Phenotypic and genotypic characterization of clinical Staphylococcus aureus isolates from Kenya.

BACKGROUND: The increase and spread of virulent-outbreak associated, methicillin and vancomycin resistant (MRSA/VRSA) Staphylococcus aureus require a better understanding of the resistance and virulence patterns of circulating and emerging strains globally. This study sought to establish the resistance phenotype, and strains of 32 non-duplicate clinical MRSA and MSSA S. aureus isolates from four Kenyan hospitals, identify their resistance and virulence genes and determine the genetic relationships of MRSA with global strains. METHODS: Antimicrobial susceptibility profiles were determined on a Vitek 2, genomic DNA sequenced on an Illumina Miseq and isolates typed in-silico. Resistance and virulence genes were identified using ARIBA and phylogenies generated using RAxML. RESULTS: The MRSA isolates were 100% susceptible to vancomycin, teicoplanin, linezolid, and tigecycline. Nine distinct CC, 12 ST and 15 spa types including the novel t17826 and STs (4705, 4707) were identified with CC8 and CC152 predominating. MRSA isolates distributed across 3 CCs; CC5-ST39 (1), CC8 - ST241 (4), a novel CC8-ST4705 (1), ST8 (1) and CC152 (1). There was > 90% phenotype-genotype concordance with key resistance genes identified only among MRSA isolates: gyrA, rpoB, and parC mutations, mecA, ant (4')-lb, aph (3')-IIIa, ermA, sat-4, fusA, mphC and msrA. Kenyan MRSA isolates were genetically diverse and most closely related to Tanzanian and UK isolates. There was a significant correlation between map, hlgA, selk, selq and cap8d virulence genes and severe infections. CONCLUSION: The findings showed a heterogeneous S. aureus population with novel strain types. Though limited by the low number of isolates, this study begins to fill gaps and expand our knowledge of S. aureus epidemiology while uncovering interesting patterns of distribution of strain types which should be further explored. Although last-line treatments are still effective, the potential for outbreaks of both virulent and resistant strains remain, requiring sustained surveillance of S. aureus populations.

Genetic divergence of Chikungunya virus plaque variants from the Comoros Island (2005).

Chikungunya virus (CHIKV) from a human sample collected during the 2005 Chikungunya outbreak in the Comoros Island, showed distinct and reproducible large (L2) and small (S7) plaques which were characterized in this study. The parent strain and plaque variants were analysed by in vitro growth kinetics in different cell lines and their genetic similarity assessed by whole genome sequencing, comparative sequence alignment and phylogenetic analysis. In vitro growth kinetic assays showed similar growth patterns of both plaque variants in Vero cells but higher viral titres of S7 compared to L2 in C6/36 cells. Amino acids (AA) alignments of the CHIKV plaque variants and S27 African prototype strain, showed 30 AA changes in the non-structural proteins (nsP) and 22 AA changes in the structural proteins. Between L2 and S7, only two AAs differences were observed. A missense substitution (C642Y) of L2 in the nsP2, involving a conservative AA substitution and a nonsense substitution (R524X) of S7 in the nsP3, which has been shown to enhance O'nyong-nyong virus infectivity and dissemination in Anopheles mosquitoes. The phenotypic difference observed in plaque size could be attributed to one of these AA substitutions. Phylogenetic analysis showed that the parent strain and its variants clustered closely together with each other and with Indian Ocean CHIKV strains indicating circulation of isolates with close evolutionary relatedness in the same outbreak. These observations pave way for important functional studies to understand the significance of the identified genetic changes in virulence and viral transmission in mosquito and mammalian hosts.

Ticks and tick-borne viruses from livestock hosts in arid and semiarid regions of the eastern and northeastern parts of Kenya.

