Characterization of embB mutations involved in ethambutol resistance in multi-drug resistant Mycobacterium tuberculosis isolates in Zambia

Background: Ethambutol (EMB) is an important anti-tuberculosis drug used in the management of multi-drug resistant tuberculosis (MDR-TB). Mutations in embB are the major mechanism of resistance. This study investigated embB mutations among MDR-TB isolates and analyzed their correlations with phenotypic drug susceptibility testing (DST) in Zambia. Method: A total of 132 MDR-TB isolates were collected from January 2014 to April 2017 and characterized using MGIT 960 systems, embB sequencing, and spoligotyping. Results: Out of 61 phenotypically EMB resistant isolates, 53 had mutations in embB. Among the 71 EMB susceptible isolates, 47 had embB mutations. Sensitivity of embB mutations was 86.9% while specificity was 33.8%. CAS1_Kili (SIT21) had high odds of having embB mutations, particularly, G918A (Met306eIl) (Odds ratio 16.7, p < 0.0001). Conclusion: Molecular EMB resistance testing by DNA sequencing can improve detection of EMB resistance among MDR-TB patients in Zambia. Additionally, CAS1_Kili was associated with embB amino acid substitution Met306Ile suggesting transmission. A detailed investigation to track and determine transmission hotspot area for MDR-TB could help optimize control strategies

Genotypic characterization of pyrazinamide resistance in Mycobacterium tuberculosis isolated from Lusaka, Zambia

Pyrazinamide forms a core part of treatment for all types of tuberculosis (TB) in Zambia. Due to challenges associated with pyrazinamide testing, little information is available to indicate the frequency of resistance to this drug in Zambia. To determine the frequency of pyrazinamide (PZA) resistance and its correlation with mutation in pncA in Mycobacterium tuberculosis isolated from patients in Lusaka, Zambia, BACTEC MGIT M960 was used for phenotypic PZA susceptibility testing while sequencing was used to determine resistance-conferring mutations in the pncA. Of the 131 isolates analyzed, 32 were phenotypically resistant to PZA. Among multidrug-resistant (MDR) M. tuberculosis isolates, the frequency of PZA resistance was 21 of 35 (58.3%). And 27 of 32 PZA resistant isolates had mutations in the pncA that seem to confer resistance. With BACTEC MGIT 960 as the reference standard, gene sequencing showed 84.4% sensitivity and 100% specificity. Nine new mutations were identified and the single nucleotide substitution T104G and C195T were the most frequent mutations. However, they were observed in both susceptible and resistant strains and indicating that they are non-resistance conferring mutations. This study has demonstrated that PZA susceptibility testing is necessary especially in patients suffering from MDR-TB as approximately half of the patients have PZA resistant TB. Similar studies will have to be carried out in other provinces to get an accurate estimate of PZA resistance in Zambia. Mutations in pncA were the major mechanism of PZA resistance with no involvement of rpsA and panD genes. However, the presence of mutations among phenotypically PZA susceptible M. tuberculosis isolates makes it challenging to independently use genotyping method for the determination of PZA resistance

Characterization of Mutations Associated with Streptomycin Resistance in Multidrug-Resistant Mycobacterium tuberculosis in Zambia

Streptomycin (STR) is recommended for the management of multidrug-resistant tuberculosis (MDR-TB). Streptomycin resistance-conferring mutation types and frequency are shown to be influenced by genotypes of circulating strains in a population. This study aimed to characterize the mutations in MDR-TB isolates and examine their relationship with the genotypes in Zambia. A total of 138 MDR-TB isolates stored at the University Teaching Hospital Tuberculosis Reference Laboratory in Zambia were analyzed using spoligotyping and sequencing of STR resistance-associated genes. Streptomycin resistance was observed in 65.9% (91/138) of MDR-TB isolates. Mutations in rpsL, rrs, and gidB accounted for 33%, 12.1%, and 49.5%, respectively. Amino acid substitution K43R in rpsL was strongly associated with the CAS1_Kili genotype (p < 0.0001). The combination of three genes could predict 91.2% of STR resistance. Clustering of isolates based on resistance-conferring mutations and spoligotyping was observed. The clustering of isolates suggests that the increase in STR-resistant MDR-TB in Zambia is largely due to the spread of resistant strains from inadequate treatment. Therefore, rapid detection of STR resistance genetically is recommended before its use in MDR-TB treatment in Zambia.</p

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

Prevalence and Characterization of Quinolone-Resistance Determinants in Escherichia coli Isolated from Food-Producing Animals and Animal-Derived Food in the Philippines

Antimicrobial resistance to quinolones, which constitutes a threat to public health, has been increasing worldwide. In this study, we investigated the prevalence of quinolone-resistant determinants in Escherichia coli not susceptible to quinolones and isolated from food-producing animals and food derived from them, in the Philippines. A total of 791 E. coli strains were isolated in 56.4% of 601 beef, chicken, pork, egg, and milk samples, as well as environmental, cloacal, and rectal swab-collected samples from supermarkets, open markets, abattoirs, and poultry, swine, and buffalo farms. Using the disc diffusion method, it was determined that 78.6% and 55.4% of the isolates were resistant to at least one antimicrobial and multiple drugs, respectively. In 141 isolates not susceptible to quinolones, 115 (81.6%) harbored quinolone-resistant determinants and had mutations predominantly in the quinolone-resistance determining regions (QRDRs) of gyrA and parC. Plasmid-mediated, quinolone resistance (PMQR) and Qnr family (qnrA1, qnrB4, and qnrS1) genes were detected in all isolates. Forty-eight sequence types were identified in isolates harboring mutations in QRDR and/or PMQR genes by multilocus sequence typing analysis. Moreover, 26 isolates harboring mutations in QRDR and/or PMQR genes belonged mostly to phylogroup B1 and Enteroaggregative E. coli. In conclusion, a high prevalence of E. coli was found in food-producing animals and products derived from them, which could potentially spread high-risk clones harboring quinolone-resistance determinants