Antibiotic resistance and virulence profiles of staphylococcus aureus and escherichia coli from rodents, humans and chicken coexisting in Karatu, Tanzania.

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Date

2022

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Sokoine University of Agriculture

Abstract

Antimicrobial resistance (AMR) is a rapidly growing multifaceted problem which threatens global security, public health and the economy. Currently, about 700 000 humans worldwide lose lives annually due to AMR infections that are difficult to treat which are also associated with higher health care costs, elongated time spent in hospitals and increased animal production costs. This study was conducted in Karatu district, northern Tanzania, to investigate antibiotic resistance and virulence profiles of Staphylococcus aureus and Escherichia coli isolated from humans, rodents, chicken and soil in households. Interaction of rodents with humans and livestock in households’ environment has frequently been reported in Karatu, facilitating wide spread of resistant bacterial infections among different hosts in the community. The main objective of this study was to determine the antibiotic resistance and virulence profiles of Staphylococcus aureus and Escherichia coli isolated from rodents, chicken, humans and their surrounding environment in Karatu District. S. aureus were isolated from 284 human nasal swabs, 101 rodents’ deep pharyngeal swabs, 286 chicken cloaca swabs and 285 household soil samples. Specimens were plated into Mannitol Salt Agar (Oxoid, Basingstoke, UK) and incubated aerobically at 37 °C for 24 h. Presumptive colonies of S. aureus were subjected to Gram staining, catalase, deoxyribonuclease (DNAse) and coagulase tests for identification. Antibiotic susceptibility testing (AST) was performed by using Kirby-Bauer disc diffusion method on Mueller-Hinton Agar (Oxoid, Basingstoke, UK). The antibiotics tested were tetracycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), ciprofloxacin (5 μg), iii clindamycin (2 μg) and amoxicillin-clavulanate (20 μg/10 μg). S. aureus strain American Type Culture Collection (ATCC) 25923 was used as a standard organism. Results were interpreted according to Clinical and Laboratory Standard Institute (CLSI) guideline of 2020. The samples used for E. coli isolation included 288 chicken cloaca swabs, 281 human stool, 101 rodents’ intestinal contents and 290 household soils. The specimens were plated onto MacConkey agar (Oxoid Ltd., Detroid, Michigan, USA) and incubated aerobically at 37 ºC for 24 h. Presumptive E. coli colonies were subjected to motility test and later indole, methyl red, Voges-Proskauer and citrate utilization (IMViC) tests for identification. E. coli strain ATCC 29522 was used as a reference organism. AST was performed by using Kirby-Bauer disc diffusion method on Mueller-Hinton Agar plates (Oxoid, Basingstoke, UK). The antibiotics tested were; tetracycline (30 μg), imipenem (10 μg), gentamicin (10 μg), ciprofloxacin (5 μg) cefotaxime (30 μg) and amoxicillin clavulanate (20 μg/10 μg). The results were interpreted by using CLSI (2020) guideline. Results of this investigation revealed high frequencies of isolation for S. aureus and E. coli in rodents, humans, and chicken and soil samples. For S. aureus, the isolation frequencies were 52.1%, 66.5%, 74.3% and 24.5% in samples from chicken, human, rodent and soil, respectively. The isolation frequencies of E. coli from chicken, humans, rodents and soil were 81.6 %, 86.5 %, 80.2 % and 31.0 %, respectively. Based on AST phenotypic results, S. aureus isolates displayed resistance to clindamycin (51%), erythromycin (50.9%) and tetracycline (62.5%) while E. coli isolates showed high resistance against tetracycline (73.7%), imipenem (79.8%) and cefotaxime (79.7%). MDR E. coli (n=50) and S. aureus (n=57) isolates that exhibited high levels of phenotypic resistance to various classes of antibiotics were subjected to molecular analysis using iv multiplex polymerase chain reaction (PCR) technique to detect presence of antibiotic resistance (ARGs) and virulence genes (VGs). ARGs detected in MDR E. coli were; blaTEM (46%), blaCTX-M (26%), blaSHV (22%), tetA (46%), tetB (14%), qnrA (24%), qnrB (8%), blaOXA-48 (12%) and blaKPC (6%) while VGs detected included; ompA (72%), traT (26%), east (18%), bfp (10%), eae (2%) and stx-1 (4%). For MDR S. aureus, ARGs were; tetK (31.6%), tetL (8.9%), ermC (1.8%) and mecA (28.1%) while VGs detected were; clfB (10.5%), coa (14.0%), clfA (1.8%), hlg (1.8%), ebpS (3.5%), fnbB (3.5%), luk-PV (10.5%) and tst (1.8%). Positive and negative correlations between resistance and virulence genes were observed. For MDR E. coli, positive correlations were found between blaTEM and traT genes (r=0.51) and qnrB and bfp genes (r=0.63), while negative correlations were found between blaOXA-48 and ompA (r= -0.05), blaSHV and traT (r=-0.44) and tetA and east (r=-0.10). For S. aureus, positive correlations were found between resistance (ermC) and clfA (r=0.57), hlg (r=1.00) and clfB (r=0.43), tetK and clfB (r=0.39); tetK and coa (r=0.36). The principal component analysis (PCA) results for S. aureus showed that, resistance genes (tetK and mecA) and virulence determinants (clfB, coa and luk-PV) were common in all sample sources. The PCA also revealed that, MDR E. coli and S. aureus isolates from rodents and chicken had more ARGs and VGs compared to isolates from soil and humans. Besides, MDR E. coli isolates harboured traT, east, eae, stx-1, bfp and ompA genes indicating ability of isolates to cause various infections. Based on findings, this study documents high levels of antimicrobial resistance including MDR in E. coli and S. aureus isolated from chicken, humans, rodents and soil samples in Karatu, northern Tanzania. According to PCA results, E. coli isolates from rodents had more ARGs and VGs while for S. aureus these genes were found more in rodents and soil v environment implying that both subjects are potential reservoirs and can be sources of transmission. The increased prevalence of both resistance and virulence genes in the isolates suggests the ability of the pathogens to cause infections that are difficult to treat. Comprehensive one health interventions, are urgently needed and should include improving; i) improving hygiene and control of rodents in household environments. ii) Future studies should base on adequate understanding of the human-livestock environment interphase using well-designed genomic studies such as whole genomic sequencing (WGS) which provides a comprehensive picture on the pattern and magnitude of AMR and virulence genes spread. The advances and accessibility of genomic sequencing and analytical methods are essential in improving our understanding of AMR transmission dynamics at the human-livestock/animal-environment interface. Genomic studies should be coupled with behavioural, epidemiological, clinical and modelling using One Health approaches. This will ensure that the key drivers of resistance and virulence transmission between human-livestock-environment are accurately identified and the most appropriate interventions adopted. It is important to understand the importance of each component of the human-livestock/animal-environment. A One Health approach should be deployed to ensure involvement of relevant multisectoral and multidisciplinary to attain an optimal public health and ensure a safe environment.

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Keywords

Virulence Profiles, Staphylococcus aureus, Escherichia coli, Antibiotic resistance

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