TY - JOUR T1 - Multinational collaboration in solving a European Braenderup outbreak linked to imported melons, 2021. JF - Euro Surveill Y1 - 2024 A1 - Hannah L Moore A1 - Martine Aabye A1 - Ann Hoban A1 - Bettina Rosner A1 - Stine K Lefevre A1 - Eva Litrup A1 - Luise Müller A1 - Ethelberg, Steen A1 - Sandra Simon A1 - Sooria Balasegaram A1 - Lesley Larkin A1 - Cecilia Jernberg A1 - Johanna Takkinen A1 - EU/EEA/UK S. Braenderup Outbreak Investigation Group A1 - Members of the EU/EEA/UK S. Braenderup Outbreak Investigation Group KW - Disease Outbreaks KW - Europe KW - Humans KW - Salmonella KW - Salmonella enterica KW - Salmonella Food Poisoning AB -

A genomic cluster of Braenderup ST22, a serovar of subsp. which causes symptoms of gastrointestinal illness, was notified by Danish authorities to the European Centre for Disease Prevention and Control (ECDC) on 3 May 2021. By 6 July 2021, Braenderup outbreak cases (n = 348) had been reported from 12 countries in the European Union/European Economic Area (EU/EEA) and the United Kingdom (UK), including 68 hospitalised cases. With support from affected EU/EEA countries, and in partnership with the European Food Safety Authority (EFSA), ECDC established an international outbreak investigation team to rapidly identify the source and prevent outbreak spread. Consumption information was shared with affected countries through a standard line list, revealing that 124 of 197 cases (63%) reported having eaten (any) melons within 7 days prior to disease onset. The speed and completeness of the investigation, which identified the outbreak vehicle as galia melons imported from Honduras in June 2021, was a direct result of extensive collaboration and information sharing between countries' national food safety and public health authorities. This article describes the outbreak and the benefits, successes, and challenges of multi-country collaboration for consideration in future large foodborne outbreaks across Europe.

VL - 29 CP - 1 M3 - 10.2807/1560-7917.ES.2024.29.1.2300273 ER - TY - JOUR T1 - Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning. JF - J Clin Microbiol Y1 - 2023 A1 - David Rodríguez-Temporal A1 - Laura Herrera A1 - Fernando Alcaide A1 - Diego Domingo A1 - Genevieve Héry-Arnaud A1 - Jakko van Ingen A1 - An Van den Bossche A1 - André Ingebretsen A1 - Clémence Beauruelle A1 - Eva Terschlüsen A1 - Samira Boarbi A1 - Vila, Neus A1 - Manuel J Arroyo A1 - Gema Méndez A1 - Patricia Muñoz A1 - Luis Mancera A1 - María Jesús Ruiz-Serrano A1 - Belén Rodriguez-Sanchez KW - Humans KW - Mycobacterium KW - Mycobacterium abscessus KW - Mycobacterium Infections, Nontuberculous KW - Nontuberculous Mycobacteria KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization AB -

Mycobacterium abscessus is one of the most common and pathogenic nontuberculous mycobacteria (NTM) isolated in clinical laboratories. It consists of three subspecies: M. abscessus subsp. , M. abscessus subsp. , and M. abscessus subsp. . Due to their different antibiotic susceptibility pattern, a rapid and accurate identification method is necessary for their differentiation. Although matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has proven useful for NTM identification, the differentiation of M. abscessus subspecies is challenging. In this study, a collection of 325 clinical isolates of M. abscessus was used for MALDI-TOF MS analysis and for the development of machine learning predictive models based on MALDI-TOF MS protein spectra. Overall, using a random forest model with several confidence criteria (samples by triplicate and similarity values >60%), a total of 96.5% of isolates were correctly identified at the subspecies level. Moreover, an improved model with Spanish isolates was able to identify 88.9% of strains collected in other countries. In addition, differences in culture media, colony morphology, and geographic origin of the strains were evaluated, showing that the latter had an impact on the protein spectra. Finally, after studying all protein peaks previously reported for this species, two novel peaks with potential for subspecies differentiation were found. Therefore, machine learning methodology has proven to be a promising approach for rapid and accurate identification of subspecies of M. abscessus using MALDI-TOF MS.

