Front Microbiol %D 2019 %T Validation of a Bioinformatics Workflow for Routine Analysis of Whole-Genome Sequencing Data and Related Challenges for Pathogen Typing in a European National Reference Center: as a Proof-of-Concept. %A Bert Bogaerts %A Raf Winand %A Fu, Qiang %A Julien Van Braekel %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Sophie Bertrand %A Sigrid C.J. De Keersmaecker %A Nancy Roosens %A Kevin Vanneste %K national reference center %K Neisseria meningitidis %K public health %K VALIDATION %K whole-genome sequencing %X

Despite being a well-established research method, the use of whole-genome sequencing (WGS) for routine molecular typing and pathogen characterization remains a substantial challenge due to the required bioinformatics resources and/or expertise. Moreover, many national reference laboratories and centers, as well as other laboratories working under a quality system, require extensive validation to demonstrate that employed methods are "fit-for-purpose" and provide high-quality results. A harmonized framework with guidelines for the validation of WGS workflows does currently, however, not exist yet, despite several recent case studies highlighting the urgent need thereof. We present a validation strategy focusing specifically on the exhaustive characterization of the bioinformatics analysis of a WGS workflow designed to replace conventionally employed molecular typing methods for microbial isolates in a representative small-scale laboratory, using the pathogen as a proof-of-concept. We adapted several classically employed performance metrics specifically toward three different bioinformatics assays: resistance gene characterization (based on the ARG-ANNOT, ResFinder, CARD, and NDARO databases), several commonly employed typing schemas (including, among others, core genome multilocus sequence typing), and serogroup determination. We analyzed a core validation dataset of 67 well-characterized samples typed by means of classical genotypic and/or phenotypic methods that were sequenced in-house, allowing to evaluate repeatability, reproducibility, accuracy, precision, sensitivity, and specificity of the different bioinformatics assays. We also analyzed an extended validation dataset composed of publicly available WGS data for 64 samples by comparing results of the different bioinformatics assays against results obtained from commonly used bioinformatics tools. We demonstrate high performance, with values for all performance metrics >87%, >97%, and >90% for the resistance gene characterization, sequence typing, and serogroup determination assays, respectively, for both validation datasets. Our WGS workflow has been made publicly available as a "push-button" pipeline for Illumina data at https://galaxy.sciensano.be to showcase its implementation for non-profit and/or academic usage. Our validation strategy can be adapted to other WGS workflows for other pathogens of interest and demonstrates the added value and feasibility of employing WGS with the aim of being integrated into routine use in an applied public health setting.

%B Front Microbiol %V 10 %8 2019 %G eng %R 10.3389/fmicb.2019.00362 %0 Journal Article %J Indian Journal of Microbiology %D 2018 %T Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria %A Céline Rens %A Pieter-Jan Ceyssens %A Françoise Laval %A Lefèvre, Philippe %A Vanessa Mathys %A Daffé, Mamadou %A Véronique Fontaine %B Indian Journal of Microbiology %8 Apr-05-2018 %G eng %R 10.1007/s12088-018-0734-0 %0 Generic %D 2018 %T Evaluation of Non-Tuberculous Mycobacteria identification with maldi-tof mass spectrometry by applying sonication: a multicenter study %A EU Mycobacteria-MALDI study group %A An Van den Bossche %A Pieter-Jan Ceyssens %B ECCMID 2018 %C Madrid, Spain %8 04/2018 %G eng %N ESCMID %0 Journal Article %J Viruses %D 2018 %T The Magistral Phage. %A Pirnay, Jean-Paul %A Verbeken, Gilbert %A Pieter-Jan Ceyssens %A Isabelle Huys %A De Vos, Daniel %A Charlotte Ameloot %A Alan Fauconnier %K Antibiotic %K Antimicrobial resistance %K compounding pharmacy %K magistral preparation %K personalized medicine %K phage therapy %K regulatory framework %X

Since time immemorial, phages-the viral parasites of bacteria-have been protecting Earth's biosphere against bacterial overgrowth. Today, phages could help address the antibiotic resistance crisis that affects all of society. The greatest hurdle to the introduction of phage therapy in Western medicine is the lack of an appropriate legal and regulatory framework. Belgium is now implementing a pragmatic phage therapy framework that centers on the magistral preparation (compounding pharmacy in the US) of tailor-made phage medicines.

%B Viruses %V 10 %G eng %N 2 %R 10.3390/v10020064 %0 Journal Article %J Methods Mol Biol %D 2018 %T Preparing cDNA Libraries from Lytic Phage-Infected Cells for Whole Transcriptome Analysis by RNA-Seq. %A Blasdel, Bob %A Pieter-Jan Ceyssens %A Lavigne, Rob %K Bacteriophage %K RNA-seq %X

Whole genome wide analysis of transcription using RNA-Seq methods is a powerful way to elucidate differential expression of gene features in bacteria across different conditions as well as for discovering previously exotic RNA species. Indeed, RNA sequencing has revolutionized the study of bacterial transcription with the diversity and quantity of small noncoding RNA elements that have been found and its ability to clearly define operons, promoters , and terminators . We discuss our experience with applying RNA sequencing technology to analyzing the lytic cycle, including extraction, processing, and a guide to the customized statistical analysis necessary for analyzing differential host and phage transcription.

%B Methods Mol Biol %V 1681 %8 2018 %G eng %N 1681 %& 185 %R 10.1007/978-1-4939-7343-9_14 %0 Journal Article %J Sci Rep %D 2018 %T Rapid detection of tetracycline resistance in bovine Pasteurella multocida isolates by MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). %A Van Driessche, Laura %A Jade Bokma %A Gille, Linde %A Pieter-Jan Ceyssens %A Sparbier, Katrin %A Haesebrouck, Freddy %A Deprez, Piet %A Boyen, Filip %A Pardon, Bart %K Antimicrobial resistance %K detection %K MALDI-TOF %X

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8-101.5) and 100% (95% CI = 100-100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 93.5% (95% CI = 86.3-100.6) and 96.3% (95% CI = 91.3-101.3) respectively, classifying 95% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

%B Sci Rep %V 8 %8 2018 Sep 11 %G eng %N 1 %R 10.1038/s41598-018-31562-8 %0 Generic %D 2018 %T RNA-based drug susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Leen Rigouts %A Jean-Yves Coppee %A Vanessa Mathys %A Pieter-Jan Ceyssens %K Mycobacterium tuberculosis; antimicrobial resistance; diagnostics %X

To counter the long turnaround time of standard phenotypic Drug Susceptibility Testing (DST) of M. tuberculosis (Mtb), multiple DNA-based methods were successfully introduced over the last years. Although these are fast and sensitive, they (a) are based on the knowledge on resistance mutations which is limited for especially 2nd-line and new drugs, (b) do not distinguish living from death cells, (c) ignore all intrinsic resistance mechanisms like efflux pump overexpression, and (d) ignore the multifactorial influence of compensatory mutations. Here, we introduce a next-generation diagnostic test based on quantification of antibiotic-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure triggers specific transcriptional responses in susceptible, but not in resistant, microbes within minutes to a few hours. A major advantage of this method is that it avoids a long culture-dependent step, yet detects the resistance phenotype, independent of the specific cause of resistance.

First, the global transcriptional response of H37Rv and clinical Mtb strains on ten anti-TB drugs including Bedaquiline, Pyrazinamide and Delamanid was determined, to identify RNA Biomarkers. In a next phase, the RNA-based DST was developed in 96 well format. In short, 200 µl of a positively flagged MGIT culture is spiked with a specific concentration of a drug, while a control sample is incubated in absence of the drug. Multiplex mRNA quantification is performed directly on crude cell lysate using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fischer) technology. Normalized, relative genes expression levels (control vs drug) are combined to one numeric value which determines the drug susceptibility of the investigated strain.

The assay was optimized for parameters as cell density, incubation time and lysis method. I will present the results, showing that following a biomarker set of 5 responsive genes and 3 normalizing genes enables to distinguish low- and high resistant Mtb strains after an incubation step of 6 hours. With a total of 8 biomarker sets under optimization, the phenotypic drug resistance profile of up to 14 drugs can be determined for any combination of antibiotics in 96 well format.

 

%B Combating resistance : microbes and vectors %C Paris, France %8 16/11/2018 %G eng %N Institute Pasteur %0 Generic %D 2018 %T RNA-based drug susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Roby Bhattacharyya %A Jean-Yves Coppee %A Leen Rigouts %A Alain Baulard %A Deborah Hung %A Vanessa Mathys %A Pieter-Jan Ceyssens %K drug susceptibility testing %K Mycobacterium tuberculosis %B 39th Annual Congress of the European Society of Mycobacteriology %C Dresden, Germany %8 07/2018 %G eng %N ESM %0 Generic %D 2018 %T RNA-based drug susceptibility testing of Mycobacterium tuberculosis %A An Van den Bossche %A Roby Bhattacharyya %A Jean-Yves Coppee %A Leen Rigouts %A Alain Baulard %A Alexandra Vodolazkaia %A Deborah Hung %A Vanessa Mathys %A Pieter-Jan Ceyssens %X

Background

Multidrug resistance of Tuberculosis strains (MDR-TB) are one of the major WHO health concerns. One of the challenges that hampers the effective response to MDR-TB is the long turnaround time of phenotypic Drug Susceptibility Testing (DST). To counter this, new fast and sensitive DNA-based methods were successfully introduced over the last years. However, these (a) are based on the knowledge on resistance mutations, (b) do not distinguish living from dead cells, (c) ignore all intrinsic resistance mechanisms, and (d) ignore the influence of compensatory mutations.