Biodiversity and relative abundance of ticks and associated arboviruses in Garissa (northeastern) and Isiolo (eastern) provinces of Kenya were evaluated. Ticks were collected from livestock, identified to species, pooled, and processed for virus isolation. In Garissa, Rhipicephalus pulchellus Gerstacker (57.8%) and Hyalomma truncatum Koch (27.8%) were the most abundant species sampled, whereas R. pulchellus (80.4%) and Amblyomma gemma Donitz (9.6%) were the most abundant in Isiolo. Forty-four virus isolates, comprising Dugbe virus (DUGV; n = 22) and Kupe virus (n = 10; Bunyaviridae: Nirovirus), Dhori virus (DHOV; n = 10; Orthomyxoviridae: Thogotovirus),and Ngari virus (NRIV; n = 2; Bunyaviridae: Orthobunyavirus), were recovered mostly from R. pulchellus sampled in Isiolo. DUGV was mostly recovered from R. pulchellus from sheep and cattle, and DHOV from R. pulchellus from sheep. All Kupe virus isolates were from Isiolo ticks, including R. pulchellus from all the livestock, A. gemma and Amblyomma variegatum F. from cattle, and H. truncatum from goat. NRIV was obtained from R. pulchellus and A. gemma sampled from cattle in Isiolo and Garissa, respectively, while all DHOV and most DUGV (n = 12) were from R. pulchellus sampled from cattle in Garissa. DUGV was also recovered from H. truncatum and Amblyomma hebraeum Koch from cattle and from Rhipicephalus annulatus Say from camel. This surveillance study has demonstrated the circulation of select tick-borne viruses in parts of eastern and northeastern provinces of Kenya, some of which are of public health importance. The isolation of NRIV from ticks is particularly significant because it is usually known to be a mosquito-borne virus affecting humans.

The first report of the mobile colistin resistance gene, mcr-10.1, in Kenya and a novel mutation in the phoQ gene (S244T) in a colistin-resistant Enterobacter cloacae clinical isolate.

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 ISKPn26 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.IMPORTANCEThis 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.

Sterile Instrument Storage in an Austere Environment: Are Sterile Peel Packaging and Cellulose Wrapping Equivalent?

BACKGROUND: Recommendations for optimal temperature and humidity for sterile instrument storage vary according to different sources. Furthermore, there are limited data comparing methods of packing smaller, lightweight, low-profile instruments. The purpose of this study was to compare sterile peel packaging and sterile cellulose wrapping for sterile instrument storage in an austere environment characterized by elevated temperature and humidity. METHODS: Stainless steel screws were sterilized and stored in either sterile peel packaging, sterile cellulose wrapping, or no packaging. Four groups were evaluated. Group 1 consisted of four screws in a sterile peelpack envelope and served as a time-zero control. Group 2 consisted of two groups of five screws, each packaged with blue sterilization cellulose wrap. Group 3 consisted of two groups of five screws, each packaged in sterile peel-pack envelopes. Group 4 consisted of 10 non-sterile unpackaged screws, which served as controls. Screws from groups 2, 3, and 4 were then cultured for 6 and 12 weeks. Temperature and humidity values were recorded in the instrument storage area. RESULTS: Average temperature was 21.3°C (SD 1.2°C; range 18.9°C-27.2°C) and average humidity was 51.7% (SD 3.9%; range 39%- 70%). Groups 1 (time-zero control) and 2 (sterile cellulose wrapping) demonstrated no growth. After 6 and 12 weeks, groups 3 (sterile peel packaging) and 4 (control) demonstrated bacterial growth. CONCLUSION: The most common culture isolates were gram-positive rods and two common nosocomial Staphylococcius species. Sterile peel packaging was not found to be equivalent to sterile cellulose wrapping in austere environmental conditions.

Displacement of Hospital-Acquired, Methicillin-Resistant Staphylococcus aureus Clones by Heterogeneous Community Strains in Kenya over a 13-Year Period.

