Molecular Epidemiology of Staphylococcus Aureus From Hospital Patients, Hiv Positive and Negative Abattoir Workers and Animals in Busia County, Kenya

Abstract

Hospitals, Human immunodeficiency virus (HIV)-infected outpatient clinics, cattle farms and abattoirs provide environments that promote acquisition and spread of Staphylococcus aureus (S. aureus), increasing risk of colonization and infection in the communities. It is documented that establishment of molecular epidemiology of S. aureus, with respect to infection control strategies, is important in preventing and controlling S. aureus transmission. The terms antimicrobial, antibiotic, and anti-infective encompass a wide variety of therapeutic agents for the treatment of bacterial, fungal, viral, and parasitic infections (Leekha et al., 2013; Roberts et al., 2012). For the purpose of this study, antibiotic will be referred to as agents against bacterial infection, while the term antimicrobial, will be used for the antibiotic sulfamethoxazole-trimethoprim used for the treatment and prophylaxis of a yeast-like fungus Pneumocystis jiroveci pneumonia. Excessive use of antibiotic empirical therapy in hospital settings has been reported as a contributor to emergence of bacterial resistance to antibiotics (Paterson and Rice, 2003; Laxminarayan et al., 2013). Information on empirical antibiotic use, antibiotic susceptibility prevalence of multi-drug resistance (MDR), virulence molecular epidemiology of S. aureus isolates in Western Kenya is limited. This cross sectional study was carried out to establish S. aureus Staph. aureus carriage in hospitalized patients, abattoir workers (HIV/AIDS positive and negative) and livestock in Busia County, Kenya.

The justification of this study was as a result of high empirically use of antimicrobials, in hospital patients and livestock rearing and high prevalence of HIV of primary meat handling personnel in Busia County, taking in consideration some of HIV-positive individuals are employed as abattoir workers in various Slaughterhouses spread across the rural areas of Busia County. It was hypothesized that there is a possibility that the HIV-positive abattoir workers form a key link of S. aureus transmission in the hospital setting, abattoir environment, general community meat consumers ,as well as among livestock, necessitating usage of a multidisciplinary approach such as One Health, with a view to mitigate the problems caused by S. aureus infections in Busia County. Main objective was to establish molecular epidemiology of Staphylococcus aureus from Inpatients; abattoir workers (HIV-positive and HIV negative), and animals in Busia County, Kenya. The specific objectives being identification and documentation of empirical antibiotic treatment administered to inpatients at Busia County Referral Hospital (BCRH). Isolation and determine prevalence of S. aureus in human inpatients, abattoir workers and livestock, and respective phenotypic susceptibility profiles, in Busia County, Kenya. Determine presence of the pvl, tsst-1, mec-A and sasX genes in the S. aureus isolates. Conduct S. aureus molecular typing and whole genome sequencing in order to establish S. aureus genotypes circulating among inpatients, abattoir workers (HIV-positive and HIV-negative) and livestock in Busia County, Kenya. Methods Study Sites, Busia county referral hospital with a bed capacity of 160, Busia County, and parts of Bungoma, Siaya and Kakamega Counties for Abattoir workers and livestock study population