Objectives

We introduce a next-generation diagnostic test based on quantification of drug-specific RNA biomarkers. The basic principle is that a brief antibiotic exposure triggers specific transcriptional responses in susceptible, but not in resistant, microbes within a few hours. This has the advantage that long culture-dependent steps are avoided, yet the resistance phenotype is detected independent of the specific cause of resistance.

Materials & Methods

First, the global transcriptional response of two TB strains to 10 anti-TB drugs was determined using RNAtaq-Seq. A set of highly responsive genes was selected for each drug and RNA-targeting probes were designed.

Next, the RNA-based DST was developed in 96 well format. In short, 200 µl of a positively flagged MGITTM (BD) culture is spiked with a drug, while a replicate is incubated in absence of the drug. Multiplex mRNA quantification is performed directly on crude cell lysates using a combination of the bead-based MagPixTM (Luminex) and QuantigeneTM Plex (Thermo Fisher) technology. The normalized expression levels are combined to one numeric value which determines the drug susceptibility of the investigated strain.

Results

We successfully developed 8 primary sets of RNA biomarkers for ten 1st-line, 2nd-line and new drugs. Taking isoniazid as proof of principle, we present a biomarker set of 5 responsive genes and 3 normalizing genes, which enables to distinguish susceptible, low- and high resistant TB strains after 6 hours incubation. Next, preliminary results demonstrate that the biomarker sets can successfully discriminate between susceptible and resistance strains for the selected drugs.

Conclusion

We present a robust, RNA-based DST without the need for RNA extraction. The assay was proven to be efficient for isoniazid. With a total of 8 biomarker sets under optimization, the drug resistance profile of up to 14 drugs can be determined.

%B 2nd St. Petersburg Symposium on Tuberculosis and Mycobacteria: Molecular Approach %C Sint Petersburg %G eng %N Institut Pasteur Sint Petersburg %0 Generic %D 2017 %T Clinical Impact of New Lab Technologies %A Pieter-Jan Ceyssens %K National Reference Centres %K Next-generation sequencing %B Seminar on Infectious Diseases %I WIV-ISP %C Brussels, Belgium %8 17/04/2017 %G eng %0 Generic %D 2017 %T Global transcriptional analyses of Mycobacterium tuberculosis using old and new drugs %A An Van den Bossche %A Pieter-Jan Ceyssens %A Roby Bhattacharyya %A Deborah Hung %A Vanessa Mathys %K antibiotics %K Mycobacterium tuberculosis %K Transcriptomics %X

Background: With 1.5 million deaths in 2014, the most recent report of the WHO ranks tuberculosis (TB) alongside HIV as a leading cause of death by infectious diseases. In combination, the spread of (multi-)drug resistant Mycobacterium tuberculosis is rising (480 000 new cases), impeding the treatment of infected patients.

Current TB drug resistance research is mainly focused on the genomic level, using WGS to search for resistance-causing mutations. However, transcriptional analyses can be a powerful tool as well. Analyses on susceptible strains provide information on the response to the stress a drug is causing, showing which mechanisms are activated to counter the effect of the drug. This might lead to new strategies for the development of new/complimentary drugs. On the other hand, this response gives an view on the working mechanism of the drug. Moreover, comparing the response of sensitive and resistant strains can yield additional information, like the activation of specific efflux pumps.

However, due to the high cost of RNAseq analyses, genome wide transcriptional analyses of drug influences on M. tuberculosis remain rather limited. Here we present a global study of the response of two pansensitive M. tuberculosis strains to eight TB-drugs. By using the approach called RNAtag-Seq, multiple samples were combined in one run, reducing time and cost.

 

Material/methods: For each drug (isoniazid, rifampicin, ethambutol, capreomycin, amikacin, linezolid, moxifloxacin and bedaquilin), twice the critical concentration was added to the cultures of two pansensitive M. tuberculosis strains. Samples were taken 0h, 2h, 6h and 24h after the administration of the drug.  RNA was extracted combining bead-beating in TRIzol and the Direct-Zol purification kit. After quality control, the extracts were fragmented, barcoded and pooled, cDNA libraries were constructed and sequenced using the HiSeq technology (Illumina®). Reads were mapped to the reference genome and normalized read counts were calculated per gene.

 

Results: For each drug a specific transcriptional response was mapped, revealing lists of up- and down-regulated genes. As an example and in accordance with previous studies, the highest induced genes in the presence of isoniazid belong to a cluster of genes that encodes components of the FAS-II (fatty acid synthase II) complex, which is targeted by isoniazid. On the other hand, a remarkable down-regulation of the NADH-dehydrogenase (ndh) cluster genes was noted. This can be assigned to the dependence of isoniazid target inhA on NADH. Lowering the ndh-activity is also seen in resistant strains.

 

Conclusions: By using RNAtag-seq, a global study of the transcriptional response of M. tuberculosis to several drugs could be made. These analyses can reveal the working mechanisms of existing and new drugs and provide new insights on the mechanism of resistance of strains.

 

%B ECCMID 2017 %I ECCMID %C Vienna, Austria %8 04/2017 %G eng %N ESCMID %0 Generic %D 2017 %T Global transcriptional analyses of Mycobacterium tuberculosis using old and new drugs %A An Van den Bossche %A Pieter-Jan Ceyssens %A Roby Bhattacharyya %A Deborah Hung %A Vanessa Mathys %K antibiotics %K Mycobacterium tuberculosis %K Transcriptomics %X

Background: With 1.5 million deaths in 2014, the most recent report of the WHO ranks tuberculosis (TB) alongside HIV as a leading cause of death by infectious diseases. In combination, the spread of (multi-)drug resistant Mycobacterium tuberculosis is rising (480 000 new cases), impeding the treatment of infected patients.

Current TB drug resistance research is mainly focused on the genomic level, using WGS to search for resistance-causing mutations. However, transcriptional analyses can be a powerful tool as well. Analyses on susceptible strains provide information on the response to the stress a drug is causing, showing which mechanisms are activated to counter the effect of the drug. This might lead to new strategies for the development of new/complimentary drugs. On the other hand, this response gives an view on the working mechanism of the drug. Moreover, comparing the response of sensitive and resistant strains can yield additional information, like the activation of specific efflux pumps.

However, due to the high cost of RNAseq analyses, genome wide transcriptional analyses of drug influences on M. tuberculosis remain rather limited. Here we present a global study of the response of two pansensitive M. tuberculosis strains to eight TB-drugs. By using the approach called RNAtag-Seq, multiple samples were combined in one run, reducing time and cost.

 

Material/methods: For each drug (isoniazid, rifampicin, ethambutol, capreomycin, amikacin, linezolid, moxifloxacin and bedaquilin), twice the critical concentration was added to the cultures of two pansensitive M. tuberculosis strains. Samples were taken 0h, 2h, 6h and 24h after the administration of the drug.  RNA was extracted combining bead-beating in TRIzol and the Direct-Zol purification kit. After quality control, the extracts were fragmented, barcoded and pooled, cDNA libraries were constructed and sequenced using the HiSeq technology (Illumina®). Reads were mapped to the reference genome and normalized read counts were calculated per gene.

 

Results: For each drug a specific transcriptional response was mapped, revealing lists of up- and down-regulated genes. As an example and in accordance with previous studies, the highest induced genes in the presence of isoniazid belong to a cluster of genes that encodes components of the FAS-II (fatty acid synthase II) complex, which is targeted by isoniazid. On the other hand, a remarkable down-regulation of the NADH-dehydrogenase (ndh) cluster genes was noted. This can be assigned to the dependence of isoniazid target inhA on NADH. Lowering the ndh-activity is also seen in resistant strains.

 

Conclusions: By using RNAtag-seq, a global study of the transcriptional response of M. tuberculosis to several drugs could be made. These analyses can reveal the working mechanisms of existing and new drugs and provide new insights on the mechanism of resistance of strains.