We determined antibiotic susceptibility and employed Oxford Nanopore whole-genome sequencing to explore strain diversity, resistance, and virulence gene carriage among methicillin-resistant Staphylococcus aureus (MRSA) strains from different infection sites and timepoints in a tertiary Kenyan hospital. Ninety-six nonduplicate clinical isolates recovered between 2010 and 2023, identified and tested for antibiotic susceptibility on the VITEK ID/AST platform, were sequenced. Molecular typing, antibiotic resistance, and virulence determinant screening were performed using the relevant bioinformatics tools. The strains, alongside those from previous studies, were stratified into two periods covering 2010-2017 and 2018-2023 and comparisons were made. Mirroring phenotypic profiles, aac(6')-aph(2″) [aminoglycosides]; gyrA (S84L) and grlA (S80Y) [fluoroquinolones]; dfrG [anti-folates]; and tet(K) [tetracycline] resistance determinants dominated the collection. While the proportion of ST239/241-t037-SCCmec III among MRSA reduced from 37.7% to 0% over the investigated period, ST4803-t1476-SCCmec IV and ST152-t355-SCCmec IV were pre-eminent. The prevalence of Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) genes was 38% (33/87) and 6.8% (6/87), respectively. We observed the displacement of HA-MRSA ST239/241-t037-SCCmec III with the emergence of ST152-t355-SCCmec IV and a greater clonal heterogeneity. The occurrence of PVL+/ACME+ CA-MRSA in recent years warrants further investigations into their role in the CA-MRSA virulence landscape, in a setting of high PVL prevalence.

Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.

Ticks are arachnid ectoparasites that rank second only to mosquitoes in the transmission of human diseases including bacteria responsible for anaplasmosis, ehrlichiosis, spotted fevers, and Lyme disease among other febrile illnesses. Due to the paucity of data on bacteria transmitted by ticks in Kenya, this study undertook a bacterial metagenomic-based characterization of ticks collected from Isiolo, a semi-arid pastoralist County in Eastern Kenya, and Kwale, a coastal County with a monsoon climate in the southern Kenyan border with Tanzania. A total of 2,918 ticks belonging to 3 genera and 10 species were pooled and screened in this study. Tick identification was confirmed through the sequencing of the Cytochrome C Oxidase Subunit 1 (COI) gene. Bacterial 16S rRNA gene PCR amplicons obtained from the above samples were sequenced using the MinION (Oxford Nanopore Technologies) platform. The resulting reads were demultiplexed in Porechop, followed by trimming and filtering in Trimmomatic before clustering using Qiime2-VSearch. A SILVA database pretrained naïve Bayes classifier was used to classify the Operational Taxonomic Units (OTUs) taxonomically. The bacteria of clinical interest detected in pooled tick assays were as follows: Rickettsia spp. 59.43% of pools, Coxiella burnetii 37.88%, Proteus mirabilis 5.08%, Cutibacterium acnes 6.08%, and Corynebacterium ulcerans 2.43%. These bacteria are responsible for spotted fevers, query fever (Q-fever), urinary tract infections, skin and soft tissue infections, eye infections, and diphtheria-like infections in humans, respectively. P. mirabilis, C. acnes, and C. ulcerans were detected only in Isiolo. Additionally, COI sequences allowed for the identification of Rickettsia and Coxiella species to strain levels in some of the pools. Diversity analysis revealed that the tick genera had high levels of Alpha diversity but the differences between the microbiomes of the three tick genera studied were not significant. The detection of C. acnes, commonly associated with human skin flora suggests that the ticks may have contact with humans potentially exposing them to bacterial infections. The findings in this study highlight the need for further investigation into the viability of these bacteria and the competency of ticks to transmit them. Clinicians in these high-risk areas also need to be appraised for them to include Rickettsial diseases and Q-fever as part of their differential diagnosis.

Mosquito-borne arbovirus surveillance at selected sites in diverse ecological zones of Kenya; 2007 - 2012.