VL - 61 CP - 1 M3 - 10.1128/jcm.01110-22 ER - TY - Generic T1 - The key role of WGS-based surveillance of Listeria monocytogenes in both human and food isolates in outbreak investigations Y1 - 2022 A1 - An Van den Bossche A1 - Sigrid C.J. De Keersmaecker A1 - Wesley Mattheus A1 - Nancy Roosens A1 - Bert Bogaerts A1 - Kevin Vanneste A1 - Koenraad Van Hoorde A1 - Bavo Verhaegen KW - cgMLST KW - Listeria monocytogenes KW - NGS KW - outbreak JF - 26th Conference on Food Microbiology PB - BSFM CY - Brussels, Belgium CP - BSFM ER - TY - JOUR T1 - Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification. JF - Sci Rep Y1 - 2022 A1 - David Rodriguez-Temporal A1 - Fernando Alcaide A1 - Ivana Mareković A1 - James Anthony O'Connor A1 - Rebecca Gorton A1 - Jakko van Ingen A1 - An Van den Bossche A1 - Genevieve Héry-Arnaud A1 - Clémence Beauruelle A1 - Dorothea Orth-Höller A1 - Juan-José Palacios-Gutiérrez A1 - Griselda Tudó A1 - Germán Bou A1 - Pieter-Jan Ceyssens A1 - Montserrat Garrigó A1 - Julià González-Martin A1 - Gilbert Greub A1 - Jaroslav Hrabak A1 - André Ingebretsen A1 - Maria Concepción Mediavilla-Gradolph A1 - Marina Oviaño A1 - Begoña Palop A1 - Arthur B Pranada A1 - Lidia Quiroga A1 - Maria Jesús Ruiz-Serrano A1 - Belén Rodriguez-Sanchez AB -

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.

VL - 12 CP - 1 M3 - 10.1038/s41598-022-05315-7 ER - TY - JOUR T1 - Coverage of the national surveillance system for human Salmonella infections, Belgium, 2016-2020. JF - PLoS One Y1 - 2021 A1 - Nina Van Goethem A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens A1 - Adrien Lajot A1 - Wim Coucke A1 - Kris Vernelen A1 - Nancy Roosens A1 - Sigrid C.J. De Keersmaecker A1 - Dieter Van Cauteren A1 - Wesley Mattheus KW - Belgium KW - Diagnostic Tests, Routine KW - Disease Outbreaks KW - Humans KW - Public Health Surveillance KW - Salmonella KW - Salmonella Food Poisoning KW - Salmonella Infections KW - whole genome sequencing AB -

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.

VL - 16 CP - 8 M3 - 10.1371/journal.pone.0256820 ER - TY - JOUR T1 - A molecular assay for rapidly distinguishing the AviPro SALMONELLA VAC T vaccine strain from wild-type field isolates JF - Journal of Microbiological Methods Y1 - 2021 A1 - Pieter-Jan Ceyssens A1 - An Van den Bossche A1 - Lac Kim Phan A1 - Koenraad Van Hoorde A1 - Wesley Mattheus KW - Luminex-based multiplex test KW - Molecular differentiation KW - Salmonella typhimurium KW - Vac T KW - vaccin AB -

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.

VL - 184 M3 - 10.1016/j.mimet.2021.106190 ER - TY - JOUR T1 - Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019 JF - European Journal of Clinical Microbiology & Infectious Diseases Y1 - 2021 A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens A1 - Sarah Denayer A1 - Naïma Hammami A1 - Maaike van den Beld A1 - Timothy J Dallman A1 - Wesley Mattheus KW - Cluster analyses KW - Next-generation sequencing KW - outbreak KW - Shigella sonnei AB -

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. 

M3 - https://doi.org/10.1007/s10096-021-04164-y ER - TY - Generic T1 - Listeria surveillance in Belgium Y1 - 2019 A1 - An Van den Bossche A1 - Wesley Mattheus KW - Listeria KW - surveillence AB -

As is the case in the rest of Europe, the annual incidence of Listeria monocytogenes infections in humans significantly increased over the years. Over the last 18 years, the incidence rates varied between 0.4 and 0.9 cases/100 000 inhabitants, which is relatively high compares to other European countries (average of 0.43/100 000 inhabitants, ECDC report 2016). This increasing trend is mainly due to an increase of non-maternal-neonatal (n-MN) cases, since maternal-neonatal (MN) cases have been decreasing over the years. This latter is a consequence of active prevention campaigns targeting pregnant women. However, in 2018 a relatively high number of 11 MN cases were reported.

In Belgium, listeriosis is a notifiable infectious disease, and strains are voluntarily sent to the National Reference Center (NRC). Identity confirmation is performed using biochemical assays and serotyping by slide agglutination. Since 2018, according to recent evolution in molecular typing, the NRC completely switched to Whole-Genome-Sequencing (WGS) for cluster detection. Therefore, an in-house Galaxy-based bio-informatic pipeline was developed. For the strain collection from 2010 until 2017 both classical data (MLST and PFGE) as cgMLST data are available.