 

%B 38th Annual Congress of the European Society of Mycobacteriology %I ESM %C Sibenik, Craotia %8 06/2017 %G eng %N ESM %0 Generic %D 2017 %T Great diversity in Mcr-1 encoding plasmids among human E. coli and Salmonella spp. isolates in Belgium %A Pieter-Jan Ceyssens %A Bogaerts, Pierre %A Sigrid C.J. De Keersmaecker %A Sophie Bertrand %A Glupzynski, Youri %K colistin resistance %K mcr-1 %K NGS %B ECCMID 2017 %I ESCMID %C Vienna, Austria %V 1 %8 17/05/2017 %G eng %N ESCMID %& 1 %0 Journal Article %J Clin Microbiol Infect %D 2017 %T How To: Identify Non-Tuberculous Mycobacterium Species By Using Maldi-Tof Mass Spectrometry. %A Fernando Alcaide %A J Amlerová %A Germán Bou %A Pieter-Jan Ceyssens %A Pere Coll %A Dan Corcoran %A Marie-Sarah Fangous %A Iván González-Álvarez %A Rebecca Gorton %A Gilbert Greub %A Geneviève Hery-Arnaud %A Jaroslav Hrábak %A André Ingebretsen %A Brigid Lucey %A Ivana Marekoviċ %A Concepción Mediavilla-Gradolph %A James O'Connor %A Jim O'Mahony %A Onya Opota %A Brendan O'Reilly %A Dorothea Orth-Höller %A Marina Oviaño %A Juan José Palacios %A Begoña Palop %A Arthur Pranada %A Lidia Quiroga %A David Rodríguez-Temporal %A María Jesús Ruiz-Serrano %A Griselda Tudó %A An Van den Bossche %A Jakko van Ingen %A Belén Rodriguez-Sanchez %K MALDI-TOF MS %K Non-Tuberculoous Mycobacteria %X

BACKGROUND: The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application on the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS AIMS: The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases and the interpretation of the results.

SOURCES: Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods or the implementation of improved databases are discussed.

CONTENT: Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided.

IMPLICATIONS: Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable and inexpensive manner.

%B Clin Microbiol Infect %8 2017 Nov 21 %G eng %R 10.1016/j.cmi.2017.11.012 %0 Generic %D 2017 %T MALDI-TOF for the Identification and Drug Sensitivity Testing of Mycobacteria %A Pieter-Jan Ceyssens %K MALDI-TOF %K Mycobacteria %B Hot Topics in Microbiology - Join the Grapevine %I BRITISH SOCIETY FOR MICROBIAL TECHNOLOGY %C PHE, Colindale, UK %V 32 %8 12/05/2017 %G eng %N BRITISH SOCIETY FOR MICROBIAL TECHNOLOGY %0 Journal Article %J J Clin Microbiol %D 2017 %T Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria. %A Pieter-Jan Ceyssens %A Karine Soetaert %A Timke, Markus %A An Van den Bossche %A Sparbier, Katrin %A Koen De Cremer %A Kostrzewa, Markus %A Marijke Hendrickx %A Vanessa Mathys %K Antitubercular Agents %K Bacteriological Techniques %K Humans %K Mycobacterium tuberculosis %K Nontuberculous Mycobacteria %K Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization %X

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

%B J Clin Microbiol %V 55 %P 624-634 %8 2017 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/28003422?dopt=Abstract %R 10.1128/JCM.02089-16 %0 Generic %D 2017 %T Multicentric study on the improved identification of Non-Tuberculous Mycobacteria with MALDI-TOF MS using three different preprocessing protocols %A EU Mycobacteria-MALDI study group %A An Van den Bossche %A Pieter-Jan Ceyssens %B ECCMID 2017 %C Vienna, Austria %8 04/2017 %G eng %N ESCMID %0 Journal Article %J Clin Infect Dis %D 2017 %T Salmonella enterica serovar Typhi Producing CTX-M-15 Extended Spectrum β-Lactamase in the Democratic Republic of the Congo. %A Phoba, Marie-France %A Barbé, Barbara %A Lunguya, Octavie %A Lysette Masendu %A Deo Lulengwa %A Gordon Dougan %A Vanessa K. Wong %A Sophie Bertrand %A Pieter-Jan Ceyssens %A Jacobs, Jan %A Sandra Van Puyvelde %A Stijn Deborggraeve %K Democratic Republic of the Congo %K ESBL %K Salmonella %K Typhi %X

We report a typhoid fever case with a Salmonella enterica serovar Typhi isolate showing extended spectrum β-lactamase (ESBL) production in the Democratic Republic of the Congo. Whole genome sequencing revealed that the strain carried a plasmid-mediated CTX-M-15 ESBL gene and did not belong to the dominant H58 Salmonella Typhi clade.

%B Clin Infect Dis %V 65 %8 2017 Oct 01 %G eng %N 7 %R 10.1093/cid/cix342 %0 Government Document %D 2017 %T Salmonella infections: identification techniques for successful investigations %A Sophie Bertrand %A Wesley Mattheus %A Pieter-Jan Ceyssens %A Mathieu Gand %A Raymond Vanhoof %A N Botteldoorn %A Sarah Denayer %A Nancy Roosens %A Sigrid C.J. De Keersmaecker %K identification %K method %K Salmonella %B Labinfo %I AFSCA-FAVV %C Belgium %V 16 %8 2017 %G eng %& 41 %0 Journal Article %J J Antimicrob Chemother %D 2016 %T Development of a Luminex xTAG® assay for cost-effective multiplex detection of β-lactamases in Gram-negative bacteria. %A Pieter-Jan Ceyssens %A Cristina Garcia-Graells %A Fux, Frédéric %A N Botteldoorn %A Wesley Mattheus %A Wuyts, Véronique %A Sigrid C.J. De Keersmaecker %A Katelijne Dierick %A Sophie Bertrand %K Bacteriological Techniques %K beta-Lactamases %K Cost-Benefit Analysis %K Genotyping Techniques %K Gram-Negative Bacteria %K Humans %K Multiplex Polymerase Chain Reaction %K RNA, Ribosomal, 16S %X

OBJECTIVES: The objective of this study was to design and validate a genotyping method for multiplex identification of ESBLs and carbapenemases in Gram-negative bacilli. This assay had to be (i) superior to traditional (multiplex) PCR/sequencing-based tests in turn-around time, gene coverage and the ability to detect multiple variants of the same allele, and (ii) significantly more cost-effective than commercial microarrays and WGS. The targeted β-lactamases include ESBLs (CTX-M families and subtypes, ESBL and non-ESBL SHV- and TEM-likes, OXA-1/2/7-likes, PER, VEB, GES), plasmid-mediated cephalosporinases (CMY, MOX, FOX, ACC, DHA, MIR/ACT) and carbapenemases (OXA-48, NDM, KPC, VIM, IMP).

METHODS: A modular multiplex oligonucleotide ligation-PCR procedure was used, with read-out on a Luminex MAGPIX(®) platform. We designed 46 xTAG(®)-compatible probes targeting β-lactamase alleles and allele variants, and one probe targeting a conserved 16S rRNA region serving as a DNA extraction control. The assay was optimized using a collection of 48 reference strains and further validated using 105 foodborne ESBL-producing Escherichia coli isolates.

RESULTS: The specificity and selectivity of the test are 100% and 99.4%, respectively. Multiple variants of the same allele were successfully discriminated, as exemplified by five E. coli strains encoding both blaTEM-1 and blaTEM-52 genes. The turn-around time from single colony to result is 5 h and total consumable costs remained <€5 per sample.

CONCLUSIONS: We designed and validated the first Luminex-compatible genotyping assay that reliably and rapidly identifies a broad range of ESBL, pAmpC and carbapenemase producers in culture.

%B J Antimicrob Chemother %V 71 %P 2479-83 %8 2016 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/27287233?dopt=Abstract %R 10.1093/jac/dkw201 %0 Generic %D 2016 %T Development of Luminex assays for identification of antimicrobial resistance genes. %A Pieter-Jan Ceyssens %A Sophie Bertrand %E i3S meeting %K Antimicrobial %K Antimicrobial resistance %K Development %K identification %K Luminex %K resistance %B i3S meeting %8 0/0/2016 %G eng %N i3S meeting %1 39258 %2 .. %0 Journal Article %J PLoS One %D 2016 %T Diversity of Listeria monocytogenes Strains of Clinical and Food Chain Origins in Belgium between 1985 and 2014. %A Sophie Bertrand %A Pieter-Jan Ceyssens %A Yde, M %A Katelijne Dierick %A Boyen, F %A Jean Vanderpas %A Vanhoof, R %A Wesley Mattheus %K Adult %K Anti-Infective Agents %K Belgium %K DNA, Bacterial %K Drug Resistance, Bacterial %K Female %K Food Chain %K Food Microbiology %K Foodborne Diseases %K Humans %K Listeria monocytogenes %K Listeriosis %K Male %K Microbial Sensitivity Tests %K Serotyping %X

Listeriosis is a rare but severe disease, mainly caused by Listeria monocytogenes. This study shows the results of the laboratory-based surveillance of Listeriosis in Belgium over the period 1985-2014. Besides the incidence and some demographic data we present also more detailed microbiological and molecular characteristics of human strains isolated since 2000. The strains from the latter period were compared to food and animal strains from the same period. Our study shows that different food matrices were commonly contaminated with L. monocytogenes presenting the same PFGE profile as in patient's isolates. Since 1985, we observed a significant decrease in incidence of the Materno-Neonatal cases (from 0.15 to 0.04 cases /100,000 inhabitants-year), which is probably to be attributed to active prevention campaigns targeting pregnant women. Despite the strengthening of different control measures by the food industry, the incidence of non-Materno-Neonatal listeriosis increased in Belgium (from 0.3 to 0.7 cases /100,000 inhabitants-year), probably due to the rise of highly susceptible patients in an aging population. This significant increase found in non-Materno-Neonatal cases (slope coefficient 7.42%/year, P<0.0001) can be attributed to significant increase in incidence of isolates belonging to serovars 1/2a (n = 393, slope coefficient 6.62%/year, P<0.0001). Although resistance to antimicrobials is rare among L. monocytogenes isolates, a trend to increasing MIC values is evident with chloramphenicol, amoxicillin, tetracycline and ciprofloxacin. We show that fluoroquinolone resistance is not linked to chromosomal mutations, but caused by a variety of efflux pumps. Our study also shows that huge majority of known underlying pathologies (426 out of 785 cases) were cancers (185/426, 43.1%) and haematological malignancies (75/185, 40.5%). Moreover the risk population is susceptible to low levels of contamination in food stressing the need of prevention campaigns specifically targeting these persons.

