Klebsiella pneumoniae is a globally significant opportunistic pathogen causing healthcare-associated and community-acquired infections. This study examined the epidemiology and the distribution of resistance and virulence genes in clinical K. pneumoniae strains in Kenya. A total of 89 K. pneumoniae isolates were collected over six years from five counties in Kenya and were analyzed using whole-genome sequencing and bioinformatics. These isolates were obtained from community-acquired (62/89) and healthcare-associated infections (21/89), and from the hospital environment (6/89). Genetic analysis revealed the presence of bla_NDM-1 and bla_OXA-181 carbapenemase genes and the armA and rmtF genes known to confer pan-aminoglycoside resistance. The most abundant extended-spectrum beta-lactamase genes identified were bla_CTX-M-15 (36/89), bla_TEM (35/89), and bla_OXA (18/89)_. In addition, one isolate had a mobile colistin resistance gene (mcr-8). Fluoroquinolone resistance-conferring mutations in gyrA and parC genes were also observed. The most notable virulence factors were those associated with hyper-virulence (rmpA/A2 and magA), yersiniabactin (ybt), salmochelin (iro), and aerobactin (iuc and iutA). A total of 38 distinct sequence types were identified, including known global lineages ST14, ST15, ST147, and ST307, and a regional clone ST17 implicated in regional outbreaks. In addition, this study genetically characterized two potential hypervirulent isolates and two community-acquired ST147 high-risk clones that contained carbapenemase genes, yersiniabactin, and other multidrug resistance genes. These results demonstrate that the resistome and virulome of Kenyan clinical and hospital environmental K. pneumoniae isolates are diverse. The reservoir of high-risk clones capable of spreading resistance, and virulence factors have the potential to cause unmanageable infection outbreaks with high morbidity and mortality.

This study describes the identification of the mcr-10.1 gene in a clinical isolate of an ST1 Enterobacter cloacae isolate cultured in 2015 in Kenya. The isolate was multidrug resistant, phenotypically non-susceptible to various antibiotics, including colistin. Whole genome sequence analyses indicated carriage of chromosomally encoded antimicrobial resistance genes and the colistin-resistant gene mcr-10.1 located on a 72-kb plasmid designated pECC011b with an IncFIA(HI1) replicon directly adjacent to tyrosine recombinase gene, xerC , and downstream of an IS KPn26 insertion sequence. Studies have shown that expression of mcr-10.1 may not be sufficient to confer colistin resistance, but a novel non-synonymous mutation (S244T) was identified in the phoQ gene known to influence colistin resistance within lipid modification pathways, which could have complemented the mcr-10.1 resistance mechanism. In silico analysis of the mutant phoQ protein shows the location of the mutation to be at the Histidine kinases, Adenyl cyclases, Methyl-accepting proteins and Phosphatases (HAMP) region, which plays a crucial role in the protein’s activity. This study and our previous report of mcr-8 in Klebsiella pneumoniae indicate the presence of mobile mcr genes in the Enterobacterales order of bacteria in Kenya. The study points to the importance of regulation of colistin in the animal industry and enhancing surveillance in both human and animal health to curb the spread of mcr genes and accurately assess the risks posed by these mobile genetic elements in both sectors. IMPORTANCE: This paper reports the detection of new colistin resistance mechanisms in Kenya in a clinical isolate of Enterobacter cloacae in a patient with a healthcare-associated infection. The plasmid-mediated resistance gene, mcr-10.1 , and a novel amino acid mutation S244T in the phoQ gene, located in a region of the protein involved in membrane cationic stability contributing to colistin resistance, were detected. Colistin is a critical last-line drug for multidrug-resistant (MDR) gram-negative human infections and is used for treatment and growth promotion in the animal industry. The emergence of the resistance mechanisms points to the potential overuse of colistin in the animal sector in Kenya, which enhances resistance, threatens the utility of colistin, and limits treatment options for MDR infections. This study highlights the need to enhance surveillance of colistin resistance across sectors and strengthen One Health policies that ensure antimicrobial stewardship and implementation of strategies to mitigate the spread of antibiotic resistance.

