Draft Genome Sequences of 77 Endemic Multidrug-Resistant Mycobacterium tuberculosis Strains of SIT41 (TUR) Spoligotype from Bulgaria.

Sequences of multidrug-resistant (MDR) Mycobacterium tuberculosis strains are of particular interest to study the molecular mechanisms of drug resistance evolution. Here, we report the draft genome sequences of 77 endemic multidrug-resistant Mycobacterium tuberculosis strains of SIT41 (TUR) spoligotype from Bulgaria. SIT41 spoligotype is dominant (>40%) among the MDR-TB strains in Bulgaria.

Detection of NDM-1/5 and OXA-48 co-producing extensively drug-resistant hypervirulent Klebsiella pneumoniae in Northern Italy.

OBJECTIVES: Using a hybrid long-read sequencing approach, we aimed to fully characterise four extensively-drug resistant (XDR) hypervirulent Klebsiella pneumoniae isolates, one of which represented the first strain isolated in Italy co-expressing NDM-1/5 and OXA-48 carbapenemases. METHODS: Whole-genome sequencing was performed using Illumina and Oxford Nanopore Technology platforms. An assembly pipeline was used to recover the structures both of the chromosome and plasmids. RESULTS: Multilocus sequence typing (MLST) showed that these strains belonged to high-risk sequence types (STs) not commonly circulating in Italy (ST383, ST147 and ST15). The hybrid sequencing approach allowed to characterise three multidrug resistance plasmids, which demonstrated high homology with previously sequenced plasmids, that were simultaneously detected in one ST383 strain carrying, respectively, blaNDM-1, blaNDM-5 and blaOXA-48. CONCLUSION: This is the first report in Italy of new hypervirulent XDR K. pneumoniae clones characterised by co-production of OXA-48, NDM-1 and NDM-5. The discovery of new high-risk clones harbouring multiple mobile elements is a growing problem that poses a great challenge for public health.

Anti-tuberculosis drug resistance in Slovakia, 2018-2019: The first whole-genome epidemiological study.

OBJECTIVE: The resistance of Mycobacterium (M.) tuberculosis to antituberculosis drugs poses a major threat to global public health. Whole genome sequencing (WGS) is an increasingly preferred method in the diagnostics and monitoring of the transmission dynamics of resistant forms of tuberculosis (TB). The aim of the study was to, for the first time, use the sequencing-based analysis to study the transmission and resistance patterns of a systematic and recent collection of extensively drug resistant (XDR) and multidrug resistant tuberculosis (MDR-TB) isolates and to expand our knowledge about drug resistant (DR) TB epidemiological dynamics in Slovakia. DESIGN: A total of 495 patients with pulmonary TB, who were referred to National Reference Laboratory for Mycobacteriology (Vysné Hágy, Slovakia) in the years 2018-2019, were studied. Out of the total of 495 patients, 4 XDR-TB (0.8%) and 8 (1.6%) MDR-TB isolates were identified by conventional drug susceptibility testing on Löwenstein-Jensen solid medium and subjected to whole genome sequencing. Sequencing data were evaluated for molecular-epidemiological analysis and identification of resistance patterns. RESULTS: Phylogenetic and cluster analysis showed extensive recent transmission events and the predominance of Euro-American lineage 4.7 in Slovakia. However, phylogenetic analysis revealed the circulation of several lineages that originally occurred in Eastern European countries. Resistance patterns for first- and second-line antituberculosis drugs characterized by whole genome sequencing were in high concordance with the results of phenotypic drug susceptibility testing. CONCLUSION: Forty percent of at least MDR-TB isolates were not genetically linked, indicating that appropriate measures should be taken to monitor and prevent the spread of drug-resistant tuberculosis within the country as well as in other regions.

Performance of the GenoType MTBDRsl V 2.0 for detecting second-line drugs resistance of Mycobacterium tuberculosis isolates in Tunisia.