%B PLoS One %V 11 %P e0164283 %8 2016 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/27723768?dopt=Abstract %& e0164283 %R 10.1371/journal.pone.0164283 %0 Journal Article %J ISME J %D 2016 %T High coverage metabolomics analysis reveals phage-specific alterations to Pseudomonas aeruginosa physiology during infection. %A De Smet, Jeroen %A Zimmermann, Michael %A Kogadeeva, Maria %A Pieter-Jan Ceyssens %A Vermaelen, Wesley %A Blasdel, Bob %A Bin Jang, Ho %A Sauer, Uwe %A Lavigne, Rob %K Genome, Viral %K Metabolomics %K Myoviridae %K Pseudomonas aeruginosa %K Pseudomonas Phages %X

Phage-mediated metabolic changes in bacteria are hypothesized to markedly alter global nutrient and biogeochemical cycles. Despite their theoretic importance, experimental data on the net metabolic impact of phage infection on the bacterial metabolism remains scarce. In this study, we tracked the dynamics of intracellular metabolites using untargeted high coverage metabolomics in Pseudomonas aeruginosa cells infected with lytic bacteriophages from six distinct phage genera. Analysis of the metabolomics data indicates an active interference in the host metabolism. In general, phages elicit an increase in pyrimidine and nucleotide sugar metabolism. Furthermore, clear phage-specific and infection stage-specific responses are observed, ranging from extreme metabolite depletion (for example, phage YuA) to complete reorganization of the metabolism (for example, phage phiKZ). As expected, pathways targeted by the phage-encoded auxiliary metabolic genes (AMGs) were enriched among the metabolites changing during infection. The effect on pyrimidine metabolism of phages encoding AMGs capable of host genome degradation (for example, YuA and LUZ19) was distinct from those lacking nuclease-encoding genes (for example, phiKZ), which demonstrates the link between the encoded set of AMGs of a phage and its impact on host physiology. However, a large fraction of the profound effect on host metabolism could not be attributed to the phage-encoded AMGs. We suggest a potentially crucial role for small, 'non-enzymatic' peptides in metabolism take-over and hypothesize on potential biotechnical applications for such peptides. The highly phage-specific nature of the metabolic impact emphasizes the potential importance of the 'phage diversity' parameter when studying metabolic interactions in complex communities.

%B ISME J %V 10 %P 1823-35 %8 2016 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/26882266?dopt=Abstract %& 1823 %R 10.1038/ismej.2016.3 %0 Generic %D 2016 %T MALDI-TOF for drug resistance testing in mycobacterium tuberculosis %A Pieter-Jan Ceyssens %A Karine Soetaert %A Koen De Cremer %A Sophie Bertrand %A Marijke Hendrickx %A Vanessa Mathys %K DRUG %K Drug Resistance %K MALDI-TOF %K Mycobacterium %K resistance %K TESTING %B ECCMID 2016 %I NA %C NA %8 9/4/2016 %G eng %N ECCMID %1 39257 %2 09/04/2016 %0 Generic %D 2016 %T MALDI-TOF for the identification of Mycobacteria and their drug resistance profiles %A An Van den Bossche %A Pieter-Jan Ceyssens %A Marijke Hendrickx %A Vanessa Mathys %K drug susceptibility testing %K identification %K MALDI-TOF %K Mycobacterium tuberculosis %X

In the last years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been increasingly introduced as a valuable method for the identification of bacteria and yeast1. This technique, which generates mass spectra that are compared to reference spectra in databases, is accurate, fast and cost-effective compared to traditional biochemical and molecular techniques.

For the identification of Mycobacteria, this method is more challenging due to the special need for inactivation and protein extraction. Although several studies have been published, there is no clear consensus on the processing of Mycobacteria for MALDI-TOF MS and outcomes vary from 55% correct identifications to 97%2-4. In this study, we optimized an extraction protocol and evaluated its efficiency using the Brüker Biotyper system v3.0 for 194 cultures. In total 88.6% (172/194) of the samples were correctly identified on the species-level using a cut-off score of 1.8 and 91.75% (178/194) using a cut-off of 1.65. All samples of the Mycobacterium tuberculosis complex were correctly classified (52/52), while 126 of the 142 Non-tuberculosis Mycobacteria were accurately identified at a 1.65 cut-off.

Recently, it was described that MALDI-TOF MS can also be used for drug-susceptibility testing (DST) in bacteria. The MS-ASTRA technology is based on the parallel incubation of a culture in presence or absence of an antibiotic. By adding an internal standard during the protein extraction, semi-quantitative measurements of the bacterial biomass can be derived from the MALDI-TOF spectra7.

In this study, we show that this technology can be applied on Mycobacteria. 34 clinical M. tuberculosis isolates were investigated for their resistance profile to four different drugs (rifampicin, isoniazid, ethambutol and linezolid), yielding a 100% concordance to the BACTEC MGIT results. Moreover, incubation with serial dilutions allowed a correct determination of the Minimal Inhibitory Concentrations of isoniazid and rifampicin. Therefore, this method has the potential to provide a cost-effective and fast alternative for classical phenotypic DST, independent of the mechanism of drug resistance. However, a current lack of automation hinders implementation in diagnostic laboratories.

%B 37th Annual Congress of the European Society of Mycobacteriology %I ESM %C Catania, Sicily, Italy %8 06/2017 %G eng %0 Journal Article %J BMC Infect Dis %D 2016 %T Microbiological, clinical and molecular findings of non-typhoidal Salmonella bloodstream infections associated with malaria, Oriental Province, Democratic Republic of the Congo. %A Falay, Dadi %A Kuijpers, Laura Maria Francisca %A Phoba, Marie-France %A De Boeck, Hilde %A Lunguya, Octavie %A Vakaniaki, Emmanuel %A Sophie Bertrand %A Wesley Mattheus %A Pieter-Jan Ceyssens %A Vanhoof, Raymond %A Devlieger, Hugo %A Van Geet, Chris %A Verheyen, Erik %A Ngbonda, Dauly %A Jacobs, Jan %K ADOLESCENT %K Adult %K Anti-Bacterial Agents %K Asian Continental Ancestry Group %K Azithromycin %K Bacteremia %K Ceftriaxone %K Child %K Child, Preschool %K Coinfection %K Democratic Republic of the Congo %K Disease Outbreaks %K Drug Resistance, Multiple, Bacterial %K Female %K Hospitalization %K Humans %K Infant %K Infant, Newborn %K Malaria %K Malaria, Falciparum %K Male %K Salmonella enteritidis %K Salmonella Infections %K Salmonella typhimurium %K Serogroup %K Tandem Repeat Sequences %X

BACKGROUND: In sub-Saharan Africa, non-typhoidal Salmonella (NTS) can cause bloodstream infections, referred to as invasive non-typhoidal Salmonella disease (iNTS disease); it can occur in outbreaks and is often preceded by malaria. Data from Central Africa is limited.

METHODS: Clinical, microbiological and molecular findings of NTS recovered in a blood culture surveillance project (2009-2014) were analyzed.

RESULTS: In March-July 2012 there was an epidemic increase in malaria infections in the Oriental Province of the Democratic Republic of the Congo (DRC). In one referral hospital, overall hospital admissions in June 2012 were 2.6 times higher as compared to the same period in the years before and after (336 versus an average of 128 respectively); numbers of malaria cases and blood transfusions were nearly three- and five-fold higher respectively (317 versus 112 and 250 versus 55). Case fatality rates (in-hospital deaths versus all admissions) peaked at 14.6 %. Salmonella Typhimurium and Salmonella Enteritidis together accounted for 88.9 % of pathogens isolated from blood cultures collected during an outreach visit to the affected districts in June 2012. Children infected with Salmonella Enteritidis (33 patient files available) tended to be co-infected with Plasmodium falciparum more often than children infected with Salmonella Typhimurium (40 patients files available) (81.8 % versus 62.5 %). Through the microbiological surveillance project (May 2009-May 2014) 113 unique NTS isolates were collected (28.5 % (113/396) of pathogens); most (95.3 %) were recovered from children < 15 years. Salmonella Typhimurium (n = 54) and Salmonella Enteritidis (n = 56) accounted for 47.8 % and of 49.6 % NTS isolates respectively. Multilocus variable-number tandem-repeat analysis (MLVA) revealed more heterogeneity for Salmonella Typhimurium than for Salmonella Enteritidis. Most (82/96, 85.4 %) NTS isolates that were available for antibiotic susceptibility testing were multidrug resistant. All isolates were susceptible to ceftriaxone and azithromycin.

