Characteristic SNPs defining the major multidrug-resistant Mycobacterium tuberculosis clusters identified by EuSeqMyTB to support routine surveillance, EU/EEA, 2017 to 2019.

BackgroundThe EUSeqMyTB project, conducted in 2020, used whole genome sequencing (WGS) for surveillance of drug-resistant Mycobacterium tuberculosis in the European Union/European Economic Area (EU/EEA) and identified 56 internationally clustered multidrug-resistant (MDR) tuberculosis (TB) clones.AimWe aimed to define and establish a rapid and computationally simple screening method to identify probable members of the main cross-border MDR-TB clusters in WGS data to facilitate their identification and track their future spread.MethodsWe screened 34 of the larger cross-border clusters identified in the EuSeqMyTB pilot study (2017-19) for characteristic single nucleotide polymorphism (SNP) signatures that could identify and define members of each cluster. We also linked this analysis with published clusters identified in previous studies and identified more distant genetic relationships between some of the current clusters.ResultsA panel of 30 characteristic SNPs is presented that can be used as an initial (routine) screen for members of each cluster. For four of the clusters, no unique defining SNP could be identified; three of these are closely related (within approximately 20 SNPs) to one or more other clusters and likely represent a single established MDR-TB clade composed of multiple recent subclusters derived from the previously described ECDC0002 cluster.ConclusionThe identified SNP signatures can be integrated into routine pipelines and contribute to the more effective monitoring, rapid and widespread screening for TB. This SNP panel will also support accurate communication between laboratories about previously identified internationally transmitted MDR-TB genotypes.

Tracking Mycobacterium tuberculosis sequencing samples using unique spikes of random DNA.

In the Netherlands, local laboratories are involved in the primary diagnosis of tuberculosis. Positive Mycobacterium tuberculosis complex cultures are sent to the National Institute for Public Health and the Environment (RIVM) for species identification, epidemiological typing, and screening for resistance by Whole Genome Sequencing (WGS). Occasional sample-swaps and cross-contaminations are known to occur in the diagnostic procedures. Such errors may lead to incorrect diagnoses resulting in the unnecessary or sub-optimal treatment of patients. Internal controls throughout the process ideally allow the early detection of such mistakes.

Use of a whole genome sequencing-based approach for Mycobacterium tuberculosis surveillance in Europe in 2017-2019: an ECDC pilot study.

Whole genome sequencing (WGS) can be used for molecular typing and characterisation of Mycobacterium tuberculosis complex (MTBC) strains. We evaluated the systematic use of a WGS-based approach for MTBC surveillance involving all European Union/European Economic Area (EU/EEA) countries and highlight the challenges and lessons learnt to be considered for the future development of a WGS-based surveillance system.WGS and epidemiological data of patients with rifampicin-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) were collected from EU/EEA countries between January 2017 and December 2019. WGS-based genetic relatedness analysis was performed using a standardised approach including both core genome multilocus sequence typing (cgMLST) and single nucleotide polymorphism (SNP)-based calculation of distances on all WGS data that fulfilled minimum quality criteria to ensure data comparability.A total of 2218 RR/MDR-MTBC isolates were collected from 25 countries. Among these, 56 cross-border clusters with increased likelihood of recent transmission (≤5 SNPs distance) comprising 316 RR/MDR-MTBC isolates were identified. The cross-border clusters included between two and 30 resistant isolates from two to six countries, demonstrating different RR/MDR-TB transmission patterns in Western and Eastern EU countries.This pilot study shows that a WGS-based surveillance system is not only feasible but can efficiently elucidate the dynamics of in-country and cross-border RR/MDR-TB transmission across EU/EEA countries. Lessons learnt from this study highlight that the establishment of an EU/EEA centralised WGS-based surveillance system for TB will require strengthening of national integrated systems performing prospective WGS surveillance and the development of clear procedures to facilitate international collaboration for the investigation of cross-border clusters.

Towards standardisation: comparison of five whole genome sequencing (WGS) analysis pipelines for detection of epidemiologically linked tuberculosis cases.