BACKGROUND: Increased frequency of arbovirus outbreaks in East Africa necessitated the determination of distribution of risk by entomologic arbovirus surveillance. A systematic vector surveillance programme spanning 5 years and covering 11 sites representing seven of the eight provinces in Kenya and located in diverse ecological zones was carried out. METHODS: Mosquitoes were sampled bi-annually during the wet seasons and screened for arboviruses. Mosquitoes were identified to species, pooled by species, collection date and site and screened for arboviruses by isolation in cell culture and/or RT-PCR screening and sequencing. RESULTS: Over 450,000 mosquitoes in 15,890 pools were screened with 83 viruses being detected/isolated that include members of the alphavirus, flavivirus and orthobunyavirus genera many of which are known to be of significant public health importance in the East African region. These include West Nile, Ndumu, Sindbis, Bunyamwera, Pongola and Usutu viruses detected from diverse sites. Ngari virus, which was associated with hemorrhagic fever in northern Kenya in 1997/98 was isolated from a pool of Anopheles funestus sampled from Tana-delta and from Aedes mcintoshi from Garissa. Insect only flaviviruses previously undescribed in Kenya were also isolated in the coastal site of Rabai. A flavivirus most closely related to the Chaoyang virus, a new virus recently identified in China and two isolates closely related to Quang Binh virus previously unreported in Kenya were also detected. CONCLUSION: Active transmission of arboviruses of public health significance continues in various parts of the country with possible undetermined human impact. Arbovirus activity was highest in the pastoralist dominated semi-arid to arid zones sites of the country where 49% of the viruses were isolated suggesting a role of animals as amplifiers and indicating the need for improved arbovirus disease diagnosis among pastoral communities.

Environmental contamination across multiple hospital departments with multidrug-resistant bacteria pose an elevated risk of healthcare-associated infections in Kenyan hospitals.

BACKGROUND: Healthcare-associated infections (HAIs) are often caused by multidrug-resistant (MDR) bacteria contaminating hospital environments which can cause outbreaks as well as sporadic transmission. METHODS: This study systematically sampled and utilized standard bacteriological culture methods to determine the numbers and types of MDR Enterococcus faecalis/faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli (ESKAPEE) from high-touch environments of five Kenyan hospitals; level 6 and 5 hospitals (A, B, and C), and level 4 hospitals (D and E), in 2018. Six hundred and seventeen high-touch surfaces across six hospital departments; surgical, general, maternity, newborn, outpatient and pediatric were sampled. RESULTS: 78/617 (12.6%) of the sampled high-touch surfaces were contaminated with MDR ESKAPEE; A. baumannii, 23/617 (3.7%), K. pneumoniae, 22/617 (3.6%), Enterobacter species, 19/617 (3.1%), methicillin resistant S. aureus (MRSA), 5/617 (0.8%), E. coli, 5/617 (0.8%), P. aeruginosa, 2/617 (0.3%), and E. faecalis and faecium, 2/617 (0.3%). Items found in patient areas, such as beddings, newborn incubators, baby cots, and sinks were the most frequently contaminated. Level 6 and 5 hospitals, B, 21/122 (17.2%), A, 21/122 (17.2%), and C, 18/136 (13.2%), were more frequently contaminated with MDR ESKAPEE than level 4 hospitals; D, 6/101 (5.9%), and E, 8/131 (6.1%). All the sampled hospital departments were contaminated with MDR ESKAPEE, with high levels observed in newborn, surgical and maternity. All the A. baumannii, Enterobacter species, and K. pneumoniae isolates were non-susceptible to piperacillin, ceftriaxone and cefepime. 22/23 (95.6%) of the A. baumannii isolates were non-susceptible to meropenem. In addition, 5 K. pneumoniae isolates were resistant to all the antibiotics tested except for colistin. CONCLUSION: The presence of MDR ESKAPEE across all the hospitals demonstrated gaps in infection prevention practices (IPCs) that should be addressed. Non-susceptibility to last-line antibiotics such as meropenem threatens the ability to treat infections.

Genomics for public health and international surveillance of antimicrobial resistance.

