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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Publisher
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),
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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
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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
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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.
Description
Keywords
Virulence Profiles, Staphylococcus aureus, Escherichia coli, Antibiotic resistance