Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the Mycobacterium avium complex and Mycobacterium abscessus using broth microdilution.

OBJECTIVE: For non-tuberculous mycobacteria (NTM), minimum inhibitory concentration (MIC) distributions of wild-type isolates have not been systematically evaluated despite their importance for establishing antimicrobial susceptibility testing (AST) breakpoints. METHODS: We gathered MIC distributions for drugs used against the Mycobacterium avium complex (MAC) and Mycobacterium abscessus (MAB) obtained by commercial broth microdilution (SLOMYCOI and RAPMYCOI) from 12 laboratories. Epidemiological cut-off values (ECOFFs) and tentative ECOFFs (TECOFFs) were determined by EUCAST methodology including quality control (QC) strains. RESULTS: The clarithromycin ECOFF was 16 mg/L for M. avium (n = 1271) whereas TECOFFs were 8 mg/L for M. intracellulare (n = 415) and 1 mg/L for MAB (n = 1014) confirmed by analysing MAB subspecies without inducible macrolide resistance (n = 235). For amikacin, the ECOFFs were 64 mg/L for MAC and MAB. For moxifloxacin, the WT spanned >8 mg/L for both MAC and MAB. For linezolid, the ECOFF and TECOFF were 64 mg/L for M. avium and M. intracellulare, respectively. Current CLSI breakpoints for amikacin (16 mg/L), moxifloxacin (1 mg/L) and linezolid (8 mg/L) divided the corresponding WT distributions. For QC M. avium and M. peregrinum, ≥95% of MIC values were well within recommended QC ranges. CONCLUSION: As a first step towards clinical breakpoints for NTM, (T)ECOFFs were defined for several antimicrobials against MAC and MAB. Broad wild-type MIC distributions indicate a need for further method refinement which is now under development within the EUCAST subcommittee for anti-mycobacterial drug susceptibility testing. In addition, we showed that several CLSI NTM breakpoints are not consistent in relation to the (T)ECOFFs.

Identification of Mycobacterium abscessus Subspecies by MALDI-TOF Mass Spectrometry and Machine Learning.

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. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. 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.

Belgian bulk tank milk surveillance program reveals the impact of a continuous vaccination protocol for small ruminants against Coxiella burnetii.

Endemic Q fever in small ruminants remains an ongoing challenge for veterinary and human public health agencies. Though surveillance programs are implemented in Belgium, infection patterns and vaccination profiles, driving variables, as well as geographical clustering were not presented until now. Based on data from a decade of bulk tank milk analysis between 2009 and 2019, shedding in dairy goat herds declined from 16% (8/50) to 6% (10/162), whereas seroprevalence remained between 32% and 40%. Merely up to two shedding dairy sheep flocks were detected until 2019; seroprevalence peaked in 2017 (43%, 12/28) and declined thereafter. The number of animals in the holding influenced significantly (p = .048) the likelihood of shedding, whereas other established risk factors such as uncovered manure, high abortion rates and diversified farm structure could not be confirmed to significantly affect infection on Belgian herd level. Intermittent, incomplete and unsynchronized vaccinated herds shed Coxiella burnetii significantly more often and longer (p < .001) than continuously, complete and synchronized vaccinated herds. Spatial analyses revealed restricted but matching, homogenous clusters with ≤35 km diameter, concentrated in the coastal region close to the border to the Netherlands from 2009 to 2012, and broadened, heterogeneous clusters with ≥45 km diameter between 2014 and 2016 spreading south-west. Though the majority of human cases was notified in this region, the animal clusters could not be allied with Q fever cases. The impact of environmental factors as well as the role of wildlife, rodents and ticks on the transmission between flocks and to humans remains to be elucidated to harness additional epidemiological drivers of Q fever in Belgium. In conclusion, attempts to reduce the burden of Q fever in Belgium should particularly focus on the timely, complete and synchronized vaccination of flocks, including the breeding sire, and particularity in high-risk areas.

Retrospective evaluation of routine whole genome sequencing of Mycobacterium tuberculosis at the Belgian National Reference Center, 2019.

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 Mycobacterium tuberculosis 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 M. tuberculosis 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.

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.

The use of whole-genome sequencing (WGS) for routine typing of bacterial isolates has increased substantially in recent years. For Mycobacterium tuberculosis (MTB), in particular, WGS has the benefit of drastically reducing the time required 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, csb/RD, hsp65), 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 in silico modified data sets, allowing us to greatly increase the set of evaluated mutations. The workflow demonstrated very high performance with performance metrics of >99% for all assays, except for spoligotyping, where sensitivity dropped to ∼90%. The validation framework for our WGS-based bioinformatics workflow can aid in the 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 nonprofit use through the Galaxy instance of our institute at https://galaxy.sciensano.be.

Laboratory Diagnosis of Bovine Abortions Caused by Non-Maintenance Pathogenic Leptospira spp.: Necropsy, Serology and Molecular Study Out of a Belgian Experience.

: Bovine leptospirosis is a bacterial zoonotic disease caused by pathogenic Leptospira spp.. The pathology and epidemiology of this infection are influenced by the numerous existing serovars and their adaptation to specific hosts. Infections by host-maintained serovars such as Hardjo are well documented, unlike those from the incidental ones. In July 2014, an emerging phenomenon of an increased incidence of icteric abortions associated with leptospiral infection occurred in southern Belgium. First-line serological analyses targeting cattle-adapted serovars failed at initial diagnosis. This study provides a comprehensive description of laboratory findings-at the level of necropsy, serology and molecular diagnosis-regarding icteric and non-icteric abortions (n = 116) recorded during this time (years 2014-2015) and associated with incidental infection by serovars such as Grippotyphosa, Australis and Icterohaemorrhagiae. Based on these tests, a diagnostic pathway is proposed for these types of infection in cattle to establish an affordable but accurate diagnosis in the future. These investigations add insights into the understanding of the pathogenesis of bovine leptospirosis associated with serovars classically described as non-maintenance.

