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.

Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya.

Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to ß-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.

Bottled water brands are contaminated with multidrug resistant bacteria in Nairobi, Kenya.

Background: The demand for drinking water has necessitated the proliferation of bottled water companies in Kenya. This study evaluated if retailed bottled water in Nairobi Kenya complies with both local and international reference criteria. Methods: A total of 42 different water brands (25 approved by Kenya Revenue Authority (KRA) and 17 banned brands) were analyzed for both physicochemical and bacteriological quality. The spread plate method was used to obtain the total plate count of bacteria, while the membrane filter method was used to obtain total coliform count (TCC) and fecal coliform count (FCC). Structured interviews were used to gather company-related information. Results: Overall, 16% of KRA-approved and 35.3% of banned bottled water were contaminated with heterotrophic bacteria. Of the approved water brands, 4% were positive for total coliforms, compared with 17% of the banned brands.  Similarly, 4% and 17% approved and banned water brands were positive for fecal coliforms, respectively. Escherichia coli (19.1%), Pseudomonas spp. (9.5%) and Klebsiella spp. (4.8%) were the most common bacterial types isolated from all water brands, most of which exhibited multidrug resistance. In multivariable analysis, water companies that cleaned pipework and bottles using chlorine-based disinfectants (OR 0.08, 95% CI 0.01 to 0.8), those that had food safety programs (OR 0.1, 95% CI 0.019 to 0.9), had standard operating procedures (SOP) for water sourcing (OR 0.1, 95% CI 0.012 to 0.9) and SOP for contamination protection (OR 0.1, 95% CI 0.02 to 0.9) remained independently associated with bottled water brands exceeding WHO TCC limits. Conclusions: A number of bottled water brands were contaminated with one or more types of indicator bacteria, some of which were multidrug-resistant. Water bottling companies' processes contribute to contamination. Rigorous regulation and monitoring will improve water quality and safety.

A six-member SNP assay on the iPlex MassARRAY platform provides a rapid and affordable alternative for typing major African Staphylococcus aureus types.

PURPOSE: Data on the clonal distribution of Staphylococcus aureus in Africa are scanty, partly due to the high costs and long turnaround times imposed by conventional genotyping methods such as spa and multilocus sequence typing (MLST), which means there is a need for alternative typing approaches. This study evaluated the discriminatory power, cost of and time required for genotyping Kenyan staphylococcal isolates using iPlex MassARRAY compared to conventional methods. METHODOLOGY: Fifty-four clinical S. aureus isolates from three counties were characterized using iPlex MassARRAY, spa and MLST typing methods. Ten single-nucleotide polymorphisms (SNPs) from the S. aureus MLST loci were assessed by MassARRAY. >RESULTS: The MassARRAY assay identified 14 unique SNP genotypes, while spa typing and MLST revealed 22 spa types and 21 sequence types (STs) that displayed unique regional distribution. spa type t355 (ST152) was the dominant type overall while t037/t2029 (ST 241) dominated among the methicillin-resistant S. aureus (MRSA) isolates. MassARRAY showed 83 % and 82 % accuracy against spa typing and MLST, respectively, in isolate classification. Moreover, MassARRAY identified all MRSA strains and a novel spa type. MassARRAY had a reduced turnaround time (<12 h) compared to spa typing (4 days) and MLST (20 days). The MassARRAY reagent and consumable costs per isolate were approximately $18 USD compared to spa typing ($30 USD) and MLST ($126 USD). CONCLUSION: This study demonstrated that iPlex MassARRAY can be adapted as a useful surveillance tool to provide a faster, more affordable and fairly accurate method for genotyping African S. aureus isolates to identify clinically significant genotypes, MRSA strains and emerging strain types.

Positive correlation of HIV infection with Giardia intestinalis assemblage B but not with assemblage A in asymptomatic Kenyan children.