Genomic surveillance is vital for detecting outbreaks and understanding the epidemiology and transmission of bacterial strains, yet it is not integrated into many national antimicrobial resistance (AMR) surveillance programmes. Key factors are that few scientists in the public health sector are trained in bacterial genomics, and the diverse sequencing platforms and bioinformatic tools make it challenging to generate reproducible outputs. In Kenya, these gaps were addressed by training public health scientists to conduct genomic surveillance on isolates from the national AMR surveillance repository and produce harmonized reports. The 2-week training combined theory and laboratory and bioinformatic experiences with Klebsiella pneumoniae isolates from the surveillance repository. Whole-genome sequences generated on Illumina and Nanopore sequencers were analysed using publicly available bioinformatic tools, and a harmonized report was generated using the HAMRonization tool. Pre- and post-training tests and self-assessments were used to assess the effectiveness of the training. Thirteen scientists were trained and generated data on the K. pneumoniae isolates, summarizing the AMR genes present consistently with the reported phenotypes and identifying the plasmid replicons that could transmit antibiotic resistance. Ninety per cent of the participants demonstrated an overall improvement in their post-training test scores, with an average increase of 14 %. Critical challenges were experienced in delayed delivery of equipment and supplies, power fluctuations and internet connections that were inadequate for bioinformatic analysis. Despite this, the training built the knowledge and skills to implement bacterial genomic surveillance. More advanced and immersive training experiences and building supporting infrastructure would solidify these gains to produce tangible public health outcomes.

Klebsiella pneumoniae is a globally significant opportunistic pathogen causing healthcare-associated and community-acquired infections. This study examined the epidemiology and the distribution of resistance and virulence genes in clinical K. pneumoniae strains in Kenya. Eighty-nine K. pneumoniae isolates were collected over six years from five counties in Kenya and were analyzed using whole genome sequencing and bioinformatics. These isolates were obtained from community-acquired (62/89) and healthcare-associated infections (21/89), and the hospital environment (6/89). Genetic analysis revealed the presence of bla _NDM-1 and bla _OXA-181 carbapenemase genes and the armA and rmtF genes known to confer pan-aminoglycoside resistance. The most abundant extended-spectrum beta-lactamase genes identified were bla _CTX-M-15 (36/89), bla _TEM (35/89), and bla _OXA (18/89). In addition, one isolate had a mobile colistin resistance gene ( mcr-8 ). Fluoroquinolone resistance-conferring mutations in gyrA and parC genes were also observed. The most notable virulence factors were those associated with hyper-virulence ( rmpA/A2 and magA ), yersiniabactin ( ybt ), salmochelin ( iro ), and aerobactin ( iuc and iutA ). Thirty-eight distinct sequence types were identified, including known global lineages ST14, ST15, ST147, and ST307, and a regional clone ST17 implicated in regional outbreaks. In addition, this study genetically characterized two potential hypervirulent isolates and two community-acquired ST147 high-risk clones that contained carbapenemase genes, yersiniabactin, and other multidrug resistance genes. These results demonstrate that the resistome and virulome of Kenyan clinical and hospital environmental K. pneumoniae isolates are diverse. The reservoir of high risk-clones capable of spreading resistance and virulence factors have the potential to cause unmanageable infection outbreaks with high morbidity and mortality. AUTHOR SUMMARY: Klebsiella pneumoniae is one of the human-disease-causing bacteria that easily acquires and spreads antibiotic resistance genes and is thus a serious threat to human health. We studied both the antibiotic resistance genes and the genes it uses to cause disease (virulence). Forty-two percent of our isolates were multidrug resistant (MDR). They carried several resistance and virulence genes bound in mobile circular DNA molecules called plasmids which easily migrate and spread the genes between bacteria. We identified 38 distinct K. pneumoniae strains (STs) distributed within the study sites. Fifteen isolates were classified under the groups of K. pneumoniae strains known to cause global infection outbreaks such as ST14, ST15 and ST147 collected from Nairobi and Kisumu, hotspot areas for spread of resistance. In particular, two ST147 isolates were resistant to carbapenems and one isolate to colistin, which are last line antibiotics. We also identified two isolates with the potential to cause high levels of disease. We concluded that the presence of highly resistant and virulent strains in the hospital and community demonstrates a need for the continuous monitoring and management of MDR K. pneumoniae infections to prevent disease outbreaks that are difficult to control and that lead to high death rate.