CONCLUSION: During the peak of an epidemic increase in malaria in the DRC in 2012, a high proportion of multidrug resistant Salmonella Typhimurium and Salmonella Enteritidis were isolated from blood cultures. Overall, the two serovars showed subtle differences in clinical presentation and genetic diversity.

%B BMC Infect Dis %V 16 %P 271 %8 2016 Jun 10 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/27286886?dopt=Abstract %& 271 %R 10.1186/s12879-016-1604-1 %0 Journal Article %J PLoS One %D 2016 %T Molecular Analysis of Rising Fluoroquinolone Resistance in Belgian Non-Invasive Streptococcus pneumoniae Isolates (1995-2014). %A Pieter-Jan Ceyssens %A Van Bambeke, Françoise %A Wesley Mattheus %A Sophie Bertrand %A Fux, Frédéric %A Van Bossuyt, Eddie %A Damée, Sabrina %A Nyssen, Henry-Jean %A Stéphane De Craeye %A Verhaegen, Jan %A Tulkens, Paul M %A Vanhoof, Raymond %K Amino Acid Substitution %K ATP-Binding Cassette Transporters %K Bacterial Proteins %K Belgium %K DNA Gyrase %K Drug Resistance, Bacterial %K Female %K Fluoroquinolones %K Humans %K Longitudinal Studies %K Male %K Mutation, Missense %K Pneumococcal Infections %K Streptococcus pneumoniae %X

We present the results of a longitudinal surveillance study (1995-2014) on fluoroquinolone resistance (FQ-R) among Belgian non-invasive Streptococcus pneumoniae isolates (n = 5,602). For many years, the switch to respiratory fluoroquinolones for the treatment of (a)typical pneumonia had no impact on FQ-R levels. However, since 2011 we observed a significant decrease in susceptibility towards ciprofloxacin, ofloxacin and levofloxacin with peaks of 9.0%, 6.6% and 3.1% resistant isolates, respectively. Resistance to moxifloxacin arised sporadically, and remained <1% throughout the entire study period. We observed classical topoisomerase mutations in gyrA (n = 25), parC (n = 46) and parE (n = 3) in varying combinations, arguing against clonal expansion of FQ-R. The impact of recombination with co-habiting commensal streptococci on FQ-R remains marginal (10.4%). Notably, we observed that a rare combination of DNA Gyrase mutations (GyrA_S81L/GyrB_P454S) suffices for high-level moxifloxacin resistance, contrasting current model. Interestingly, 85/422 pneumococcal strains display MICCIP values which were lowered by at least four dilutions by reserpine, pointing at involvement of efflux pumps in FQ-R. In contrast to susceptible strains, isolates resistant to ciprofloxacin significantly overexpressed the ABC pump PatAB in comparison to reference strain S. pneumoniae ATCC 49619, but this could only be linked to disruptive terminator mutations in a fraction of these. Conversely, no difference in expression of the Major Facilitator PmrA, unaffected by reserpine, was noted between susceptible and resistant S. pneumoniae strains. Finally, we observed that four isolates displayed intermediate to high-level ciprofloxacin resistance without any known molecular resistance mechanism. Focusing future molecular studies on these isolates, which are also commonly found in other studies, might greatly assist in the battle against rising pneumococcal drug resistance.

%B PLoS One %V 11 %P e0154816 %8 2016 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/27227336?dopt=Abstract %& e0154816 %R 10.1371/journal.pone.0154816 %0 Journal Article %J Elife %D 2016 %T Structural elucidation of a novel mechanism for the bacteriophage-based inhibition of the RNA degradosome. %A An Van den Bossche %A Hardwick, Steven W %A Pieter-Jan Ceyssens %A Hendrix, Hanne %A Voet, Marleen %A Dendooven, Tom %A Bandyra, Katarzyna J %A De Maeyer, Marc %A Aertsen, Abram %A Noben, Jean-Paul %A Luisi, Ben F %A Lavigne, Rob %K Binding Sites %K Crystallography, X-Ray %K Endoribonucleases %K Host-Parasite Interactions %K Models, Molecular %K Multienzyme Complexes %K Polyribonucleotide Nucleotidyltransferase %K Protein Binding %K Protein Conformation %K Protein Multimerization %K Pseudomonas aeruginosa %K Pseudomonas Phages %K RNA Helicases %K Viral Proteins %X

In all domains of life, the catalysed degradation of RNA facilitates rapid adaptation to changing environmental conditions, while destruction of foreign RNA is an important mechanism to prevent host infection. We have identified a virus-encoded protein termed gp37/Dip, which directly binds and inhibits the RNA degradation machinery of its bacterial host. Encoded by giant phage фKZ, this protein associates with two RNA binding sites of the RNase E component of the Pseudomonas aeruginosa RNA degradosome, occluding them from substrates and resulting in effective inhibition of RNA degradation and processing. The 2.2 Å crystal structure reveals that this novel homo-dimeric protein has no identifiable structural homologues. Our biochemical data indicate that acidic patches on the convex outer surface bind RNase E. Through the activity of Dip, фKZ has evolved a unique mechanism to down regulate a key metabolic process of its host to allow accumulation of viral RNA in infected cells.

%B Elife %V 5 %8 2016 Jul 22 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/27447594?dopt=Abstract %R 10.7554/eLife.16413 %0 Generic %D 2016 %T WIV-ISP's experiences in the use of MALDI-TOF for the identification of Mycobacteria and their drug resistance profiles %A An Van den Bossche %A Pieter-Jan Ceyssens %K drug susceptibility testing %K identification %K MALDI-TOF %K Mycobacterium tuberculosis %X

An Van den Bossche presented WIV-ISP's experiences in the use of Brüker’s Biotyper MALDI-TOF for the identification of Mycobacteria and their drug resistance profiles. By using an optimized extraction protocol, they successfully identified 88.14 % of 194 Mycobacterial strains at a species-level and 3.09 % at genus-level, using the Brüker Database v3.0. For organisms of the Mycobacterium tuberculosis complex, identifications were 100% correct, while the concordance with standard methods was a bit lower for Nontuberculous Mycobacteria, 88.03%. Five organisms were misidentified. A novel method of drug resistance testing was also presented, which uses semi-quantitative measurement of bacterial biomass by creating MALDI-TOF spectra in the presence of an internal standard.

%I FARES/VRGT %C FARES/VRGT Brussels, Belgium %8 2016 %G eng %0 Generic %D 2015 %T 20 jaar surveillance van antibioticaresistentie in niet invasieve pneumokokkeninfecties. %A R. Vanhoof %A Pieter-Jan Ceyssens %K Klinische biologie %K Surveillance %B VUB-UZ Ma-na-Ma navormingsprogramma Klinische Biologie: 20 jaar pneumokokken surveillance in België. %I NA %C NA %8 0/0/2015 %G eng %N VUB-UZ Ma-na-Ma navormingsprogramma Klinische Biologie %1 37022 %2 02/06/2015 %0 Generic %D 2015 %T Antimicrobial resistance testing in 2020: Will we still be looking at the phenotype? %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Vanessa Mathys %A R. Vanhoof %A Michael Kalai %A Sophie Bertrand %E Institut Pasteur %K Antimicrobial %K Antimicrobial resistance %K at %K Phenotype %K resistance %K Still %K TESTING %B RIIP Meeting %8 0/0/2015 %G eng %N Institut Pasteur %1 37023 %2 04/2015 %0 Generic %D 2015 %T Blueprint of serotype distribution and antimicrobial resistance in human salmonellosis in Belgium (2009-2013) %A Pieter-Jan Ceyssens %A Wesley Mattheus %A R. Vanhoof %A Sophie Bertrand %K Antimicrobial %K Antimicrobial resistance %K Belgium %K conference %K distribution %K Human %K International %K ON %K Research %K resistance %B International Conference on Antimicrobial Research %I ICAR %C Madrid, Spain %V 4 %8 17/5/2015 %G eng %N ICAR %1

37037

%2 1/10/2014 ; 3/10/2014 %6 4 %R 10.1128/AAC.04203-14 %0 Generic %D 2015 %T Development of xTAG®-based assays as a tailor-made solution in the genotyping of antimicrobial resistance in Enterobacteriaceae. %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Sophie Bertrand %K a %K Antimicrobial %K Antimicrobial resistance %K AS %K Development %K Enterobacteriaceae %K resistance %B xMAP connect meeting %I NA %C NA %8 0/0/2015 %G eng %N xMAP connect %1 39152 %2 11/2015 %0 Generic %D 2015 %T Evolution of beta-lactam resistance in non-invasive clinical isolates of Streptococcus pneumoniae collected in Belgium in a 20 year survey (1995 to 2014). %A R. Vanhoof %A Sophie Bertrand %A Pieter-Jan Ceyssens %A Damee,S. %A Fux,F. %A Wesley Mattheus %A Nyssen,H.J. %A Van Bossuyt,E. %A Van Eldere,J. %A Jan Verhaegen %A The Belgian Streptococcus pneumoniae study group %K a %K Belgium %K beta-Lactam Resistance %K Clinical %K de %K Evolution %K non-invasive %K resistance %K Streptococcus pneumoniae %K survey %B 35 e Réunion Interdisciplinaire de chimiothérapie anti-infectieuse. %I RICAI %C Paris, France %8 0/0/2015 %G eng %N XX %1