Rapid detection of the second-line drug (SLD) resistant tuberculosis (TB) strains is challenging to prescribe an immediate adequate treatment and limit the transmission of SLD resistant strains. The study aimed to evaluate the performance of GenoType MTBDRsl V2.0 compared to phenotypic drug susceptibility testing (pDST:MGIT960) to detect resistance to SLD of Mycobacterium tuberculosis (MTB) isolates in Tunisia, between May 2015 and December 2019. As a matter of fact, 103 rifampicin-resistant and multidrug-resistant MTB strains were included. Discrepancies between pDST and MTBDRsl were solved by whole genome sequencing. Compared to pDST, MTBDRsl V2.0 showed a sensitivity of 92.8% (68.5%-98.7%) in detecting resistance to fluoroquinolones. As for second-line injectable drugs, it presented a sensitivity of 80.0% (49.0%-94.3%). MTBDRsl had sensitivities of 100.0% (67.5%-100.0%), 75.0% (40.9%-92.8%) and 100.0% (60.9%-100.0%) respectively for kanamycin, capreomycin and amikacin. The specificity was 100.0% for all the drugs evaluated. As for diagnosing XDR-TB, it had a sensitivity of 57.1% (25.0%-84.1%) and a specificity of 100.0% (96.1%-100.0%). MTBDRsl V2.0 showed a high performance in detecting SLD resistance with a short turnaround time compared with pDST, which made it possible to start an early treatment and to maintain a low prevalence of SLD resistance and XDR-TB in Tunisia.

Mycobacterium helveticum sp. nov., a novel slowly growing mycobacterial species associated with granulomatous lesions in adult swine.

The occurrence of nontuberculous mycobacteria in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. Mycobacteriosis in pigs is usually associated with members of the Mycobacterium avium complex and, in particular, with 'Mycobacterium avium subsp. hominissuis'. Here we describe a novel slow-growing mycobacterial species isolated from lymph nodes obtained from two sows housed in different Swiss farms. The animals presented chronic inappetence and mild diarrhoea. Gross pathology revealed focal caseous lymphadenopathy of the mesenteric lymph nodes. Complete genome sequencing of the two isolates from the two sows was performed. The genomes comprised 5.76 Mb and an average nucleotide identity score of 99.97 %. Whole genome sequence, mycolic acid and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the two isolates were not related to any previously described Mycobacterium species. The closest related species was Mycobacterium parmense, a slow-growing scotochromogenic mycobacterium first isolated from a cervical lymph node of a 3-year-old child. The name proposed for the new species is Mycobacterium helveticum sp. nov. and 16-83T (=DSM 109965T= LMG 2019-02457T) is the type strain.

Molecular characterization of Mycobacterium abscessus subspecies isolated from patients attending an Italian Cystic Fibrosis Centre.

Mycobacterium abscessus (MABS) infection represents significant management challenge in cystic fibrosis (CF) patients. This retrospective study (2005-2016) aims to determine the prevalence of the subspecies of MABS isolated from CF patients, to evaluate the persistence over the years of a single subspecies of MABS and to correlate mutations responsible for macrolides and amikacin resistance with MIC values. We investigated 314 strains (1 isolate/patient/year) isolated from the lower respiratory tract of 51 chronically infected CF patients. Sequencing of rpoB gene was performed to identify the MABS subspecies. The erm(41) gene was sequenced to differentiate the strains with and without inducible macrolide resistance. Regions of 23S and 16S rRNA were sequenced to investigate mutations responsible for constitutive resistance to macrolides and aminoglycosides, respectively. Antibiotic susceptibility, using commercial microdilution plates, was evaluated according to CLSI. M. abscessus subsp. abscessus accounted for 64% of the isolates, bolletii subspecies for 16% and massiliense subspecies for 20%. All the massiliense strains presented truncated erm(41) gene while 12 abscessus strains presented the mutation T28->C in the erm(41) gene, which makes it inactive. The 23S rRNA analysis did not show constitutive resistance to macrolides in any strain. Mutation of the 16S rRNA gene was highlighted in 2 strains out of 314, in agreement with high MIC values. The correct identification at the subspecies level and the molecular analysis of 23S rRNA, 16S rRNA and erm gene is useful to guide the treatment strategy in patients with M. abscessus lung infection.

