High genetic heterogeneity of Mycobacterium intracellulare isolated from respiratory specimens

Background M. intracellulare is a frequent causative pathogen of nontuberculous mycobacteria infection that causes infections in the respiratory tract, whose incidence is increasing in many countries. This study aimed at determining the VNTR-based genetic diversity of a collection of 39 M. intracellulare human strains isolated from respiratory specimens over the last 5 years. Results The VNTR analysis showed that M. intracellulare strains displayed a high genetic diversity, indicating that the M. intracellulare genotypes are quite heterogeneous in our geographical area. Moreover, a comparison with VNTR profiles of strains from other countries confirmed that genotypes of clinical strains of M. intracellulare are not related to geographical origin. Conclusions VNTR typing has proved to be a highly discriminatory method for better understanding the molecular epidemiology of M. intracellulare.

High-resolution genotyping of Lymphogranuloma Venereum (LGV) strains of Chlamydia trachomatis in London using multi-locus VNTR analysis-ompA genotyping (MLVA-ompA)

Background Lymphogranuloma venereum (LGV) is caused by Chlamydia trachomatis strains with ompA genotypes L1 to L3. An LGV epidemic associated with the L2b genotype has emerged in the past few decades amongst men who have sex with men (MSM). C. trachomatis genotypes can be discriminated by outer membrane protein A gene (ompA) sequencing, however this method has limited resolution. This study employed a high-resolution genotyping method, namely, multi-locus tandem repeat (VNTR) analysis with ompA sequencing (MLVA-ompA), to assess the distribution of LGV MLVA-ompA genotypes amongst individuals attending genitourinary medicine (GUM) clinics in London. Methods Clinical specimens were collected from individuals attending eight London-based GUM clinics. Specimens that tested positive for C. trachomatis by commercial nucleic acid amplification test (NAAT) were confirmed as LGV by pmpH real-time PCR. LGV-positive DNA extracts were subsequently genotyped using MLVA-ompA. Results Two hundred and thirty DNA extracts were confirmed as LGV, and 162 (70%) yielded complete MLVA-ompA genotypes. Six LGV MLVA-ompA genotypes were identified: 1.9.2b-L2, 1.9.3b-L2b, 1.9.2b-L2b, 1.9.2b-L2b/D, 1.4a.2b-L2b, and 5.9.2b-L1. The following LGV ompA genotypes were identified (in descending order of abundance): L2, L2b, L2b/D, and L1. Eight ompA sequences with the hybrid L2b/D profile were detected. The hybrid sequence was identical to the ompA of a recombinant L2b/D strain detected in Portugal in 2017. Conclusions The L2 ompA genotype was found to predominate in the London study population. The study detected an unusual hybrid L2b/D ompA profile that was previously reported in Portugal. We recommend further monitoring and surveillance of LGV strains within the UK population.

High Prevalence and New Genotype of Coxiella burnetii in Ticks Infesting Camels in Somalia

Coxiella burnetii is the causative agent of Q fever. It can infect animals, humans, and birds, as well as ticks, and it has a worldwide geographical distribution. To better understand the epidemiology of C. burnetii in Somalia, ticks infesting camels were collected from five different regions, including Bari, Nugaal, Mudug, Sool, and Sanaag, between January and March 2018. Collected ticks were tested for C. burnetii and Coxiella-like endosymbiont DNA by using IS1111, icd, and Com1-target PCR assays. Moreover, sequencing of the 16S-rRNA was conducted. Molecular characterization and typing were done by adaA-gene analysis and plasmid-type identification. Further typing was carried out by 14-marker Multi-Locus Variable-Number Tandem Repeats (MLVA/VNTR) analysis. The investigated ticks (n = 237) were identified as Hyalomma spp. (n = 227, 95.8%), Amblyomma spp. (n = 8, 3.4%), and Ripicephalus spp. (n = 2, 0.8%), and 59.1% (140/237) of them were positive for Coxiella spp. While Sanger sequencing and plasmid-type identification revealed a C. burnetii that harbours the QpRS-plasmid, MLVA/VNTR genotyping showed a new genotype which was initially named D21. In conclusion, this is the first report of C. burnetii in ticks in Somalia. The findings denote the possibility that C. burnetii is endemic in Somalia. Further epidemiological studies investigating samples from humans, animals, and ticks within the context of “One Health” are warranted.

Insight into population structure of Mycobacterium tuberculosis isolates in the multiethnic province of Alborz, Iran

Background and Objectives: Genetic diversity of Mycobacterium tuberculosis clinical isolates from tuberculosis patients in the multiethnic province of Alborz, Iran was assessed. Materials and Methods: A total of 17 isolates in the period of 2012-2013 were collected and subjected to a Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) consisted of 6 variable numbers of tandem repeats (VNTRs) including ETR-A, ETR-B, ETR-C, ElTR-D, ETR-E, ETR-F, 5 Mycobacterial Interspersed Repetitive Units including MIRU10, MIRU16, MIRU26, MIRU39, MIRU40, and 1 Queen University of Belfast locus, QUB11. Results: This classified all isolates into 17 distinct MIRU-VNTR types, a reflection of a highly heterogenic population. Within the 12 used VNTR loci, ten proved highly or moderately discriminant according to the calculated HGDI scores. No cluster of isolates was identified in the study panel, giving a clustering rate of 0%, several events of SVL (N=5) and DVL (N=4) and TVL (N=3) were detected. Conclusion: The greater heterogeneity observed here by MLVA-VNTR analysis is most likely due to limited background data in the study region rather than a genuine more heterogeneous population compared to other provinces of the country.