Historically, epidemiological investigation and surveillance for bacterial antimicrobial resistance (AMR) has relied on low-resolution isolate-based phenotypic analyses undertaken at local and national reference laboratories. Genomic sequencing has the potential to provide a far more high-resolution picture of AMR evolution and transmission, and is already beginning to revolutionise how public health surveillance networks monitor and tackle bacterial AMR. However, the routine integration of genomics in surveillance pipelines still has considerable barriers to overcome. In 2022, a workshop series and online consultation brought together international experts in AMR and pathogen genomics to assess the status of genomic applications for AMR surveillance in a range of settings. Here we focus on discussions around the use of genomics for public health and international AMR surveillance, noting the potential advantages of, and barriers to, implementation, and proposing recommendations from the working group to help to drive the adoption of genomics in public health AMR surveillance. These recommendations include the need to build capacity for genome sequencing and analysis, harmonising and standardising surveillance systems, developing equitable data sharing and governance frameworks, and strengthening interactions and relationships among stakeholders at multiple levels.

Phage banks as potential tools to rapidly and cost-effectively manage antimicrobial resistance in the developing world.

Lower and middle-income countries seldom develop vaccines and therapeutics for their own populations and are dependent on supplies from industrialized countries, which are often hampered by financial or supply chain limitations. This has resulted in major delays in delivery with significant loss of life, as seen with the coronavirus pandemic. Since the vast majority of deaths from the antimicrobial resistance crisis are expected to occur in developing countries, there is an urgent need for in-country production of antibacterial therapies such as phages. Nationally controlled phage banks might provide such a solution since locally developed phage therapies tailored to endemic bacterial strains could offer cost-effective antibiotic alternatives.

Phenotypic and Genotypic Characteristics of Uropathogenic Escherichia coli Isolates from Kenya.

Introduction: Uropathogenic Escherichia coli (UPECs) are a significant cause of urinary tract infections (UTIs). In Kenya, UTIs are typically treated with ß-lactam antibiotics without antibiotic susceptibility testing, which could accelerate antibiotic resistance among UPEC strains. Aim: This study determined the occurrence of UPEC producing extended-spectrum ß-lactamases (ESBLs), the genes conferring resistance to ß-lactams, and the phylogenetic groups associated with ESBLs in Kenyan UPECs. Methodology: Ninety-five UPEC isolates from six Kenyan hospitals were tested for ESBL and plasmid-mediated AmpC ß-lactamase (pAmpC) production by combined disk diffusion and disk approximation tests, respectively. Real-time and conventional polymerase chain reactions (PCRs) were used to detect three ESBL and six pAmpC genes, respectively, and phylogenetic groups were assigned by a quadruplex PCR method. Results: Twenty-four percent UPEC isolates were ESBL producers with blaCTX-M (95.6%), blaTEM (95.6%), and blaSHV (21.7%) genes detected. Sixteen isolates had blaCTX-M/TEM, whereas five had blaTEM/CTX-M/SHV. A total of 5/23 ESBLs were cefoxitin resistant, but no AmpC genes were detected. The UPECs belonged predominantly to phylogenetic groups B2 (31/95; 32.6%) and D (30/95; 31.6%), while groups B2 and A had the most ESBL producers. Conclusions: ß-Lactam antibiotics have reduced utility for treating UTIs as a quarter of UPECs were ESBL producing. Single or multiple ESBL genes were present in UPECs, belonging primarily to phylogenetic groups B2 and A.

Antimicrobial Resistance and Virulence Characteristics of Klebsiella pneumoniae Isolates in Kenya by Whole-Genome Sequencing.

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 blaNDM-1 and blaOXA-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 blaCTX-M-15 (36/89), blaTEM (35/89), and blaOXA (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.

Immunization of Rabbits with a Quadrivalent Shigella Bioconjugate Vaccine Induces Functional Antibodies Reactive with Shigella Isolates from Kenya.