Comparison of core-genome MLST, coreSNP and PFGE methods for Klebsiella pneumoniae cluster analysis.

In this work we compared the most frequently used Klebsiella pneumoniae typing methods: PFGE, cgMLST and coreSNP. We evaluated the discriminatory power of the three methods to confirm or exclude nosocomial transmission on K. pneumoniae strains isolated from January to December 2017, in the framework of the routine surveillance for multidrug-resistant organisms at the San Raffaele Hospital, in Milan. We compared the results of the different methods to the results of epidemiological investigation. Our results showed that cgMLST and coreSNP are more discriminant than PFGE, and that both approaches are suitable for transmission analyses. cgMLST appeared to be inferior to coreSNP in the K. pneumoniae CG258 phylogenetic reconstruction. Indeed, we found that the phylogenetic reconstruction based on cgMLST genes wrongly clustered ST258 clade1 and clade2 strains, conversely properly assigned by coreSNP approach. In conclusion, this study provides evidences supporting the reliability of both cgMLST and coreSNP for hospital surveillance programs and highlights the limits of cgMLST scheme genes for phylogenetic reconstructions.

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings.

Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex (MTBC) isolates, based on 24 loci, is still widely used as the standard for routine molecular surveillance of tuberculosis (TB). QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods regarding MIRU-VNTR typing of MTBC. We aimed to evaluate the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories. A total of 173 DNA were used for the study. Results obtained with QIAxcel were compared to the reference results obtained with an ABI 3730 DNA analyzer. In Albania, the overall agreement with the reference method was 97.92%. A complete agreement result was obtained for 17 loci. In Tunisia, the overall agreement with the reference method was 98.95%. A complete agreement result was obtained for 17 loci. Overall agreement in both centers was 98.43%. In our opinion, use of QIAxcel technology has the potential to be reliable, given an optimized algorithm. Inaccuracies in sizing of long fragments should be solved, especially regarding locus 4052.

Peculiar features of the Mycobacterium tuberculosis population structure in Albania.

Albania is a Balkan country with moderate to low incidence of tuberculosis (TB) and very low prevalence of drug resistant TB. Here, we analyzed a country-wide multi-year Mycobacterium tuberculosis collection in order to detect possible dynamic trends of TB in Albania, with a focus on drug resistance and endemic/epidemic clones. In total, 743 isolates collected in 2007 to 2011 were divided into 107 spoligotypes and 351 MIRU-types. Based on the MIRU-VNTR phylogenetic analysis, the isolates were assigned to the following lineages/families: animal ecotypes (5 M. bovis and 2 M. caprae isolates), Lineage 2 (5 Beijing isolates), Lineage 3 (1 CAS-Delhi isolate) and, mostly and overwhelmingly, Lineage 4 (Cameroon, Uganda, Ghana and related; NEW-1-related; Ural, Haarlem, LAM, S, TUR; and unclassified isolates). Most of the isolates (452/743) were intermediately located on the global VNTR tree and did not cluster with any reference profile; they were distantly related to different families within Lineage 4 and we designated them as "unclassified L4" isolates. The significantly higher proportion of drug resistance was observed in (i) Beijing genotype compared to all other isolates (60%, P = .008), (ii) "unclassified L4" compared to all other isolates (13.9%, P = .04) and (iii) SIT2936 compared to other "unclassified L4" (34.3%, P = .0006). Analysis of the yearly collections revealed (i) some decrease of the large heterogeneous "unclassified L4" from 65% to 57%; (ii) steadily increasing gradient of LAM from 3.4 to 13.3%; (iii) stable prevalence of Haarlem (15-20%); and (iv) decrease of TUR with only 1.1% in 2011. Most of the LAM (33/49) and Beijing (3/5) isolates belonged to the VNTR types specific for Russia and former Soviet Union countries. To conclude, our results highlight a peculiar nature of M. tuberculosis population in Albania that is dominated by local and unclassified genotypes within Lineage 4, and also features European genotypes and epidemically relevant clones originating from the former Soviet Union countries. At the same time, these imported clones remain drug susceptible and prevalence of drug resistance on a whole is low.