A cross-sectional molecular epidemiological study of Giardia intestinalis infection was conducted among asymptomatic Kenyan children with (n = 123) and without (n = 111) HIV infection. G. intestinalis assemblage B infection was positively correlated with HIV infection [HIV (+), 18.7% vs. HIV (-), 11.7%; P = 0.013], whereas assemblage A infection was not [HIV (+), 4.1% vs. HIV (-), 6.3%; P = 0.510]. Thus, HIV infection is a risk factor for G. intestinalis assemblage B infection but not for assemblage A infection.

Lower prevalence of Entamoeba species in children with vertically transmitted HIV infection in Western Kenya.

A cross-sectional molecular epidemiological study of Entamoeba species was conducted among asymptomatic Kenyan children with (n = 123) and without (n = 111) HIV infection. The prevalence of E. histolytica was low (0.4%). Entamoeba species infection was inversely related with HIV infection [HIV(+): 29.3% vs. HIV(-): 55.0%, P < 0.001]: multiple-species infection was related to higher CD4 T-cell counts. Thus, HIV infection is not a risk factor for amebic infection, and multiple-species infection can be an indicator of better immune status.

Virulence factors in environmental and clinical Vibrio cholerae from endemic areas in Kenya.

BACKGROUND: Since 1971, Kenya has had repeated cholera outbreaks. However, the cause of seasonal epidemics of cholera is not fully understood and neither are the factors that drive epidemics, both in Kenya and globally. OBJECTIVES: The objectives of the study were to determine the environmental reservoirs of V. cholerae during an interepidemic period in Kenya and to characterise their virulence factors. METHODS: One hundred (50 clinical, 50 environmental) samples were tested for V. cholerae isolates using both simplex and multiplex polymerase chain reaction. RESULTS: Both sediments and algae from fishing and landing bays yielded isolates of V. cholerae. Clinical strains were characterised along with the environmental strains for comparison. All clinical strains harboured ctxA, tcpA (El Tor), ompU, zot, ace, toxR, hylA (El Tor) and tcpI genes. Prevalence for virulence genes in environmental strains was hylA (El Tor) (10%), toxR (24%), zot (22%), ctxA (12%), tcpI (8%), hylA (26%) and tcpA (12%). CONCLUSION: The study sites, including landing bays and beaches, contained environmental V. cholerae, suggesting that these may be reservoirs for frequent epidemics. Improved hygiene and fish-handling techniques will be important in reducing the persistence of reservoirs.

Prevalence and antibiotic resistance of bacterial pathogens isolated from childhood diarrhoea in four provinces of Kenya.

INTRODUCTION: Diarrhoea is one of the main causes of morbidity and mortality among children in sub-Saharan Africa, and one of the main causes of hospital admissions in rural areas of Kenya. In Kenya, antimicrobial resistance surveillance has been conducted only at the institutional levels, with limited sharing of information and analysis of data. As a result, the actual scale of regional or national antimicrobial drug resistance is not well defined. METHODOLOGY: Stool samples were collected between 1 October 2007 and 30 September 2008 from a total of 651 outpatients with diarrhoea who were under five years of age in four provinces of Kenya.  Conventional, biochemical methods, multiplex PCR and antimicrobial susceptibility were conducted to identify the bacterial causes and virulence factors in the isolates, respectively.  RESULTS: Of the 651 patients screened, we identified the causes of 115 cases (17.7%) as follows: Pathogenic E. coli (11.2%) [enteroaggregative (8.9%), enterotoxigenic (1.2%), enteroinvasive (0.6%), shigatoxigenic (0.5%)], Salmonella (3.5%), Shigella (2%) and Vibrio cholera O1 (0.7%). The highest levels of resistance among the E. coli isolates were observed in ampicillin and trimethoprim/sulphamethoxazole each at 95% followed by tetracycline at 81%. Shigella isolate levels of resistance ranged from 80% to 100% for ampicillin, tetracycline and trimethoprim/sulphamethoxazole. CONCLUSION: The highest prevalence of antimicrobial resistance was to ampicillin followed by trimethoprim/sulphamethoxazole and tetracycline. Though still at low levels, the major concern from our findings is the emerging resistance of enteric pathogens that was observed to quinolones (ciprofloxacin, nalidixic acid, norfloxacin) and gentamycin.