39226

%2 14/12/2015;15/12/2015 %0 Journal Article %J Clin Infect Dis %D 2015 %T Invasive Salmonella Infections at Multiple Surveillance Sites in the Democratic Republic of the Congo, 2011-2014. %A Lisette Mbuyi Kalonji %A Post, Annelies %A Phoba, Marie-France %A Falay, Dadi %A Ngbonda, Dauly %A Muyembe, Jean-Jacques %A Sophie Bertrand %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Verhaegen, Jan %A Barbé, Barbara %A Kuijpers, Laura %A Van Geet, Chris %A Lunguya, Octavie %A Jacobs, Jan %K ADOLESCENT %K Adult %K Aged %K Anti-Bacterial Agents %K Azithromycin %K Bacteremia %K beta-Lactamases %K Child %K Child, Preschool %K Ciprofloxacin %K Democratic Republic of the Congo %K Drug Resistance, Multiple, Bacterial %K Epidemiological Monitoring %K Female %K Humans %K Infant %K Male %K Microbial Sensitivity Tests %K middle aged %K Salmonella %K Salmonella enteritidis %K Salmonella Infections %K Salmonella typhi %K Salmonella typhimurium %K Seasons %K Young adult %X

BACKGROUND: This study reports the microbiological landscape of Salmonella Typhi and invasive nontyphoidal Salmonella (iNTS) in the Democratic Republic of the Congo (DRC).

METHODS: Blood cultures obtained from hospital-admitted patients suspected of bloodstream infection (BSI) in 4 of 11 provinces in DRC (Kinshasa, Bas-Congo, Equateur, and Orientale) were processed. Sampling had started in 2007; the results for the period 2011-2014 are reported.

RESULTS: Salmonella Typhi and iNTS were cultured from 194 (1.4%) and 840 (5.9%), respectively, of 14,110 BSI episodes and ranked first among BSI pathogens in adults (65/300 [21.7%]) and children (783/1901 [41.2%]), respectively. A total of 948 of 1034 (91.7%) isolates were available for analysis (164 Salmonella Typhi and 784 iNTS). Salmonella Typhimurium and Salmonella Enteritidis represented 386 (49.2%) and 391 (49.9%), respectively, of iNTS isolates, fluctuating over time and geography and increasing during the rainy season. Adults accounted for <5% of iNTS BSI episodes. Children <5 years accounted for 20.3% of Salmonella Typhi BSI episodes. Among Salmonella Typhi, rates of multidrug resistance and decreased ciprofloxacin susceptibility (DCS) were 37.8% and 37.2%, respectively, and 18.3% displayed combined multidrug resistance and DCS; rates of azithromycin and ceftriaxone resistance were 0.6% and absent, respectively. Among NTS isolates, ≥80% (79.7% of Salmonella Enteritidis and 90.2% of Salmonella Typhimurium isolates) showed multidrug resistance, and <2.5% showed DCS. Combined extended-spectrum β-lactamase production (blaTEM-1 gene) and azithromycin resistance was noted in 12.7% of Salmonella Typhimurium isolates, appearing in Bas-Congo from 2013 onward.

CONCLUSIONS: Salmonella Typhi and NTS are major causes of BSI in DRC; their antimicrobial resistance is increasing.

%B Clin Infect Dis %V 61 Suppl 4 %P S346-53 %8 2015 Nov 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26449951?dopt=Abstract %R 10.1093/cid/civ713 %0 Generic %D 2015 %T Macrolide and tetracycline resistance and the presence of mef A, erm B and tet M genes in non-invasive clinical isolates of Streptococcus pneumoniae collected in Belgium in a 20 year survey (1995 to 2014). %A R. Vanhoof %A Sophie Bertrand %A Pieter-Jan Ceyssens %A Damee,S. %A Fux,F. %A Wesley Mattheus %A Nyssen,H.J. %A Van Bossuyt,E. %A Van Eldere,J. %A Jan Verhaegen %A The Belgian Streptococcus pneumoniae study group %K non-invasive %K resistance %K Streptococcus pneumoniae %K survey %B 35 e Réunion Interdisciplinaire de chimiothérapie anti-infectieuse. %I RICAI 2015 %C Paris, France %8 0/0/2015 %G eng %N XX %1

37043

%2 14/12/2015;15/12/2015 %0 Generic %D 2015 %T Microbial and clinical findings of an outbreak of non-typhoid Salmonella bloodstream infection associated with severe anemia, Oriental Province, Democratic Republic of Congo. %A Falay,D. %A Kuijpers,L. %A Phoba,M.F. %A De Boeck,H. %A Lunguya,O. %A E. Vakanyaki %A Sophie Bertrand %A Pieter-Jan Ceyssens %A R. Vanhoof %A Devlieger,H. %A C. Van Geet %A E. Verheyen %A Ngbonda,D. %A J. Jacobs %K an %K ANEMIA %K Bloodstream infection %K Clinical %K conference %K disease %K INFECTION %K International %K ON %K outbreak %K Salmonella %B 9 th International Conference on Typhoid and Invasive NTS Disease. %8 0/0/2015 %G eng %N XX %1 39165 %2 30/04/2015 ; 3/05/2015 %0 Journal Article %J Plos One %D 2015 %T Molecular surveillance of rising fluoroquinolone resistance in non-invasive S. pneumoniae isolates in Belgium collected in a survey (1995-2014) %A Pieter-Jan Ceyssens %A F. Van Bambeke %A Sophie Bertrand %A Damee,S. %A Fux,F. %A Wesley Mattheus %A Nyssen,H.J. %A Van Bossuyt,E. %A Jan Verhaegen %A The Belgian Streptococcus pneumoniae study group %A Tulkens,P.M. %A R. Vanhoof %K fluoroquinolone %K longitudinal study %K non-invasive infections %K Pat A/B %K Streptococcus pneumoniae %X

We present the results of a longitudinal surveillance study (1995-2014) on fluoroquinolone resistance (FQ-R) among Belgian non-invasive Streptococcus pneumoniae isolates (n = 5,602). For many years, the switch to respiratory fluoroquinolones for the treatment of (a)typical pneumonia had no impact on FQ-R levels. However, since 2011 we observed a significant decrease in susceptibility towards ciprofloxacin, ofloxacin and levofloxacin with peaks of 9.0%, 6.6% and 3.1% resistant isolates, respectively. Resistance to moxifloxacin arised sporadically, and remained <1% throughout the entire study period. We observed classical topoisomerase mutations in gyrA (n = 25), parC (n = 46) and parE (n = 3) in varying combinations, arguing against clonal expansion of FQ-R. The impact of recombination with co-habiting commensal streptococci on FQ-R remains marginal (10.4%). Notably, we observed that a rare combination of DNA Gyrase mutations (GyrA_S81L/GyrB_P454S) suffices for high-level moxifloxacin resistance, contrasting current model. Interestingly, 85/422 pneumococcal strains display MICCIP values which were lowered by at least four dilutions by reserpine, pointing at involvement of efflux pumps in FQ-R. In contrast to susceptible strains, isolates resistant to ciprofloxacin significantly overexpressed the ABC pump PatAB in comparison to reference strain S. pneumoniae ATCC 49619, but this could only be linked to disruptive terminator mutations in a fraction of these. Conversely, no difference in expression of the Major Facilitator PmrA, unaffected by reserpine, was noted between susceptible and resistant S. pneumoniae strains. Finally, we observed that four isolates displayed intermediate to high-level ciprofloxacin resistance without any known molecular resistance mechanism. Focusing future molecular studies on these isolates, which are also commonly found in other studies, might greatly assist in the battle against rising pneumococcal drug resistance.

%B Plos One %V 11 %8 26/05/2016 %G eng %N 5 %1

37042

%2 14/12/2015;15/12/2015 %& e0154816 %R 10.1371/journal.pone.0154816 %0 Journal Article %J PLoS One %D 2015 %T The search for therapeutic bacteriophages uncovers one new subfamily and two new genera of Pseudomonas-infecting Myoviridae. %A Henry, Marine %A Bobay, Louis-Marie %A Chevallereau, Anne %A Saussereau, Emilie %A Pieter-Jan Ceyssens %A Debarbieux, Laurent %K Animals %K DNA, Viral %K Genome, Viral %K mice %K Myoviridae %K Phylogeny %K Pseudomonas aeruginosa %K Pseudomonas Infections %K Pseudomonas Phages %K Tandem Mass Spectrometry %X

In a previous study, six virulent bacteriophages PAK_P1, PAK_P2, PAK_P3, PAK_P4, PAK_P5 and CHA_P1 were evaluated for their in vivo efficacy in treating Pseudomonas aeruginosa infections using a mouse model of lung infection. Here, we show that their genomes are closely related to five other Pseudomonas phages and allow a subdivision into two clades, PAK_P1-like and KPP10-like viruses, based on differences in genome size, %GC and genomic contents, as well as number of tRNAs. These two clades are well delineated, with a mean of 86% and 92% of proteins considered homologous within individual clades, and 25% proteins considered homologous between the two clades. By ESI-MS/MS analysis we determined that their virions are composed of at least 25 different proteins and electron microscopy revealed a morphology identical to the hallmark Salmonella phage Felix O1. A search for additional bacteriophage homologs, using profiles of protein families defined from the analysis of the 11 genomes, identified 10 additional candidates infecting hosts from different species. By carrying out a phylogenetic analysis using these 21 genomes we were able to define a new subfamily of viruses, the Felixounavirinae within the Myoviridae family. The new Felixounavirinae subfamily includes three genera: Felixounalikevirus, PAK_P1likevirus and KPP10likevirus. Sequencing genomes of bacteriophages with therapeutic potential increases the quantity of genomic data on closely related bacteriophages, leading to establishment of new taxonomic clades and the development of strategies for analyzing viral genomes as presented in this article.