Diarrheal diseases are a leading cause of global morbidity and mortality, disproportionately affecting children in resource-limited settings. Although improvements in hygiene and access to clean water are helpful, vaccines are considered essential due to the low infectious dose of Shigella species and increasing antibiotic resistance. Building on achievements with conjugate vaccines, a safe and immunogenic novel bioconjugate vaccine linking Shigella O-antigen to Pseudomonas aeruginosa exoprotein A has been developed to induce immunity against Shigella flexneri 2a, 3a, and 6 and S. sonnei. This study evaluated the breadth of reactivity and functionality of pooled serum from rabbits immunized with monovalent and quadrivalent Shigella bioconjugates formulated with or without an adjuvant against Shigella serotypes isolated in Kenya. Rabbit sera were assayed by colony blot for reactivity with 67 isolates of Shigella serotypes targeted by the vaccine, S. flexneri (2a, 3a, and 6) and S. sonnei, and 42 isolates of Shigella serotypes not targeted by the vaccine, S. flexneri (1b, 2b, 4a, and 4b), S. boydii, and S. dysenteriae. Shigella isolates testing positive in the colony blot assay were then used to assess functional activity using a bactericidal assay. Of the 41 Shigella isolates targeted by the vaccine, 22 were reactive with the adjuvanted quadrivalent and the respective monovalent rabbit sera. The S. flexneri 2a and 3a monovalent rabbit serum cross-reacted with S. flexneri 3a, 2b, and 2a, respectively. Immunization with the adjuvanted quadrivalent vaccine also induced cross-reactivity with isolates of S. flexneri 2b, 4a, and 4b. Collectively, these results suggest that the Shigella quadrivalent vaccine may be more broadly protective than designed, offering a promising solution to Shigella infections. IMPORTANCE Diarrheal diseases are the third leading cause of death globally, disproportionally affecting low- to middle-income countries like Kenya, with Shigella species being the leading cause of bacterial diarrhea, especially in children. The low infectious dose and high antibiotic resistance levels complicate treatment, leading to long-term sequelae that necessitate control measures such as vaccines to reduce morbidity and mortality rates, especially among children under 5 years of age. A quadrivalent bioconjugate Shigella vaccine was recently developed to safely and effectively induce immunity against four important Shigella spp. This study demonstrates the breadth of reactivity and functionality of the parenterally administered bioconjugate vaccine by evaluating the ability of rabbit sera to bind and kill Shigella isolates recently collected in Kenya. These results suggest that the Shigella quadrivalent vaccine may be more broadly protective than designed and may offer a promising solution to the morbidity and mortality associated with Shigella infections.

Genetic Diversity, Distribution, and Genomic Characterization of Antibiotic Resistance and Virulence of Clinical Pseudomonas aeruginosa Strains in Kenya.

Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide. It can produce a range of debilitating infections, have a propensity for developing antimicrobial resistance, and present with a variety of potent virulence factors. This study investigated the sequence types (ST), phenotypic antimicrobial susceptibility profiles, and resistance and virulence genes among clinical isolates from urinary tract and skin and soft tissue infections. Fifty-six P. aeruginosa clinical isolates were obtained from six medical centers across five counties in Kenya between 2015 and 2020. Whole-genome sequencing (WGS) was performed to conduct genomic characterization, sequence typing, and phylogenetic analysis of the isolates. Results showed the presence of globally distributed high-risk clones (ST244 and ST357), local high-risk clones (ST2025, ST455, and ST233), and a novel multidrug-resistant (MDR) clone carrying virulence genes (ST3674). Furthermore, 31% of the study isolates were found to be MDR with phenotypic resistance to a variety of antibiotics, including piperacillin (79%), ticarcillin-clavulanic acid (57%), meropenem (34%), levofloxacin (70%), and cefepime (32%). Several resistance genes were identified, including carbapenemases VIM-6 (ST1203) and NDM-1 (ST357), fluoroquinolone genes, crpP, and qnrVCi, while 14 and 22 different chromosomal mutations were detected in the gyrA and parC genes, respectively. All isolates contained at least three virulence genes. Among the virulence genes identified, phzB1 was the most abundant (50/56, 89%). About 21% (12/56) of the isolates had the exoU+/exoS- genotype, while 73% (41/56) of the isolates had the exoS+/exoU- genotype. This study also discovered 12 novel lineages of P. aeruginosa, of which one (ST3674) demonstrated both extensive antimicrobial resistance and the highest number of virulence genes (236/242, 98%). Although most high-risk clones were detected in Nairobi County, high-risk and clones of interest were found throughout the country, indicating the local spread of global epidemic clones and the emergence of new strains. Thus, this study illustrates the urgent need for coordinated local, regional, and international antimicrobial resistance surveillance efforts.