Molecular characterization of virulence factors in diarrhoeagenic Escherichia coli isolates from children in Nairobi, Kenya.

INTRODUCTION: Among the bacterial causes, diarrheagenic Escherichia coli (DEC) is the most important etiologic agent of childhood diarrhoea and represents a major public health problem in developing countries. New evidence suggests that major differences in virulence among groups of DEC pathotypes may be related to the presence of specific pathogenicity islands (PAIs). METHODOLOGY: Multiplex and conventional PCR assays were used to identify the DEC pathotypes and PAIs respectively from 207 E. coli isolates. RESULTS: The predominant DEC pathotype isolated was EPEC 19.3% (40/207), followed by ETEC 7.25% (15/207), EAEC 3.86% (8/207), STEC 0.97% (2/207) and EIEC 0.48% (1/207). The PAIs detected were enteropathogenic secreted protein C (EspC) 12.2% (8/66), locus of enterocyte effacement (LEE) 62.1% (41/66), and high pathogenicity island (HPI) 57.6% (38/66). Six percent (4/66) expressed only fyuA gene, 12.2% (8/66) irp2 only, and 39.4% (26/66) expressed both fyuA and irp2 genes. SHI-2 39.4% (26/66), she 6% (4/66) and O island 33.3% (22/66), 19.8% (13/66) expressed only efa/lifA gene, 7.6% (5/66) pagC gene only and 6.1% (4/66) expressed both efa/lifA and pagC genes. Toxigenic invasion A (TIA) PAI was not detected. CONCLUSION: This study revealed that in addition to eaeA, stx, aat, einv, st and lt virulence genes exhibited in the different DEC pathotypes there are numerous PAIs in the DEC pathotypes. The PAIs can increase gene mobility within various motile elements, which has implications for the spread of virulence factors from DEC to commensal E. coli.

Prevalence and genetic characteristics of Shigatoxigenic Escherichia coli from patients with diarrhoea in Maasailand, Kenya.

INTRODUCTION: Shigatoxigenic Escherichia coli strains are food-borne bacterial pathogens that may cause haemorrhagic colitis (HC) in humans which can lead to life-threatening systemic complication, including haemolytic uremic syndrome (HUS). This study aimed to characterize and analyze virulence properties of pathogenic E. coli isolates among patients with diarrhoea from a Maasai community in Kenya. METHODOLOGY: Stool samples from 380 patients of all ages from the Kajiado and Narok districts of Kenya were investigated for the presence of enteric bacterial pathogens by conventional and molecular methods. RESULTS: Bacterial diarrhoea was diagnosed in 141/380 (37.1%) cases, of which enterotoxigenic E. coli (ETEC) compromised 29.8%, shigatoxigenic E. coli (STEC) 24.1%, enteroaggregative E. coli (EAEC) 14.2%, enteroinvasive E. coli (EIEC) 12.8% and enteropathogenic E. coli (EPEC) 3.5%. Gene analysis for STEC virulence factors showed that 52.9% isolates carried stx1, 29.4% possessed stx2, 14.7% carried both stx1 and stx2, and 2.9% had stx2e. 23.5% isolates carried enterohaemolysin and 20.5% isolates possessed the Intimin gene. From 9 strains that exhibited adherence, 7 contained both Intimin and Haemolysin genes. Infections with Intimin-positive STEC strains (46%) were more frequent in patients with bloody diarrhoea, especially in children under 5 years of age, whereas Intimin-negative STEC infections dominated in adults. CONCLUSION: Although STEC infection as a cause of bloody diarrhoea has not attracted much attention as a medical problem in Kenya, our findings indicate that this is a problem that must be investigated. The 24.1% isolation rate of STEC among the Maasai is one of the highest reported rates worldwide.