Genome-based taxonomic revision detects a number of synonymous taxa in the genus Mycobacterium.

The aim of this study was to clarify the taxonomic status of named species within the genus Mycobacterium. The analysis of genomes belonging to 174 taxa (species or subspecies) of the genus Mycobacterium was conducted using both the Average Nucleotide Identity and the Genome to Genome Distance. A number of synonymous taxa were detected. The list of synonyms includes: two subspecies of M. chelonae (M. chelonae subsp. bovis and M. chelonae subsp. gwanakae), two subspecies of M. fortuitum (M. fortuitum subsp. fortuitum and M. fortuitum subsp. acetamidolyticum), four subspecies of M. avium (M. avium subsp. avium, M. avium subsp. silvaticum, M. avium subsp. paratuberculosis and "M. avium subsp. hominissuis"), two couples of subspecies of M. intracellulare (M. intracellulare subsp. intracellulare/M. intracellulare subsp. paraintracellulare and M. intracellulare subsp. chimaera/M. intracellulare subsp. yongonense), the species M. austroafricanum and M. vanbaalenii, the species M. senegalense and M. conceptionense, the species M. talmoniae and M. eburneum and the species M. marinum, M. ulcerans and M. pseudoshottsii. Furthermore one species were reclassified as subspecies of another mycobacterium: M. lepraemurium was reclassified as a subspecies of M. avium (M. avium subsp. lepraemurium). The updates to nomenclature are proposed basing on the priority of names according the Code of nomenclature of prokaryotes. For two species (M. bouchedurhonense and M. marseillense) the loss of standing in nomenclature is proposed because of unavailability of respective type strains in culture collections.

Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan.

We report on the first six cases of acquired resistance to bedaquiline in Pakistan. Seventy sequential isolates from 30 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing and by the detection of mutations in relevant genes. We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

Lack of association of novel mutation Asp397Gly in aftB gene with ethambutol resistance in clinical isolates of Mycobacterium tuberculosis.

To discover additional genotypic indicators for ethambutol (EMB) resistant M. tuberculosis, we studied polymorphisms in arabinofuranosyl transferase encoding genes aftA (Rv3792), aftB (Rv3805) and aftC (Rv2673) in 38 EMB resistant and 34 EMB susceptible isolates from India and a repository established by the World Health Organization (WHO) Special Programme for Research and Training in Tropical Disease (TDR) by DNA sequencing. The results were correlated with the minimum inhibitory concentration (MIC) of EMB and mutations in embB (Rv3795). The most common non-synonymous polymorphism identified in aftB was Asp397Gly in 12/38 (31.6%) EMB resistant and 3/34 (8.8%) EMB susceptible isolates. Interestingly, 10/12 (83.3%) EMB resistant isolates with aftB Asp397Gly mutation also carried embB306, embB402 or embB497 mutations. Association of Asp397Gly polymorphism with EMB resistance was statistically significant (p 0.0216). However, overexpression of the mutant aftB in M. tuberculosis H37Rv did not exhibit any change in the MIC. Whole genome sequencing of a panel of Indian isolates and SNP cluster grouping (SCG) of TDR strains revealed an association between aftB mutation Asp397Gly and Beijing genotype or SCG2, a cluster group representing the Beijing genotype. To conclude, though aftBAsp397Gly mutation is not associated with EMB resistance, this mutation may be a phylogenetic marker for the Beijing clade.

Drug-Resistant Tuberculosis, Lebanon, 2016 - 2017.