Study Population included 738 abattoir workers, 261 Inpatients and 103 livestock (Cattle, goats, sheep and pigs). Sample Collection, Handling, Bacterial Isolation and Identification, All participants were informed of the project objectives and protocol by medical and clinical officers who collected signed informed consent. Nasal samples were collected by rotating a sterile swab five times in both anterior nares, from consenting inpatients, abattoir workers and livestock. The swabs were inoculated in tryptone soya broth/6% salt and transported in cool boxes to the lab for culturing. The swabs were streaked on mannitol salt agar (MSA) and incubated at 37 °C overnight. Suspect S. aureus colonies (those that fermented mannitol, producing yellow colonies) were stocked in tryptone soya broth with 10% glycerol and stored at −40 °C; they were later transported on dry ice to Kenya Medical Research Institute (KEMRI) laboratories in Nairobi. The presumptive S. aureus isolates were further cultured onto MSA and repeatedly sub-cultured to get pure culture. The S. aureus isolates were identified using Gram reaction (Gram-positive cocci in clumps), catalase, and coagulase (tube method using rabbit plasma) and DNase tests. Phenotypic antimicrobial Susceptibility Testing was also performed using the VITEK 2 instrument (bioMerieux, Marcy-l‘Étoile, France), for benzylpenicillin, cefoxitin, oxacillin, ciprofloxacin, erythromycin, chloramphenicol, daptomycin, fusidic acid, gentamicin, linezolid, mupirocin, nitrofurantoin, rifampicin, teicoplanin, tetracycline, tigecycline, trimethoprim, vancomycin, clindamycin,and inducible resistance to clindamycin. Multi-drug resistant S. aureus were defined as isolates that were resistant to three or more antimicrobial. When stocking the pure S. aureus growth; a long sweep of the colonies was done to allow preservation of genetic diversity of nasal carriage of the participant. During whole genome sequencing of the sample, if

sequences from multiple isolates were detected, these samples were cultured and single colonies selected for sequencing. Molecular Genotype Testing included, DNA extraction from S. aureus isolates was performed using QIAGEN DNeasy ® Blood & Tissue Kit,(Staphylococcal cassette chromosome (SCC) mec typing was performed Isolates were screened for pvl and tsst-1 genes using PCR. Genomic libraries were generated and sequenced on an Illumina HiSeq 2000, Illumina reads were analyzed based on S. aureus MLST database, analysis of virulence and antimicrobial resistance genes were conducted using the virulence finder database. Paired-end Illumina reads were mapped to the S. aureus reference genome ST22 strain HO 5096 0412 using Snippy v4.6.0. Whole-genome alignments were created by keeping a version of the reference genome using Snippy‘s.consensus.subs.faoutput files. The resulting core-genome phylogeny was plotted with isolate metadata using ggtree v.3.0.4 and ggtreeExtra v.1.2.3 on R v4.1.0. HIV testing was performed on whole blood using the SD Bioline HIV 1/2 Fast 3.0 test strips Statistical analysis was performed using the chi-squared test. A p-value <0.05 was considered an indication of significant difference. Ethical approval was issued Kenya Medical Research Institute scientific steering Committee and Ethical Review Committee . This study produced a number of key findings: Cefotaxime was found to be the most commonly prescribed empiric antibiotic therapy in patients admitted at Busia County Referral Hospital (BCRH), accounting for 60.0%, 37.7% and 78% in pediatric, medical and surgical wards, respectively. Metronidazole was the second most prescribed at 23.3%, 22.6% and 76.3 in the

pediatric, medical and surgical wards respectively. Among patients admitted at BCRH, the prevalence of methicillin-sensitive S. aureus (MSSA) was 10.7 %; it was higher than that of methicillin-resistant isolates (MRSA; 0.8%).The hospital isolates were susceptible to Chloramphenicol, Daptomycin , Fusidic Acid, Linezolid , Mupirocin, Nitrofurantoin, Rifampicin, and Teicoplanin. The MSSA isolates were highly resistant to Penicillin (96.6%), Trimethoprim (73.3%), Cefotaxime (40.0%), but resistant to Ciprofloxacin, Clindamycin, Vancomycin, Trimethoprim/Sulfamethoxazole, Erythromycin, Gentamicin, and Tetracycline, with some isolates, exhibiting multi-drug resistance (MDR). Eleven MLST ST strains were identified in BCRH; they included: ST1633, ST22, ST152, ST188, ST80, ST8, MRSA ST140-MRSA, ST580, ST25 (new ST), ST573 and ST5. Additional new STs (sequence types) identified in BCRH included ST241 –MRSA and ~ST508. These sequence types were clustered into 8 clonal complexes (CC): CC125, CC22, CC1, CC80, CC8, CC508, CC25 and CC5. For abattoir workers, nasopharyngeal prevalence of S. aureus was 16.0 %; HIV - positive workers had a higher S. aureus colonization rate (25.8%) than their HIV - negative counterparts (14.6%). Low percentages of antibiotic resistance were detected in the abattoir workers‘ isolates: The S. aureus isolates from this study were susceptible to Chloramphenicol, Daptomycin, Fusidic Acid, Linezolid, Mupirocin, Nitrofurantoin, Rifampicin, and Teicoplanin. In contrast to isolates from the hospital, the S. aureus isolated from abattoir workers were susceptible to Linezolid, Gentamicin, Ciprofloxacin and inducible Clindamycin resistance at 99.2 % , 98.4 % , 98.4 and 96.8 % , respectively. Increased antibiotic drug resistance was identified towards Penicillin (97.7%), Trimethoprim (65.1 %) and Tetracycline (25.6%). HIV-positive abattoir worker populations significantly carried more MDR- S. aureus isolates compared to HIV-negative ones (p< 0.000418); two MDR S. aureus isolated from HIV-positive abattoir workers had