%B PLoS One %V 10 %P e0117163 %8 2015 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25629728?dopt=Abstract %& e0117163 %R 10.1371/journal.pone.0117163 %0 Journal Article %J Antimicrob Agents Chemother %D 2015 %T Trends in serotype distribution and antimicrobial susceptibility in Salmonella enterica isolates from humans in Belgium, 2009 to 2013. %A Pieter-Jan Ceyssens %A Wesley Mattheus %A Vanhoof, Raymond %A Sophie Bertrand %K Bacterial Proteins %K Belgium %K beta-Lactam Resistance %K beta-Lactamases %K Cefotaxime %K Ciprofloxacin %K DNA Gyrase %K DNA Topoisomerase IV %K Drug Resistance, Bacterial %K Humans %K Microbial Sensitivity Tests %K Salmonella enterica %K Salmonella Infections %K Serogroup %X

The Belgian National Reference Centre for Salmonella received 16,544 human isolates of Salmonella enterica between January 2009 and December 2013. Although 377 different serotypes were identified, the landscape is dominated by S. enterica serovars Typhimurium (55%) and Enteritidis (19%) in a ratio which is inverse to European Union averages. With outbreaks of Salmonella serotypes Ohio, Stanley, and Paratyphi B variant Java as prime examples, 20 serotypes displayed significant fluctuations in this 5-year period. Typhoid strains account for 1.2% of Belgian salmonellosis cases. Large-scale antibiotic susceptibility analyses (n = 4,561; panel of 12 antibiotics) showed declining resistance levels in S. Enteritis and Typhimurium isolates for 8 and 3 tested agents, respectively. Despite low overall resistance to ciprofloxacin (4.4%) and cefotaxime (1.6%), we identified clonal lineages of Salmonella serotypes Kentucky and Infantis displaying rising resistance against these clinically important drugs. Quinolone resistance is mainly mediated by serotype-specific mutations in GyrA residues Ser83 and Asp87 (92.2% not wild type), while an additional ParC_Ser80Ile mutation leads to ciprofloxacin resistance in 95.5% S. Kentucky isolates, which exceeds European averages. Plasmid-mediated quinolone resistance (PMQR) alleles qnrA1 (n = 1), qnrS (n = 9), qnrD1 (n = 4), and qnrB (n = 4) were found in only 3.0% of 533 isolates resistant to nalidixic acid. In cefotaxime-resistant isolates, we identified a broad range of Ambler class A and C β-lactamase genes (e.g., bla(SHV-12), blaTEM-52, bla(CTX-M-14), and bla(CTX-M-15)) commonly associated with members of the family Enterobacteriaceae. In conclusion, resistance to fluoroquinolones and cefotaxime remains rare in human S. enterica, but clonal resistant serotypes arise, and continued (inter)national surveillance is mandatory to understand the origin and routes of dissemination thereof.

%B Antimicrob Agents Chemother %V 59 %P 544-52 %8 2015 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25385108?dopt=Abstract %& 544 %R 10.1128/AAC.04203-14 %0 Journal Article %J Journal of Virology %D 2014 %T Development of giant bacteriophage phiKZ is independent of the host transcription apparatus %A Pieter-Jan Ceyssens %A Minakhin,L. %A Yakunina,M. %A Klimuk,E. %A Blasdel,B. %A J. De Smet %A J.P. Noben %A Blasi,U. %A Severinov,K. %A Lavigne,R. %K 0 %K Absence %K Activity %K article %K Bacterial Proteins %K Bacteriophages %K Belgium %K Beta %K Biology %K Cell %K data %K Development %K DNA-Directed RNA Polymerases %K electronic %K Enzymes %K enzymology %K Functional %K gene %K Gene Expression Regulation,Viral %K Genes %K Genetic %K genetics %K Genome %K Genome,Viral %K growth & development %K Host-Pathogen Interactions %K im %K INFECTION %K Institute %K IS %K IT %K journal %K Laboratories %K map %K metabolism %K microbiology %K Molecular %K ON %K Operon %K Paper %K physiology %K protein %K Proteins %K Pseudomonas %K Pseudomonas aeruginosa %K Pseudomonas Phages %K Research %K Research Support %K Rna %K Russia %K SB - IM %K Science %K State %K technology %K time %K transcription,genetic %K Type %K Universities %K university %K USA %K Viral Proteins %K virology %K Virus Replication %X

Pseudomonas aeruginosa bacteriophage varphiKZ is the type representative of the giant phage genus, which is characterized by unusually large virions and genomes. By unraveling the transcriptional map of the approximately 280-kb varphiKZ genome to single-nucleotide resolution, we combine 369 varphiKZ genes into 134 operons. Early transcription is initiated from highly conserved AT-rich promoters distributed across the varphiKZ genome and located on the same strand of the genome. Early transcription does not require phage or host protein synthesis. Transcription of middle and late genes is dependent on protein synthesis and mediated by poorly conserved middle and late promoters. Unique to varphiKZ is its ability to complete its infection in the absence of bacterial RNA polymerase (RNAP) enzyme activity. We propose that transcription of the varphiKZ genome is performed by the consecutive action of two varphiKZ-encoded, noncanonical multisubunit RNAPs, one of which is packed within the virion, another being the product of early genes. This unique, rifampin-resistant transcriptional machinery is conserved within the diverse giant phage genus. IMPORTANCE: The data presented in this paper offer, for the first time, insight into the complex transcriptional scheme of giant bacteriophages. We show that Pseudomonas aeruginosa giant phage varphiKZ is able to infect and lyse its host cell and produce phage progeny in the absence of functional bacterial transcriptional machinery. This unique property can be attributed to two phage-encoded putative RNAP enzymes, which contain very distant homologues of bacterial beta and beta'-like RNAP subunits

%B Journal of Virology %V 88 %P 10501 - 10510 %8 2/9/2014 %G eng %N 18 %1

6

%& 10501 %R JVI.01347-14 [pii];10.1128/JVI.01347-14 [doi] %0 Journal Article %J Cell Microbiol. %D 2014 %T Functional elucidation of antibacterial phage ORFans targeting Pseudomonas aeruginosa %A Wagemans,J. %A Blasdel,B.G. %A Uytterhoeven,B. %A J. De Smet %A Paeshuyse,J. %A Cenens,W. %A Aertsen,A. %A Uetz,P. %A Delattre,A.S. %A Pieter-Jan Ceyssens %A Lavigne,R. %K a %K an %K analysi %K analysis %K article %K AS %K bacteria %K Bacteriophages %K Belgium %K Climate %K Database %K Databases %K Dna %K electronic %K function %K Functional %K functions %K future %K gene %K Genes %K Genome %K identification %K im %K INFECTION %K interaction %K IS %K IT %K journal %K KNOWLEDGE %K metabolism %K Microscopy %K Molecular %K ON %K pattern %K protein %K Proteins %K Pseudomonas %K Pseudomonas aeruginosa %K region %K Research %K Research Support %K Role %K SB - IM %K SELECTED %K Target %K Targets %K technology %K work %X

Immediately after infection, virulent bacteriophages hijack the molecular machinery of their bacterial host to create an optimal climate for phage propagation. For the vast majority of known phages, it is completely unknown which bacterial functions are inhibited or coopted. Early expressed phage genome regions are rarely identified, and often filled with small genes with no homology in databases (so-called ORFans). In this work, we first analysed the temporal transcription pattern of the N4-like Pseudomonas-infecting phages and selected 26 unknown, early phage ORFans. By expressing their encoded proteins individually in the host bacterium Pseudomonas aeruginosa, we identified and further characterized six antibacterial early phage proteins using time-lapse microscopy, radioactive labelling and pull-down experiments. Yeast two-hybrid analysis gaveclues to their possible role in phage infection. Specifically, we show that the inhibitory proteins may interact with transcriptional regulator PA0120, the replicative DNA helicase DnaB, the riboflavin metabolism key enzyme RibB, the ATPase PA0657and the spermidine acetyltransferase PA4114. The dependency of phage infection on spermidine was shown in a final experiment. In the future, knowledge of how phages shut down their hosts as well ass novel phage-host interaction partners could be very valuable in the identification of novel antibacterial targets