Drug-resistant Mycobacterium tuberculosis and its genotypes isolated from an outbreak in western Thailand

Background Multidrug-resistant TB (MDR-TB) outbreaks have occurred in the Thamaka district, Kanchanaburi province in Thailand. Methods Seventy-two isolates, which included 7% mono-, 30.6% MDR and extensively drug-resistant TB (XDR-TB), were genotyped by spoligotyping, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) and single nucleotide polymorphism genotyping, and their drug resistance was analysed. Results The spoligotyping results showed that Beijing spoligo-international type (SIT)1 was predominant (n=38; 52.8%) while the remaining were non-Beijing sublineages (n=34). The MIRU-VNTR analysis showed that Beijing isolates, most of which belonged to the modern type (n=37), formed 5 clusters and 13 individual patterns. In katG, only mutation Ser315Thr was identified. In rpoB, Ser531Leu was predominant, except for His526Arg and Leu533Pro, which were found in two isolates. A cluster of 14 Beijing strains contained these common mutations and shared the MIRU-VNTR genotype with isolates in the Thamaka district that had spread previously. Two U SIT523 isolates contained the mutations A1400G in rrs and Asp94Gly in gyrA genes, indicating a spread of XDR-TB. Conclusions Most mutations were associated with drug resistance and the specific MDR Beijing and XDR-TB in U SIT523 isolates remain. This genotyping is a key tool for tracking TB transmission in the Thamaka district of Thailand.

Transmission Network of Deer-Borne Mycobacterium bovis Infection Revealed by a WGS Approach

Bovine tuberculosis (TB) is a zoonotic disease, mainly caused by Mycobacterium bovis. France was declared officially TB free in 2001, however, the disease persists in livestock and wildlife. Among wild animals, deer are particularly susceptible to bovine TB. Here, a whole genome sequence (WGS) analysis was performed on strains with the same genetic profile—spoligotype SB0121, Multiple Loci VNTR Analysis (MLVA) 6 4 5 3 11 2 5 7—isolated from different types of outbreaks, including from deer or cattle herds, or zoological or hunting parks where the presence of infected deer was a common trait in most of them. The results of the phylogeny based on the SNP calling shows that two sub-clusters co-exist in France, one related to deer bred to be raised as livestock, and the other to hunting parks and zoos. The persistence over almost 30 years of sporadic cases due to strains belonging to these clusters highlights the deficiency in the surveillance of captive wildlife and the need for better monitoring of animals, especially before movement between parks or herds.

Distribution of Mycobacterium leprae genotypes from Surabaya and Bandung Clinical Isolates by Multiple Locus Variable Number of Tandem Repeat Analysis

Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis has been proposed as a means of genotyping for tracking leprosy transmission due to tandem repeats’ potential as genetic markers to differentiate M. leprae strains. However, characteristics of polymorphism can vary depending on the population. This study aimed to compare the copy number of repeats in four genetic markers: TTC, AC8a, AC9 and 6-7 in leprosy patients from Surabaya and Bandung. Twenty three patients from Dr. Soetomo General Hospital and 21 from Hasan Sadikin Hospital were recruited. Multiple locus VNTR analysis was applied using total DNA extracts from Slit Skin Smear (SSS). From Surabaya, 7 samples showed the same copy number of four genetic markers (TTC=15; AC8a=10; AC9=10 and 6-7=6) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6); as for samples from Bandung, 2 showed the same copy number (TTC=15; AC8a=8; AC9=10 and 6-7=8) and 2 showed another (TTC=16; AC8a=10; AC9=11 and 6-7=6). The multiple locus VNTR analysis showed two identical M. leprae VNTR profiles from Bandung and Surabaya which supports the use of VNTR loci for transmission studies.

Improvement of Multiple-Locus VNTR Analysis Typing Scheme for Helicobacter pylori

Aims: To improve a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Helicobacter pylori typing. Materials and Methods: Polymorphic VNTRs were searched by Gene Expert. The distribution and polymorphism of each VNTR locus were analyzed in 18 H. pylori genomes from the NCBI genome database by BLAST and were compared with a collection of 15 clinical H. pylori strains. The MLVA assay was compared with MLST-typing for discriminating H. pylori isolates. Results: Twelve VNTR loci were identified by bioinformatic screening of H. pylori genomes, and five of them were highly polymorphic. Therefore, an MLVA assay composed of five VNTR loci was developed with greatest discriminatory power. Conclusion: MLVA typing is a faster and more standardized method for studying the genetic relatedness of H. pylori isolates. At preliminary stage it is sufficient to use only 3 VNTR loci for the differentiation of H. pylori strains.