combination patterns of Penicillin-Erythromycin- Trimethoprim- Inducible Clindamycin resistance - Trimethoprim/Sulfamethoxazole (PEN-E-TMP-ICR-SXT) and Penicillin-Ciprofloxacin-Erythromycin-Trimethroprim-Inducible Clindamycin resistance - Trimethoprim/Sulfamethoxazole (PEN-CIP-E-TMP-ICR-SXT); one, with a combination of Penicillin-Erythromycin-Trimethroprim—Gentamycin-Inducible Clindamycin resistance (PEN-E-TMP-Gent-ICR), was isolated from a HIV –negative abattoir worker. There was a strong association between members of ST 8 genotype with higher phenotypic resistance to Trimethoprim/Sulfamethoxazole (SXT) and HIV infection ((P > 0.00001). Forty three percent (43%) of S. aureus isolates, majority of which were PVL gene-positive, belonged to the ST 152 genotype, which accounted for 9.0% of the S. aureus isolates in study HIV-positive abattoir workers and 7.1% of S. aureus isolates in study HIV - negative abattoir workers. Other ST-type S. aureus that carried PVL genes were ST1633, ST30, ST22, ST80, ST2430 and ST1. Toxic shock syndrome toxin-1 gene (TSST-1) was associated with ST 72 (Crossley et al., 2009); these strains were exclusively found among HIV-negative abattoir workers. Phylogenetic analysis of ST8 and ST152 S. aureus strains showed a possible transmission of these strains among BCRH inpatients and that two distinct clones of ST152 were circulating in Western Kenya; ST152 S. aureus strains had dfrG (Trimethoprim resistance gene) only in Clade A, while all but one of the ST8 isolates had the dfrG gene. A novel ST661, assigned to a new clonal complex 661 was identified in one isolate, four new MSSA sequence type variants: 88~(CC8), 1290~ (CC221), 1292~ (CC9) and 8~ (CC80) were recovered from abattoir workers; a new MSSA ST 508~ variant and ST241-MRSA-III, hosting a virulence-associated gene sasX, with a single-locus variation from ST239-MRSA-III, were isolated from inpatients in BCRH. These new variant strains had slight mutations in allele of their known housekeeping gene. There was presence of

human genotypes ST152, ST8, ST1290, and ST30 among isolates from livestock nares, indicating the dispersal of human genotypes ST152, ST8, ST1290, and ST30 in livestock and identified ST1925-MSSA as a possible livestock associated S. aureus, implying that some MSSA clones have ability to cross the species barrier and jump from humans to several livestock species (Ostojić and Hukić, 2015). Findings of this study will directly improve clinical management and diagnosis of MDR MSSA and MRSA infections among HIV/ AIDS patients, hospital patients, abattoir workers and cattle in Kenya and prevent zoonotic transmission in Kenya and possibly in other low- and middle-income countries in similar situations.