Plasmid mediated penicillin and tetracycline resistance among Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.

gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae isolates from Kenya.

BACKGROUND: Phenotypic fluoroquinolone resistance was first reported in Western Kenya in 2009 and later in Coastal Kenya and Nairobi. Until recently gonococcal fluoroquinolone resistance mechanisms in Kenya had not been elucidated. The aim of this paper is to analyze mutations in both gyrA and parC responsible for elevated fluoroquinolone Minimum Inhibitory Concentrations (MICs) in Neisseria gonorrhoeae (GC) isolated from heterosexual individuals from different locations in Kenya between 2013 and 2017. METHODS: Antimicrobial Susceptibility Tests were done on 84 GC in an ongoing Sexually Transmitted Infections (STI) surveillance program. Of the 84 isolates, 22 resistant to two or more classes of antimicrobials were chosen for analysis. Antimicrobial susceptibility tests were done using E-test (BioMerieux) and the results were interpreted with reference to European Committee on Antimicrobial Susceptibility Testing (EUCAST) standards. The isolates were sub-cultured, and whole genomes were sequenced using Illumina platform. Reads were assembled de novo using Velvet, and mutations in the GC Quinolone Resistant Determining Regions identified using Bioedit sequence alignment editor. Single Nucleotide Polymorphism based phylogeny was inferred using RaxML. RESULTS: Double GyrA amino acid substitutions; S91F and D95G/D95A were identified in 20 isolates. Of these 20 isolates, 14 had an additional E91G ParC substitution and significantly higher ciprofloxacin MICs (p = 0.0044*). On the contrary, norfloxacin MICs of isolates expressing both GyrA and ParC QRDR amino acid changes were not significantly high (p = 0.82) compared to MICs of isolates expressing GyrA substitutions alone. No single GyrA substitution was found in the analyzed isolates, and no isolate contained a ParC substitution without the simultaneous presence of double GyrA substitutions. Maximum likelihood tree clustered the 22 isolates into 6 distinct clades. CONCLUSION: Simultaneous presence of amino acid substitutions in ParC and GyrA has been reported to increase gonococcal fluoroquinolone resistance from different regions in the world. Our findings indicate that GyrA S91F, D95G/D95A and ParC E91G amino acid substitutions mediate high fluoroquinolone resistance in the analyzed Kenyan GC.

Fecal contamination of drinking water in Kericho District, Western Kenya: role of source and household water handling and hygiene practices.

Inadequate protection of water sources, and poor household hygienic and handling practices have exacerbated fecal water contamination in Kenya. This study evaluated the rate and correlates of thermotolerant coliform (TTC) household water contamination in Kericho District, Western Kenya. Culture and multiplex polymerase chain reaction (PCR) techniques were used to characterize TTCs. The disk diffusion method was used for antibiotic susceptibility profiling of pathogenic Escherichia coli. Out of the 103 households surveyed, 48 (46.6%) had TTC contaminated drinking water (TTC levels of >10 cfu/100 mL). Five of these households were contaminated with pathogenic E. coli, including 40% enteroaggregative E. coli, 40% enterotoxigenic E. coli, and 20% enteropathogenic E. coli. All these pathogenic E. coli strains were multidrug resistant to sulfamethoxazole/trimethoprim, ampicillin, tetracycline and ampicillin/sulbactam. Rural household locality, drinking water hand contact, water storage container cleaning practice, hand washing before water withdrawal, water source total coliforms <10 cfu/100 mL, temperature, and free chlorine levels were associated with TTC contamination of household drinking water. Significant proportions of household drinking water in Kericho District are contaminated with TTCs including with pathogenic multidrug-resistant E. coli. Source and household hygiene and practices contribute significantly to drinking water contamination.