In a 12-month nationwide study on the prevalence of drug-resistant tuberculosis (TB) in Lebanon, we identified 3 multidrug-resistant cases and 3 extensively drug-resistant TB cases in refugees, migrants, and 1 Lebanon resident. Enhanced diagnostics, particularly in major destinations for refugees, asylum seekers, and migrant workers, can inform treatment decisions and may help prevent the spread of drug-resistant TB.

Whole-Genome Sequencing of Drug-Resistant Mycobacterium tuberculosis Strains, Tunisia, 2012-2016.

To investigate transmission of drug-resistant strains of Mycobacterium tuberculosis in Tunisia, we performed whole-genome sequencing on 46 multidrug-resistant strains isolated during 2012-2016. Core-genome multilocus sequence typing grouped 30 strains (65.2%) into 3 clusters, indicating extensive recent transmission and Haarlem clone predominance. Whole-genome sequencing might help public health services undertake appropriate control actions.

Mycobacterium decipiens sp. nov., a new species closely related to the Mycobacterium tuberculosis complex.

Two mycobacterial strains with close similarity to the Mycobacterium tuberculosis complex (MTBC) were isolated from cutaneous lesions of patients in the USA and Italy. At the phenotypic level, similarities to the MTBC included slow growth rate, rough morphotype of the unpigmented colonies and nearly identical high-performance liquid chromatography profiles of mycolic acids. In contrast to the MTBC, the strains were niacin- and nitrate-negative, and catalase-positive both at 68 °C and in semi-quantitative tests. The clinical isolates were more closely related to M. tuberculosis than to any other known mycobacterium and scored positive with commercial DNA probes (Hologic AccuProbe M. tuberculosis). Both average nucleotide identity and genome-to-genome distance suggested the strains are different from the MTBC. Therefore, given the distinguishing phenotypic and genomic-scale differences, we submit that the strains belong to a new species we have named Mycobacteriumdecipiens with type strain TBL 1200985T (=ATCC TSD-117T=DSM 105360T).

The new phylogeny of the genus Mycobacterium: The old and the news.

BACKGROUND: Phylogenetic studies of bacteria have been based so far either on a single gene (usually the 16S rRNA) or on concatenated housekeeping genes. For what concerns the genus Mycobacterium these approaches support the separation of rapidly and slowly growing species and the clustering of most species in well-defined phylogenetic groups. The advent of high-throughput shotgun sequencing leads us to revise conventional taxonomy of mycobacteria on the light of genomic data. For this purpose we investigated 88 newly sequenced species in addition to 60 retrieved from GenBank and used the Average Nucleotide Identity pairwise scores to reconstruct phylogenetic relationships within this genus. RESULTS: Our analysis confirmed the separation of slow and rapid growers and the intermediate position occupied by the M. terrae complex. Among the rapid growers, the species of the M. chelonae-abscessus complex belonged to the most ancestral cluster. Other major clades of rapid growers included the species related to M. fortuitum and M. smegmatis and a large grouping containing mostly environmental species rarely isolated from humans. The members of the M. terrae complex appeared as the most ancestral slow growers. Among slow growers two deep branches led to the clusters of species related to M. celatum and M. xenopi and to a large group harboring most of the species more frequently responsible of disease in humans, including the major pathogenic mycobacteria (M. tuberculosis, M. leprae, M. ulcerans). The species previously grouped in the M. simiae complex were allocated in a number of sub-clades; of them, only the one including the species M. simiae identified the real members of this complex. The other clades included also species previously not considered related to M. simiae. The ANI analysis, in most cases supported by Genome to Genome Distance and by Genomic Signature-Delta Difference, showed that a number of species with standing in literature were indeed synonymous. CONCLUSIONS: Genomic data revealed to be much more informative in comparison with phenotype. We believe that the genomic revolution enabled by high-throughput shotgun sequencing should now be considered in order to revise the conservative approaches still informing taxonomic sciences.