Assessment of independent comorbidities and comorbidity measures in predicting healthcare facility-onset Clostridioides difficile infection in Kenya.

INTRODUCTION: Clostridioides difficile is primarily associated with hospital-acquired diarrhoea. The disease burden is aggravated in patients with comorbidities due to increased likelihood of polypharmacy, extended hospital stays and compromised immunity. The study aimed to investigate comorbidity predictors of healthcare facility-onset C. difficile infection (HO-CDI) in hospitalized patients. METHODOLOGY: We performed a cross sectional study of 333 patients who developed diarrhoea during hospitalization. The patients were tested for CDI. Data on demographics, admission information, medication exposure and comorbidities were collected. The comorbidities were also categorised according to Charlson Comorbidity Index (CCI) and Elixhauser Comorbidity Index (ECI). Comorbidity predictors of HO-CDI were identified using multiple logistic regression analysis. RESULTS: Overall, 230/333 (69%) patients had comorbidities, with the highest proportion being in patients aged over 60 years. Among the patients diagnosed with HO-CDI, 63/71(88.7%) reported comorbidities. Pairwise comparison between HO-CDI patients and comparison group revealed significant differences in hypertension, anemia, tuberculosis, diabetes, chronic kidney disease and chronic obstructive pulmonary disease. In the multiple logistic regression model significant predictors were chronic obstructive pulmonary disease (odds ratio [OR], 9.51; 95% confidence interval [CI], 1.8-50.1), diabetes (OR, 3.56; 95% CI, 1.11-11.38), chronic kidney disease (OR, 3.88; 95% CI, 1.57-9.62), anemia (OR, 3.67; 95% CI, 1.61-8.34) and hypertension (OR, 2.47; 95% CI, 1.-6.07). Among the comorbidity scores, CCI score of 2 (OR 6.67; 95% CI, 2.07-21.48), and ECI scores of 1 (OR, 4.07; 95% CI, 1.72-9.65), 2 (OR 2.86; 95% CI, 1.03-7.89), and ≥ 3 (OR, 4.87; 95% CI, 1.40-16.92) were significantly associated with higher odds of developing HO-CDI. CONCLUSION: Chronic obstructive pulmonary disease, chronic kidney disease, anemia, diabetes, and hypertension were associated with an increased risk of developing HO-CDI. Besides, ECI proved to be a better predictor for HO-CDI. Therefore, it is imperative that hospitals should capitalize on targeted preventive approaches in patients with these underlying conditions to reduce the risk of developing HO-CDI and limit potential exposure to other patients.

Determination of Enterococcus faecalis and Enterococcus faecium Antimicrobial Resistance and Virulence Factors and Their Association with Clinical and Demographic Factors in Kenya.