BackgroundWhole genome sequencing (WGS) is a reliable tool for studying tuberculosis (TB) transmission. WGS data are usually processed by custom-built analysis pipelines with little standardisation between them.AimTo compare the impact of variability of several WGS analysis pipelines used internationally to detect epidemiologically linked TB cases.MethodsFrom the Netherlands, 535 Mycobacterium tuberculosis complex (MTBC) strains from 2016 were included. Epidemiological information obtained from municipal health services was available for all mycobacterial interspersed repeat unit-variable number of tandem repeat (MIRU-VNTR) clustered cases. WGS data was analysed using five different pipelines: one core genome multilocus sequence typing (cgMLST) approach and four single nucleotide polymorphism (SNP)-based pipelines developed in Oxford, United Kingdom; Borstel, Germany; Bilthoven, the Netherlands and Copenhagen, Denmark. WGS clusters were defined using a maximum pairwise distance of 12 SNPs/alleles.ResultsThe cgMLST approach and Oxford pipeline clustered all epidemiologically linked cases, however, in the other three SNP-based pipelines one epidemiological link was missed due to insufficient coverage. In general, the genetic distances varied between pipelines, reflecting different clustering rates: the cgMLST approach clustered 92 cases, followed by 84, 83, 83 and 82 cases in the SNP-based pipelines from Copenhagen, Oxford, Borstel and Bilthoven respectively.ConclusionConcordance in ruling out epidemiological links was high between pipelines, which is an important step in the international validation of WGS data analysis. To increase accuracy in identifying TB transmission clusters, standardisation of crucial WGS criteria and creation of a reference database of representative MTBC sequences would be advisable.

WGS more accurately predicts susceptibility of Mycobacterium tuberculosis to first-line drugs than phenotypic testing.

BACKGROUND: Drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide. METHODS: DST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT. RESULTS: In total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance. CONCLUSIONS: Applying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST.

SNP-IT Tool for Identifying Subspecies and Associated Lineages of Mycobacterium tuberculosis Complex.

The clinical phenotype of zoonotic tuberculosis and its contribution to the global burden of disease are poorly understood and probably underestimated. This shortcoming is partly because of the inability of currently available laboratory and in silico tools to accurately identify all subspecies of the Mycobacterium tuberculosis complex (MTBC). We present SNPs to Identify TB (SNP-IT), a single-nucleotide polymorphism-based tool to identify all members of MTBC, including animal clades. By applying SNP-IT to a collection of clinical genomes from a UK reference laboratory, we detected an unexpectedly high number of M. orygis isolates. M. orygis is seen at a similar rate to M. bovis, yet M. orygis cases have not been previously described in the United Kingdom. From an international perspective, it is possible that M. orygis is an underestimated zoonosis. Accurate identification will enable study of the clinical phenotype, host range, and transmission mechanisms of all subspecies of MTBC in greater detail.

Prevalence and Characterization of Heterogeneous Variable-Number Tandem-Repeat Clusters Comprising Drug-Susceptible and/or Variable Resistant Mycobacterium tuberculosis Complex Isolates in the Netherlands from 2004 to 2016.

The variable-number tandem-repeat (VNTR) typing method is used to study tuberculosis (TB) transmission. Clustering of Mycobacterium tuberculosis isolates with identical VNTR patterns is assumed to reflect recent transmission. Hence, clusters are thought to be homogeneous regarding antibiotic resistance. In practice, however, heterogeneous clusters are also identified. This study investigates the prevalence and characteristics of heterogeneous VNTR clusters and assesses whether isolates in these clusters remain clustered when subjected to whole-genome sequencing (WGS). In the period from 2004 to 2016, 9,072 isolates were included. Demographic and epidemiological linkage data were obtained from the Netherlands Tuberculosis Register. VNTR clusters were defined as homogeneous when isolates shared identical resistance profiles or as heterogeneous if both susceptible and (variable) resistant isolates were found. Multivariate logistic regression analysis was performed to identify factors associated with heterogeneous clustering. Isolates from 2016 were subjected to WGS, and a genetic distance of 12 single nucleotide polymorphisms (SNPs) was used as the cutoff for WGS clustering. In total, 4,661/9,072 (51%) isolates were clustered into 985 different VNTR clusters, of which 217 (22%) were heterogeneous. Patient characteristics associated with heterogeneous clustering were non-Dutch ethnicity (odds ratio [OR], 1.46 [95% confidence interval {CI}, 1.22 to 1.75]), asylum seeker (OR, 1.51 [95% CI, 1.24 to 1.85]), extrapulmonary TB (OR, 1.26 [95% CI, 1.09 to 1.46]), previous TB diagnosis (OR, 1.38 [95% CI, 1.04 to 1.82]), and not being a contact of a TB patient (OR, 1.35 [95% CI, 1.08 to 1.69]). With WGS, 34% of heterogeneous and 78% of homogeneous isolates from 2016 remained clustered. Heterogeneous VNTR clusters are common but seem to be explained by a substantial degree of false clustering by VNTR typing compared to WGS.