Of all confirmed L. monocytogenes cases in humans, serotypes 1/2a and 4b are most frequently observed. During the period 2000-2018, they comprise 47.1% and 36.2% of the cases, respectively. Notably, the incidence of serotype 4b remains rather stable, while the increase of serotype 1/2a is in relation with the increase of the overall incidence of Listeria cases. The number of sporadic 1/2a cases (unique pulsovars or cgMLST profiles) remained stable, whereas the proportion of cases related to clusters corresponded with the fluctuating annual incidence. Antibiotic resistance to antimicrobials remains a rare event among L. monocytogenes isolates, although a significant increase of MIC50 and MIC90 values for ciprofloxacin resistance were noted recently (Bertrand et al, 2016).

JF - ISOPOL PB - ISOPOL CY - Toronto, Canada CP - International Symposium on Problems with Listeria and Listeriosis ER - TY - BOOK T1 - Phage–Host Protein–Protein Interactions Using Strep-tag® II Purifications T2 - Phage–Host Protein–Protein Interactions Using Strep-tag® II Purifications Y1 - 2019 A1 - De Smet, Jeroen A1 - Hendrix, Hanne A1 - An Van den Bossche ED - Martha R.J. Clokie ED - Andrew Kropinski ED - Lavigne, Rob KW - affinity purifications; bacteriophage; host protein; mass spectrometry; pseudomonas aeruginosa; phage; protein interactions; AB -

After injecting their genome into the bacterial host cell, bacteriophages need to convert the host metabolism toward efficient phage production. For this, specific proteins have evolved which interact with key host proteins to inhibit, activate or redirect the function of these proteins. Since 70% of the currently annotated phage genes are hypothetical proteins of unknown function, the identification and characterization of these phage proteins involved in host–phage protein–protein interactions remains challenging. Here, we describe a method to identify phage proteins involved in host–phage protein–protein interactions using a combination of affinity purifications and mass spectrometry analyses. A bacterial strain is engineered in which a bacterial target protein is fused to a Strep-tag® II at the C-terminal end. This strain is infected with a specific bacteriophage, followed by an affinity purification of the tagged protein which allows the copurification of all bacterial and phage specific interacting proteins. After SDS-PAGE analysis and an in-gel trypsin digestion, the purified interacting proteins are identified by mass spectrometry analysis. The identification of phage proteins involved in interactions provides first hints toward the elucidation of the biological function of these proteins.

JF - Phage–Host Protein–Protein Interactions Using Strep-tag® II Purifications PB - Springer New York CY - New York, NY VL - 1898 SN - 978-1-4939-8939-3 CP - 10 M3 - 10.1007/978-1-4939-8940-9_10 ER - TY - Generic T1 - RNA-Based susceptibility testing of Mycobacterium tuberculosis Y1 - 2019 A1 - An Van den Bossche A1 - Roby Bhattacharyya A1 - Jean-Yves Coppee A1 - Alain Baulard A1 - Deborah Hung A1 - Vanessa Mathys A1 - Pieter-Jan Ceyssens JF - 29th ECCMID CY - Amsterdam, The Netherlands CP - ESCMID ER - TY - JOUR T1 - Transcriptional profiling of a laboratory and clinical Mycobacterium tuberculosis strain suggests respiratory poisoning upon exposure to delamanid JF - Tuberculosis Y1 - 2019 A1 - An Van den Bossche A1 - Hugo Varet A1 - Amandine Sury A1 - Odile Sismeiro A1 - Rachel Legendre A1 - Jean-Yves Coppee A1 - Vanessa Mathys A1 - Pieter-Jan Ceyssens KW - Delamanid KW - Mycobacterium tuberculosis KW - RNA sequencing KW - Transciptomics AB -

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.

VL - 117 M3 - 10.1016/j.tube.2019.05.002 ER - TY - Generic T1 - Transcriptional profiling of Mycobacterium tuberculosis suggests respiratory poisoning upon exposure to delamanid Y1 - 2019 A1 - An Van den Bossche A1 - Hugo Varet A1 - Amandine Sury A1 - Odile Sismeiro A1 - Rachel Legendre A1 - Jean-Yves Coppee A1 - Vanessa Mathys A1 - Pieter-Jan Ceyssens AB -

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.