Phylogeography of Human and Animal Coxiella burnetii Strains: Genetic Fingerprinting of Q Fever in Belgium.

Q fever is a zoonotic disease caused by the bacteria Coxiella burnetii. Domestic ruminants are the primary source for human infection, and the identification of likely contamination routes from the reservoir animals the critical point to implement control programs. This study shows that Q fever is detected in Belgium in abortion of cattle, goat and sheep at a different degree of apparent prevalence (1.93%, 9.19%, and 5.50%, respectively). In addition, and for the first time, it is detected in abortion of alpaca (Vicugna pacos), raising questions on the role of these animals as reservoirs. To determine the relationship between animal and human strains, Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) (n=146), Single-Nucleotide Polymorphism (SNP) (n=92) and Whole Genome Sequencing (WGS) (n=4) methods were used to characterize samples/strains during 2009-2019. Three MLVA clusters (A, B, C) subdivided in 23 subclusters (A1-A12, B1-B8, C1-C3) and 3 SNP types (SNP1, SNP2, SNP6) were identified. The SNP2 type/MLVA cluster A was the most abundant and dispersed genotype over the entire territory, but it seemed not responsible for human cases, as it was only present in animal samples. The SNP1/MLVA B and SNP6/MLVA C clusters were mostly found in small ruminant and human samples, with the rare possibility of spillovers in cattle. SNP1/MLVA B cluster was present in all Belgian areas, while the SNP6/MLVA C cluster appeared more concentrated in the Western provinces. A broad analysis of European MLVA profiles confirmed the host-species distribution described for Belgian samples. In silico genotyping (WGS) further identified the spacer types and the genomic groups of C. burnetii Belgian strains: cattle and goat SNP2/MLVA A isolates belonged to ST61 and genomic group III, while the goat SNP1/MLVA B strain was classified as ST33 and genomic group II. In conclusion, Q fever is widespread in all Belgian domestic ruminants and in alpaca. We determined that the public health risk in Belgium is likely linked to specific genomic groups (SNP1/MLVA B and SNP6/MLVA C) mostly found in small ruminant strains. Considering the concordance between Belgian and European results, these considerations could be extended to other European countries.

Imported human brucellosis in Belgium: Bio and molecular typing of bacterial isolates, 1996-2015.

OBJECTIVES: The aim of this study was to characterize by classical biotyping and Multi-Locus variable number tandem repeats (VNTR) Analysis (MLVA) all Brucella spp. derived from human cases in Belgium from 1996 to 2015. Final goals were to determine the species and biovar, to trace-back on genetic grounds the origin of each strain when patient history and risk factors were missing, and to survey for particular trends at the national level. METHODS: A total of 37 Brucella strains, isolated from 37 patients in Belgium, were analyzed by both classical biotyping and MLVA, and the genetic patterns compared to those of human strains isolated worldwide. RESULTS: Classical biotyping revealed that isolates were mainly Brucella melitensis. Most of them belonged to biovar 3, the most abundant biovar in the Mediterranean region. MLVA confirmed that Brucella melitensis is too diverse in VNTRs to be able to make clusters associated to each biovar, but it allowed retrieving precious epidemiological information. The analysis highlighted the imported nature of the strains from all over the world with a dominant part from the Mediterranean countries. Findings of the MLVA11 testing were in line with the travel history of patients coming from Italy, Turkey, Lebanon and Peru. The analysis was particularly useful because it suggested the geographical origin of the infection for 12/16 patients for whom no case history was available. CONCLUSION: Classical biotyping and MLVA analysis are not exclusive but remain complementary tools for Brucella melitensis strain surveillance. MLVA11 is sufficient for Brucella-free countries such as Belgium to trace the geographical origin of infection, but complete MLVA16 is needed to search for links with endemic areas.

[Not Available]. / Coxiella burnetii, agent de la fièvre Q.

Q fever is a zoonosis of worldwide distribution with the exception of New Zealand. It is caused by an intracellular bacterium, Coxiella burnetii. The disease often goes underdiagnosed because the main manifestation of its acute form is a general self-limiting flu-like syndrome. The Dutch epidemics renewed attention to this disease, which was less considered before. This review summarizes the description of C. burnetii (taxonomy, intracellular cycle, and genome) and Q fever disease (description, diagnosis, epidemiology, and pathogenesis). Finally, vaccination in humans and animals is also considered.

Prevalence and molecular typing of Coxiella burnetii in bulk tank milk in Belgian dairy goats, 2009-2013.

Q fever, a worldwide zoonosis, is an arousing public health concern in many countries since the recent Dutch outbreak. An emerging C. burnetii clone, genotype CbNL01, was identified as responsible for the Dutch human Q fever cluster cases. Since 2009, Q fever surveillance in the goat industry was implemented by the Belgian authorities. The herd prevalence (December 2009-March 2013) ranged between 6.3 and 12.1%. Genotypic analysis highlighted the molecular diversity of the Belgian strains from goats and identified an emerging CbNL01-like genotype. This follow-up allowed the description of shedding profiles in positive farms which was either continuous (type I) and associated to the CbNL01-like genotype; or intermittent (type II) and linked to other genotypes. Despite the circulation of a CbNL01-like strain, the number of notified Belgian human cases was very low. The mandatory vaccination (in June 2011) on positive dairy goat farms in Belgium, contributed to a decrease in shedding.

In vitro and in vivo infectious potential of coxiella burnetii: a study on Belgian livestock isolates.

Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one--the goat isolate--being identical to the predominant strain circulating in the Netherlands during the 2007-2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.

Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence.

By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.