Correction: Epidemiological links between tuberculosis cases identified twice as efficiently by whole genome sequencing than conventional molecular typing: A population-based study.

[This corrects the article DOI: 10.1371/journal.pone.0195413.].

Epidemiological links between tuberculosis cases identified twice as efficiently by whole genome sequencing than conventional molecular typing: A population-based study.

BACKGROUND: Patients with Mycobacterium tuberculosis isolates sharing identical DNA fingerprint patterns can be epidemiologically linked. However, municipal health services in the Netherlands are able to confirm an epidemiological link in only around 23% of the patients with isolates clustered by the conventional variable number of tandem repeat (VNTR) genotyping. This research aims to investigate whether whole genome sequencing (WGS) is a more reliable predictor of epidemiological links between tuberculosis patients than VNTR genotyping. METHODS: VNTR genotyping and WGS were performed in parallel on all Mycobacterium tuberculosis complex isolates received at the Netherlands National Institute for Public Health and the Environment in 2016. Isolates were clustered by VNTR when they shared identical 24-loci VNTR patterns; isolates were assigned to a WGS cluster when the pair-wise genetic distance was ≤ 12 single nucleotide polymorphisms (SNPs). Cluster investigation was performed by municipal health services on all isolates clustered by VNTR in 2016. The proportion of epidemiological links identified among patients clustered by either method was calculated. RESULTS: In total, 535 isolates were genotyped, of which 25% (134/535) were clustered by VNTR and 14% (76/535) by WGS; the concordance between both typing methods was 86%. The proportion of epidemiological links among WGS clustered cases (57%) was twice as common than among VNTR clustered cases (31%). CONCLUSION: When WGS was applied, the number of clustered isolates was halved, while all epidemiologically linked cases remained clustered. WGS is therefore a more reliable tool to predict epidemiological links between tuberculosis cases than VNTR genotyping and will allow more efficient transmission tracing, as epidemiological investigations based on false clustering can be avoided.

A Predominant Variable-Number Tandem-Repeat Cluster of Mycobacterium tuberculosis Isolates among Asylum Seekers in the Netherlands and Denmark, Deciphered by Whole-Genome Sequencing.

In many countries, Mycobacterium tuberculosis isolates are routinely subjected to variable-number tandem-repeat (VNTR) typing to investigate M. tuberculosis transmission. Unexpectedly, cross-border clusters were identified among African refugees in the Netherlands and Denmark, although transmission in those countries was unlikely. Whole-genome sequencing (WGS) was applied to analyze transmission in depth and to assess the precision of VNTR typing. WGS was applied to 40 M. tuberculosis isolates from refugees in the Netherlands and Denmark (most of whom were from the Horn of Africa) that shared the exact same VNTR profile. Cluster investigations were undertaken to identify in-country epidemiological links. Combining WGS results for the isolates (all members of the central Asian strain [CAS]/Delhi genotype), from both European countries, an average genetic distance of 80 single-nucleotide polymorphisms (SNPs) (maximum, 153 SNPs) was observed. The few pairs of isolates with confirmed epidemiological links, except for one pair, had a maximum distance of 12 SNPs. WGS divided this refugee cluster into several subclusters of patients from the same country of origin. Although the M. tuberculosis cases, mainly originating from African countries, shared the exact same VNTR profile, most were clearly distinguished by WGS. The average genetic distance in this specific VNTR cluster was 2 times greater than that in other VNTR clusters. Thus, identical VNTR profiles did not represent recent direct M. tuberculosis transmission for this group of patients. It appears that either these strains from Africa are extremely conserved genetically or there is ongoing transmission of this genotype among refugees on their long migration routes from Africa to Europe.

The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

'Inverted' analogs of the antibiotic gramicidin S with an improved biological profile.

A series of gramicidin S derivatives 4-15 are presented that have four ornithine residues as polar protonated side chains and two central hydrophobic amino acids with unaltered turn regions. These peptides were screened against human erthrocytes and our standard panel of Gram negative- and Gram positive bacteria, including four MRSA strains. Based on the antibacterial- and hemolytic data, peptides 13 and 14 have an improved biological profile compared to the clinically applied topical antibiotic gramicidin S.

High genetic diversity of Staphylococcus aureus strains colonizing patients with epidermolysis bullosa.