Background: Enterococci are clinically significant because of their increasing antibiotic resistance and their ability to cause severe infections due to an arsenal of virulence genes. Few studies in the developing world have examined virulence factors that may significantly impact patient outcomes. This study describes the antimicrobial resistance profiles and prevalence of five key Enterococcal virulence genes gelE, asa, cylA, esp, and hyl in forty-four clinical Enterococcus faecalis and E. faecium isolates in Kenya and their association with patients' demographic and clinical characteristics. Results: All E. faecium isolates were obtained from hospital-acquired skin and soft tissue infections. While E. faecalis was associated with community-acquired urinary tract infections. All isolates were resistant to erythromycin, whereas 11/44 (27.5%), 25/44 (56.8%), 28/44 (63.6%), 37/44 (84.1%), 40/44 (90.0%), and 43/44 (97.5%) were susceptible to tetracycline, levofloxacin, gentamicin, ampicillin, nitrofurantoin, and teicoplanin, respectively. All isolates were susceptible to tigecycline, vancomycin, and linezolid. There was little difference in the antibiotic resistance profiles between E. faecalis and E. faecium. The prevalence of the virulence genes among the 44 isolates were 27 (61.4%) for gelE, 26 (59.1%) for asa1, 16 (36.3%) for esp, 11 (25.0%) for cylA, and 1 (2.3%) for hyl. 72.9% of E. faecalis isolates had multiple virulence genes compared to 57% of E. faecium isolates with no virulence genes. The hyl gene was only detected in E. faecium, while cylA and asa1 were only detected in E. faecalis. A significant correlation was observed between the presence of asa1 and esp virulence genes and tetracycline resistance (P=0.0305 and 0.0363, respectively). A significant correlation was also observed between the presence of virulence genes gelE and asa1 and nitrofurantoin resistance (P=0.0175 and 0.0225, respectively) and ampicillin resistance (P=0.0005 and 0.0008, respectively). Conclusion: The study highlights the high levels of erythromycin resistance in E. faecalis and E. faecium, the demographic factors influencing the species distribution among patients, and the accumulation of multiple virulence genes in E. faecalis. The significant association of gelE, asa1, and esp virulence genes with drug resistance could explain the pathogenic success of E. faecalis and provides a guide for future studies.

Seroprevalence and distribution of arboviral infections among rural Kenyan adults: a cross-sectional study.

BACKGROUND: Arthorpod-borne viruses (arboviruses) cause wide-spread morbidity in sub-Saharan Africa, but little research has documented the burden and distribution of these pathogens. METHODS: Using a population-based, cross-sectional study design, we administered a detailed questionnaire and used ELISA to test the blood of 1,141 healthy Kenyan adults from three districts for the presence of anti-viral Immunoglobulin G (IgG) antibodies to the following viruses: dengue (DENV), West Nile (WNV), yellow fever (YFV), Chikungunya (CHIKV), and Rift Valley fever (RVFV). RESULTS: Of these, 14.4% were positive for DENV, 9.5% were WNV positive, 9.2% were YFV positive, 34.0% were positive for CHIKV and 0.7% were RVFV positive. In total, 46.6% had antibodies to at least one of these arboviruses. CONCLUSIONS: For all arboviruses, district of residence was strongly associated with seropositivity. Seroprevalence to YFV, DENV and WNV increased with age, while there was no correlation between age and seropositivity for CHIKV, suggesting that much of the seropositivity to CHIKV is due to sporadic epidemics. Paradoxically, literacy was associated with increased seropositivity of CHIKV and DENV.

Ability of selected Kenyan mosquito (Diptera: Culicidae) species to transmit West Nile virus under laboratory conditions.

West Nile virus (WNV) is currently active in Kenya as evidenced by the detection of antibodies in birds bled as part of an avian influenza surveillance program in 2009. Although WNV has been isolated from several mosquito species in Kenya, no studies have ever been conducted to determine which of these species are competent vectors of this virus. Therefore, we allowed Kenyan mosquitoes to feed on 2- or 3-d-old chickens that had been infected with a Lineage one strain of WNV 24-48 h earlier. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. All five species [Culex quinquefasciatus Say, Culex univittatus Theobald, Culex vansomereni Edwards, Mansonia africana (Theobald), and Mansonia uniformis (Theobald)] were susceptible to infection, but disseminated infections were detected only in the three Culex, and not the two Mansonia species. Culex mosquitoes with a disseminated infection readily transmitted virus by bite, but even when inoculated with WNV, the two Mansonia failed to transmit virus, indicating a salivary gland barrier. These studies indicate that the three Culex species may play a role in the transmission of WNV in Kenya.