Insight into the genetic diversity of Mycobacterium bovis isolated from cattle in Malawi.

We describe the genetic diversity and phylogenetic relationships of Mycobacterium bovis, isolated from cattle in Malawi. Deletion analysis, spoligotyping, and MIRU-VNTR typing were used to genotype the isolates. Combined with a larger dataset from neighboring countries, the overall M. bovis diversity in Southern Africa was contextualized. From the southern and northern regions of Malawi, 24 isolates were confirmed as M. bovis. We pooled data for the central region (60 isolates) from our recent publication to conceptualize the genetic and phylogenetic relationships of M. bovis in Malawi. European 1 was the dominant M. bovis clonal complex, with 10 unique spoligotype patterns, and SB0131 was ubiquitous. High genetic diversity, a low clustering rate, and many singletons, coupled with a low mutation transmission index, infer a low level of recent transmission, and suggest an endemic status of bovine tuberculosis (bTB) in Malawi. M. bovis isolates from Zambia, Mozambique, and South Africa were genetically related to Malawian isolates, whereas Tanzanian isolates were distantly related. The diversity and phylogenetic analysis suggest earlier introductions and maintenance of M. bovis by constant reinfection from reservoir animals. These findings are fundamental to understanding the source and route of infection in order to establish alternative management strategies for bTB.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Detection of Mutations in pncA in Mycobacterium tuberculosis Clinical Isolates from Nepal in Association with Pyrazinamide Resistance.

Without the proper information on pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis (MTB), PZA is inappropriately recommended for the treatment of both susceptible and multidrug-resistant tuberculosis (MDR-TB) in Nepal. This study aimed to collect information regarding PZA susceptibility in MTB isolates from Nepal by analyzing pncA and its upstream regulatory region (URR). A total of 211 MTB isolates were included in this study. Sequence analysis of pncA and its URR was performed to assess PZA resistance. First-line drug susceptibility testing, spoligotyping, and sequence analysis of rpoB, katG, the inhA regulatory region, gyrA, gyrB, and rrs were performed to assess their association with pncA mutation. Sequencing results reveal that 125 (59.2%) isolates harbored alterations in pncA and its URR. A total of 57 different mutation types (46 reported and 11 novel) were scattered throughout the whole length of the pncA gene. Eighty-seven isolates (41.2%) harbored mutations in pncA, causing PZA resistance in MTB. There was a more significant association of pncA alterations in MDR/pre-extensively drug-resistant (Pre-XDR) TB than in mono-resistant/pan-susceptible TB (p < 0.005). This first report on the increasing level of PZA resistance in DR-TB in Nepal highlights the importance of PZA susceptibility testing before DR-TB treatment.

Genetic Diversity and Transmission of Multidrug-Resistant Mycobacterium tuberculosis strains in Lusaka, Zambia.

OBJECTIVE: Zambia is among the 30 high tuberculosis burden countries in the world. Despite increasing reports of multidrug-resistant tuberculosis (MDR-TB) in routine surveillance, information on the transmission of MDR Mycobacterium tuberculosis strains is largely unknown. This study elucidated the genetic diversity and transmission of MDR M. tuberculosis strains in Lusaka, Zambia. METHODS: Eighty-five MDR M. tuberculosis samples collected from 2013 to 2017 at the University Teaching Hospital were used. Drug-resistance associated gene sequencing, spoligotyping, 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and multiplex PCR for RD-Rio sub-lineage identification were applied. RESULTS: The identified clades were LAM (48%), CAS (29%), T (14%), X (6%) and Harlem (2%). Strains belonging to SITs 21/CAS1-Kili and 20/LAM1 formed the largest clonal complexes. Combined spoligotyping and 24 loci-MIRU-VNTR revealed 47 genotypic patterns with a clustering rate of 63%. Ninety-five percent of LAM strains belonged to the RD-Rio sub-lineage. CONCLUSION: The high clustering rate suggested that a large proportion of MDR-TB was due to recent transmission rather than the independent acquisition of MDR. This spread was attributed to clonal expansion of SIT21/CAS1-Kili and SIT20/LAM1 strains. Therefore, TB control programs recommending genotyping coupled with conventional epidemiological methods can guide measures for stopping the spread of MDR-TB.

Evaluation of IS1245 LAMP in Mycobacterium avium and the influence of host-related genetic diversity on its application.

Early detection and treatment are paramount for the timely control of Mycobacterium avium infections. Herein, we designed a LAMP assay targeting a widely used species-specific marker IS1245 for the rapid detection of M. avium and evaluated its applicability using human (n = 137) and pig (n = 91) M. avium isolates from Japan. The developed assay could detect as low as 1 genome copy of M. avium DNA within 30 minutes. All 91 (100%) M. avium isolates from pigs were detected positive while all other tested bacterial species were negative. Interestingly, among the 137 clinical M. avium isolates, 41 (30%) were undetectable with this LAMP assay as they lacked IS1245, the absence of which was revealed by PCR and whole-genome sequencing. These findings highlighted genotypic differences in M. avium strains from humans and pigs in Japan and how this diversity can influence the applicability of a detection tool across different geographic areas and hosts.

Genetic characterization of coliform bacterial isolates from environmental water in Thailand.

INTRODUCTION: In contrast to the study in other part of the world, information about characteristics of plasmids carrying antimicrobial resistance genes (ARGs) in Enterobacteriaceae derived from environmental water in tropical Asian countries including Thailand is limited. This study, therefore, aimed to gain insight into genetic information of antimicrobial resistance in environmental water in Thailand. METHODS: Coliform bacteria were isolated from environmental water collected at 20 locations in Thailand and identified. Then, susceptibility profiles to ampicillin, cefazoline, cefotaxime, kanamycin, ciprofloxacin, sulfamethoxazole, tetracycline, and nalidixic acid were assessed. In addition, antimicrobial resistant genes integrons, and replicon types were analyzed. And furthermore, plasmids carrying blaTEM and tetM were identified by S1-PFGE analysis and confirmed transmissibility by transconjugation experiments. RESULTS: In 130 coliform bacteria isolated, 89 were resistant to cefazoline while 41 isolates were susceptible. Cefazoline-resistant coliform bacteria were found to be significantly resistant to cefotaxime and tetracycline as compared to susceptible isolates. Hence, blaTEM and tetM correlating with ß-lactam antibiotics and tetracycline, respectively, were analyzed found to co-localize on the IncFrepB plasmids in isolates from pig farms' wastewater by S1-PFGE analysis. And furthermore, transmissibility of the plasmids was confirmed. CONCLUSIONS: Results obtained in this study suggested that ARGs in coliform bacteria may have been spreading on the farm via IncFrepB plasmids. Hence, appropriate use of antimicrobials and good hygiene management on the farm are required to prevent the emergence and spread of resistant bacteria.

Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis.

Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.