Patients with the blistering disease, epidermolysis bullosa (EB), frequently suffer from chronic wounds that become colonized by pathogenic bacteria, such as Staphylococcus aureus. To determine S. aureus colonization rates in patients with EB, swabs were collected from the anterior nares, throats and wounds of 52 Dutch patients with EB. Swabs were also collected from nares and throats of 13 healthcare workers who occasionally meet the sampled patients with EB. All EB patients with chronic wounds and 75% of the patients without chronic wounds were colonized with S. aureus. In contrast, 39% of the sampled healthcare workers were colonized with S. aureus. Typing revealed a high degree of genetic diversity of 184 collected S. aureus isolates. Autoinoculation of S. aureus in individual patients with EB was shown to occur frequently, whereas transmission of S. aureus between patients with EB is apparently rare. There was no evidence for S. aureus transmission between patients with EB and healthcare workers.

New statistical technique for analyzing MIC-based susceptibility data.

Seventeen laboratories participated in a cooperative study to validate the regional susceptibility testing of Neisseria gonorrhoeae in The Netherlands. International reference strains were distributed. Each laboratory determined the MICs of ciprofloxacin, penicillin, and tetracycline, for each strain by Etest. To explore a more transparent assessment of quality and comparability, a statistical regression model was fitted to the data that accounted for the censoring of the MICs. The mean MICs found by all of the laboratories except three were closer than one 2-fold dilution step to the overall mean, and the mean MICs of each antimicrobial agent were close to the MICs for the international reference strains. This approach provided an efficient tool to analyze the performance of the Dutch decentralized gonococcal resistance monitoring system and confirmed good and comparable standards.

Synthesis and evaluation of strand and turn modified ring-extended gramicidin S derivatives.

In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.

Studies on Prn variation in the mouse model and comparison with epidemiological data.

The virulence factor pertactin (Prn) is a component of pertussis vaccines and one of the most polymorphic Bordetella pertussis antigens. After the introduction of vaccination shifts in predominant Prn types were observed and strains with the Prn vaccine type (Prn1) were replaced by strains carrying non-vaccine types (Prn2 and Prn3), suggesting vaccine-driven selection. The aim of this study was to elucidate the shifts observed in Prn variants. We show that, although Prn2 and Prn3 circulated in similar frequencies in the 1970s and 1980s, in the 1990s Prn2 strains expanded and Prn3 strains disappeared, suggesting that in vaccinated populations Prn2 strains are fitter than Prn3 strains. We established a role for Prn in the mouse model by showing that a Prn knock-out (Prn-ko) mutation reduced colonization in trachea and lungs. Restoration of the mutation resulted in a significant increase in colonization compared to the knock-out mutant. The ability of clinical isolates with different Prn variants to colonize the mouse lung was compared. Although these isolates were also polymorphic at other loci, only variation in the promoter for pertussis toxin (ptxP) and Prn were found to contribute significantly to differences in colonization. Analysis of a subset of strains with the same ptxP allele revealed that the ability to colonize mice decreased in the order Prn1>Prn2 and Prn3. Our results are consistent with the predominance of Prn1 strains in unvaccinated populations. Our results show that ability to colonize mice is practically the same for Prn2 and Prn3. Therefore other factors may have contributed to the predominance of Prn2 in vaccinated populations. The mouse model may be useful to assess and predict changes in the B. pertussis population due to vaccination.

Exploring the conformational and biological versatility of ß-turn-modified gramicidin S by using sugar amino acid homologues that vary in ring size.

Monobenzylated sugar amino acids (SAAs) that differ in ether ring size (containing an oxetane, furanoid, and pyranoid ring) were synthesized and incorporated in one of the ß-turn regions of the cyclo-decapeptide gramicidin S (GS). CD, NMR spectroscopy, modeling, and X-ray diffraction reveal that the ring size of the incorporated SAA moieties determines the spatial positioning of their cis-oriented carboxyl and aminomethyl substituents, thereby subtly influencing the amide linkages with the adjacent amino acids in the sequence. Unlike GS itself, the conformational behavior of the SAA-containing peptides is solvent dependent. The derivative containing the pyranoid SAA is slightly less hydrophobic and displays a diminished haemolytic activity, but has similar antimicrobial properties as GS.

Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids.

Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the amphipathicity of these compounds is correlated to their biological activity. Several bacterial strains including MRSA strains were shown to be killed by the novel peptides. The most potent antibacterial peptides are tetradecameric GS analogues containing six positives charges and two adamantane moieties.