%B Cell Microbiol. %V 16 %P 1822 - 1835 %8 0/12/2014 %G eng %N 12 %1

39231

%& 1822 %R 10.1111/cmi.12330 [doi] %0 Journal Article %J Journal of Proteome Research %D 2014 %T Systematic identification of hypothetical bacteriophage proteins targeting key protein complexes of pseudomonas aeruginosa %A Pieter-Jan Ceyssens %A J. De Smet %A Hendrix,H. %A Bellon,H. %A Leimer,N. %A Wagemans,J. %A Delattre,A.S. %A Cenens,W. %A Aertsen,A. %A Landuyt,B. %A Minakhin,L. %A Severinov,K. %A J.P. Noben %A Lavigne,R. %K Bacteriophages %K MALDI-TOF MS %K Pseudomonas aeruginosa %X

Addressing the functionality of predicted genes remains an enormous challenge in the postgenomic era. A prime example of genes lacking functional assignments are the poorly conserved, early expressed genes of lytic bacteriophages, whose products are involved in the subversion of the host metabolism. In this study, we focused on the composition of important macromolecular complexes of Pseudomonas aeruginosa involved in transcription, DNA replication, fatty acid biosynthesis, RNA regulation, energy metabolism, and cell division during infection with members of seven distinct clades of lytic phages. Using affinity purifications of these host protein complexes coupled to mass spectrometric analyses, 37 host complex-associated phage proteins could be identified. Importantly, eight of these show an inhibitory effect on bacterial growth upon episomal expression, suggesting that these phage proteins are potentially involved in hijacking the host complexes. Using complementary protein-protein interaction assays, we further mapped the inhibitory interaction of gp12 of phage 14-1 to the alpha subunit of the RNA polymerase. Together, our data demonstrate the powerful use of interactomics to unravel the biological role of hypothetical phage proteins, which constitute an enormous untapped source of novel antibacterial proteins. (Data are available via ProteomeXchange with identifier PXD001199.)

%B Journal of Proteome Research %V 13 %P 4446 - 4456 %8 3/10/2014 %G eng %N 10 %1

23

%& 4446 %R 10.1021/pr500796n %0 Journal Article %J MBio. %D 2013 %T A multifaceted study of Pseudomonas aeruginosa shutdown by virulent podovirus LUZ19 %A Lavigne,R. %A Lecoutere,E. %A Wagemans,J. %A Cenens,W. %A Aertsen,A. %A Schoofs,L. %A Landuyt,B. %A Paeshuyse,J. %A Scheer,M. %A Schobert,M. %A Pieter-Jan Ceyssens %K Bacteriophages %K Gene Expression Profiling %K Microarray Analysis %K mRNA %K RNA-seq %X

In contrast to the rapidly increasing knowledge on genome content and diversity of bacterial viruses, insights in intracellular phage development and its impact on bacterial physiology are very limited. We present a multifaceted study combining quantitative PCR (qPCR), microarray, RNA-seq, and two-dimensional gel electrophoresis (2D-GE), to obtain a global overview of alterations in DNA, RNA, and protein content in Pseudomonas aeruginosa PAO1 cells upon infection with the strictly lytic phage LUZ19. Viral genome replication occurs in the second half of the phage infection cycle and coincides with degradation of the bacterial genome. At the RNA level, there is a sharp increase in viral mRNAs from 23 to 60% of all transcripts after 5 and 15 min of infection, respectively. Although microarray analysis revealed a complex pattern of bacterial up- and downregulated genes, the accumulation of viral mRNA clearly coincides with a general breakdown of abundant bacterial transcripts. Two-dimensional gel electrophoretic analyses shows no bacterial protein degradation during phage infection, and seven stress-related bacterial proteins appear. Moreover, the two most abundantly expressed early and late-early phage proteins, LUZ19 gene product 13 (Gp13) and Gp21, completely inhibit P. aeruginosa growth when expressed from a single-copy plasmid. Since Gp13 encodes a predicted GNAT acetyltransferase, this observation points at a crucial but yet unexplored level of posttranslational viral control during infection. IMPORTANCE: Massive genome sequencing has led to important insights into the enormous genetic diversity of bacterial viruses (bacteriophages). However, for nearly all known phages, information on the impact of the phage infection on host physiology and intracellular phage development is scarce. This aspect of phage research should be revitalized, as phages evolved genes which can shut down or redirect bacterial processes in a very efficient way, which can be exploited towards antibacterial design. In this context, we initiated a study of the human opportunistic pathogen Pseudomonas aeruginosa under attack by one its most common predators, the Phikmvlikevirus. By analyzing various stages of infection at different levels, this study uncovers new features of phage infection, representing a cornerstone for future studies on members of this phage genus

%B MBio. %V 4 %P e00061 - 13 %8 19/3/2013 %G eng %N 2 %1

45

%& e00061 %R mBio.00061-13 [pii];10.1128/mBio.00061-13 [doi] %0 Journal Article %J PLoS One %D 2011 %T Microbiological and molecular assessment of bacteriophage ISP for the control of Staphylococcus aureus %A Vandersteegen,K. %A Wesley Mattheus %A Pieter-Jan Ceyssens %A Bilocq,F. %A De Vos,D. %A Pirnay,J.P. %A J.P. Noben %A Merabishvili,M. %A Lipinska,U. %A Hermans,K. %A Lavigne,R. %K Antibiotic resistance %K phage %K phage therapy %K Staphylococcus aureus %X

The increasing antibiotic resistance in bacterial populations requires alternatives for classical treatment of infectious diseases and therefore drives the renewed interest in phage therapy. Methicillin resistant Staphylococcus aureus (MRSA) is a major problem in health care settings and live-stock breeding across the world. This research aims at a thorough microbiological, genomic, and proteomic characterization of S. aureus phage ISP, required for therapeutic applications. Host range screening of a large batch of S. aureus isolates and subsequent fingerprint and DNA microarray analysis of the isolates revealed a substantial activity of ISP against 86% of the isolates, including relevant MRSA strains. From a phage therapy perspective, the infection parameters and the frequency of bacterial mutations conferring ISP resistance were determined. Further, ISP was proven to be stable in relevant in vivo conditions and subcutaneous as well as nasal and oral ISP administration to rabbits appeared to cause no adverse effects. ISP encodes 215 gene products on its 138,339 bp genome, 22 of which were confirmed as structural proteins using tandem electrospray ionization-mass spectrometry (ESI-MS/MS), and shares strong sequence homology with the 'Twort-like viruses'. No toxic or virulence-associated proteins were observed. The microbiological and molecular characterization of ISP supports its application in a phage cocktail for therapeutic purposes

%B PLoS One %V 6 %P e24418 %8 0/0/2011 %G eng %N 9 %1

36579

%& e24418 %R http://dx.doi.org/10.1371/journal.pone.0024418 %0 Journal Article %J J Bacteriol %D 2006 %T Genomic analysis of Pseudomonas aeruginosa phages LKD16 and LKA1: establishment of the phiKMV subgroup within the T7 supergroup. %A Pieter-Jan Ceyssens %A Lavigne, Rob %A Wesley Mattheus %A Chibeu, Andrew %A Hertveldt, Kirsten %A Jan Mast %A Robben, Johan %A Volckaert, Guido %K Dna %K Electron %K Gene Order %K Mass Spectrometry %K Microscopy %K Molecular Sequence Data %K Nucleic Acid %K Podoviridae %K Pseudomonas aeruginosa %K Pseudomonas Phages %K Repetitive Sequences %K Sequence Analysis %K Sequence Homology %K Terminal Repeat Sequences %K Transmission %K Viral %K Viral Genes %K Viral Genome %K Viral Proteins %X

Lytic Pseudomonas aeruginosa phages LKD16 and LKA1 were locally isolated and morphologically classified as Podoviridae. While LKD16 adsorbs weakly to its host, LKA1 shows efficient adsorption (ka = 3.9 x 10(-9) ml min(-1)). LKA1, however, displays a narrow host range on clinical P. aeruginosa strains compared to LKD16. Genome analysis of LKD16 (43,200 bp) and LKA1 (41,593 bp) revealed that both phages have linear double-stranded DNA genomes with direct terminal repeats of 428 and 298 bp and encode 54 and 56 genes, respectively. The majority of the predicted structural proteins were experimentally confirmed as part of the phage particle using mass spectrometry. Phage LKD16 is closely related to bacteriophage phiKMV (83% overall DNA homology), allowing a more thoughtful gene annotation of both genomes. In contrast, LKA1 is more distantly related, lacking significant DNA homology and showing protein similarity to phiKMV in 48% of its gene products. The early region of the LKA1 genome has diverged strongly from phiKMV and LKD16, and intriguing differences in tail fiber genes of LKD16 and LKA1 likely reflect the observed discrepancy in infection-related properties. Nonetheless, general genome organization is clearly conserved among phiKMV, LKD16, and LKA1. The three phages carry a single-subunit RNA polymerase gene adjacent to the structural genome region, a feature which distinguishes them from other members of the T7 supergroup. Therefore, we propose that phiKMV represents an independent and widespread group of lytic P. aeruginosa phages within the T7 supergroup.

%B J Bacteriol %V 188 %8 2006 Oct %G eng %N 19 %R 10.1128/JB.00831-06