JF - 40th ESM conference CY - Valencia, Spain CP - European Society of Mycobacteria ER - TY - Generic T1 - Evaluation of Non-Tuberculous Mycobacteria identification with maldi-tof mass spectrometry by applying sonication: a multicenter study Y1 - 2018 A1 - EU Mycobacteria-MALDI study group A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens JF - ECCMID 2018 CY - Madrid, Spain CP - ESCMID ER - TY - Generic T1 - RNA-based drug susceptibility testing of Mycobacterium tuberculosis Y1 - 2018 A1 - An Van den Bossche A1 - Roby Bhattacharyya A1 - Jean-Yves Coppee A1 - Leen Rigouts A1 - Alain Baulard A1 - Deborah Hung A1 - Vanessa Mathys A1 - Pieter-Jan Ceyssens KW - drug susceptibility testing KW - Mycobacterium tuberculosis JF - 39th Annual Congress of the European Society of Mycobacteriology CY - Dresden, Germany CP - ESM ER - TY - Generic T1 - RNA-based drug susceptibility testing of Mycobacterium tuberculosis Y1 - 2018 A1 - An Van den Bossche A1 - Roby Bhattacharyya A1 - Jean-Yves Coppee A1 - Leen Rigouts A1 - Alain Baulard A1 - Alexandra Vodolazkaia A1 - Deborah Hung A1 - Vanessa Mathys A1 - Pieter-Jan Ceyssens AB -

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.

JF - 2nd St. Petersburg Symposium on Tuberculosis and Mycobacteria: Molecular Approach CY - Sint Petersburg CP - Institut Pasteur Sint Petersburg ER - TY - Generic T1 - Global transcriptional analyses of Mycobacterium tuberculosis using old and new drugs Y1 - 2017 A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens A1 - Roby Bhattacharyya A1 - Deborah Hung A1 - Vanessa Mathys KW - antibiotics KW - Mycobacterium tuberculosis KW - Transcriptomics AB -

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.

 

JF - ECCMID 2017 PB - ECCMID CY - Vienna, Austria CP - ESCMID ER - TY - Generic T1 - Global transcriptional analyses of Mycobacterium tuberculosis using old and new drugs Y1 - 2017 A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens A1 - Roby Bhattacharyya A1 - Deborah Hung A1 - Vanessa Mathys KW - antibiotics KW - Mycobacterium tuberculosis KW - Transcriptomics AB -

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.

 

JF - 38th Annual Congress of the European Society of Mycobacteriology PB - ESM CY - Sibenik, Craotia CP - ESM ER - TY - JOUR T1 - How To: Identify Non-Tuberculous Mycobacterium Species By Using Maldi-Tof Mass Spectrometry. JF - Clin Microbiol Infect Y1 - 2017 A1 - Fernando Alcaide A1 - J Amlerová A1 - Germán Bou A1 - Pieter-Jan Ceyssens A1 - Pere Coll A1 - Dan Corcoran A1 - Marie-Sarah Fangous A1 - Iván González-Álvarez A1 - Rebecca Gorton A1 - Gilbert Greub A1 - Geneviève Hery-Arnaud A1 - Jaroslav Hrábak A1 - André Ingebretsen A1 - Brigid Lucey A1 - Ivana Marekoviċ A1 - Concepción Mediavilla-Gradolph A1 - James O'Connor A1 - Jim O'Mahony A1 - Onya Opota A1 - Brendan O'Reilly A1 - Dorothea Orth-Höller A1 - Marina Oviaño A1 - Juan José Palacios A1 - Begoña Palop A1 - Arthur Pranada A1 - Lidia Quiroga A1 - David Rodríguez-Temporal A1 - María Jesús Ruiz-Serrano A1 - Griselda Tudó A1 - An Van den Bossche A1 - Jakko van Ingen A1 - Belén Rodriguez-Sanchez KW - MALDI-TOF MS KW - Non-Tuberculoous Mycobacteria AB -

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.

M3 - 10.1016/j.cmi.2017.11.012 ER - TY - Generic T1 - Multicentric study on the improved identification of Non-Tuberculous Mycobacteria with MALDI-TOF MS using three different preprocessing protocols Y1 - 2017 A1 - EU Mycobacteria-MALDI study group A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens JF - ECCMID 2017 CY - Vienna, Austria CP - ESCMID ER - TY - Generic T1 - MALDI-TOF for the identification of Mycobacteria and their drug resistance profiles Y1 - 2016 A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens A1 - Marijke Hendrickx A1 - Vanessa Mathys KW - drug susceptibility testing KW - identification KW - MALDI-TOF KW - Mycobacterium tuberculosis AB -

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.

JF - 37th Annual Congress of the European Society of Mycobacteriology PB - ESM CY - Catania, Sicily, Italy ER - TY - Generic T1 - WIV-ISP's experiences in the use of MALDI-TOF for the identification of Mycobacteria and their drug resistance profiles Y1 - 2016 A1 - An Van den Bossche A1 - Pieter-Jan Ceyssens KW - drug susceptibility testing KW - identification KW - MALDI-TOF KW - Mycobacterium tuberculosis AB -

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.

PB - FARES/VRGT CY - FARES/VRGT Brussels, Belgium ER -