Domestic cats are potential reservoirs of multidrug-resistant human enteropathogenic E. coli strains in Bangladesh

Companion animals serve as our best friends, confidants, and family members. Thus, disease and antibiotic resistance gene transmission in pets and humans must be sought out. The study aimed to identify the common pathogenic Escherichia coli (E.coli) in pet cats and the antibiotic resistance patterns and resistant gene distribution. Samples (n = 210) were collected from different veterinary clinics in Bangladesh’s cities of Mymensingh and Dhaka. Pathogenic E. coli was identified using conventional and molecular approaches. The disc diffusion method assessed the resistance profile against 12 antibiotics, and PCR was used to identify the beta-lactam resistance genes. The prevalence of the stx-1 gene was found to be 2.86%, whereas the rfbO157 prevalence was found to be 1.90% in cats. The stx-1 gene (n = 6) was 100% resistant to erythromycin and imipenem, whereas 100% sensitive to chloramphenicol. In turn, the rfbO157 gene (n = 4) exhibited 100% resistance to erythromycin, imipenem, cefixime, and azithromycin. In addtion, we identified genes that exhibit resistance to beta-lactam antibiotics (100% blaTEM, 40% blaCTX-M, 40% blaSHV2). This study found shiga-toxin producing and extended-spectrum beta-lactamase (ESBL) producing E. coli for the first time in pet cats of Bangladesh. Furthermore, the antimicrobial resistance (AMR) profile of the isolated strains refers to the occurrence of multidrug, which concerns cats and their owners. The existence of these genes in non-diarrheic pet animal isolates indicates that domestic pets may serve as a reservoir for human infection. Thus, one health strategy comprising animal and human health sectors, governments, together with stakeholders is needed to confront multidrug-resistant E. coli infections in Bangladesh.


Introduction
Escherichia coli (E.coli) is one of the commonest members of gut microbiota that can affect both humans and animals.There are sixcategories of diarrheagenic E. coli as Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Shiga toxin-producing (STEC), Enteroaggregative (EAEC), diffuse-adhering E. coli (DAEC) and Enteropathogenic (EPEC) E. coli (Puño-Sarmiento et al., 2013).EPEC E. coli causes infant diarrhea (Ochoa and Contreras 2011;Hennessey et al., 2021).Strains of E. coli that produce STEC are the reason for an array of infections in people and animals, encompassing gastrointestinal and extra-intestinal difficulties like urinary tract infections.(Bentancor et al., 2007;Hu et al., 2021;Pan et al., 2021).Extra-intestinal pathogenic E. coli's (ExPEC) involvement in causing severe infections has recently attracted attention regarding its zoonosis from a veterinary clinical standpoint (Bélanger et al., 2011;Pitout, 2012;Najafi et al., 2019).Hemolytic urinary syndrome (HUS), a worldwide distributed disease, is identified in a child closely aligning with a pet cat as a carrier of STEC O15:H7 strains (Rumi et al., 2012).Moreover, E. coli that produces Vero toxin (Shiga-like toxin) emerges to be predominant in the faces of animals, including cats that show no illness (Beutin et al., 1993).ETEC's (Enterotoxigenic E. coli) presence in pets and their significance in diarrheal disease are poorly understood.Despite their widespread prevalence in dogs, a limited number of ETEC strains are currently studied in cats (Beutin, 1999).Enteroinvasive (EIEC) and Enteroaggregative (EAEC) strains are mainly tested for human infections, and the pathogenic significance of strains in diarrhea is unknown and requires further investigation.(Puño-Sarmiento et al., 2013).
The upsurge in antimicrobial resistance (AMR) is one of the imminent risks to medicine (Moo et al., 2020).Drug-resistant zoonotic illnesses can spread quickly to human populations through animals due to the growing usage of antibiotics in animals (WHO).Enterobacteriaceae, which produces the antibioticresistant enzyme ESBL, is a bit of a nightmare worldwide (Larramendy et al., 2021;Salinas et al., 2021., Ullah et al., 2023).ESBL enzymes hydrolyze third-generation of cephalosporins and aztreonam, although clavulanic acid inhibits them.This is now considered one of the most severe public health hazards.Moreover, the most frequent ESBL-causing strain is E. coli carrying CTX-M genes (Peirano and Pitout, 2019).
Cats were found to be present in 31% of U.K. residences, according to a survey conducted in 2007 (Murray et al., 2010).There are companion animals in over 58 million homes in the United States, with cats (59.1 million) being the most preferred (Enriquez et al., 2001).Similarly, petting animals, especially dogs, and cats, has become popular in Bangladesh, notably for youngsters' and owners' emotional and social well-being (Robertson et al., 2000).Due to this, more pets need treatments, and more significant amounts of antimicrobial are prescribed.E. coli is the principal repository of the resistance genes; therefore, inadequate constraints on antimicrobial have a significant role in the development of MDR strains posing a zoonotic risk to human health (Morato et al., 2009, Cui et al., 2022).
ESBL-producing E. coli (ESBL-EC) infections are becoming more prevalent in pets and humans (Ho et al., 2011;Cui et al., 2022).The feces of healthy cats and dogs are a significant reserver ESBL-EC (Cui et al., 2022).The overall development of antibiotic resistance rates among E. coli strains from farm and pet animals, as well as fish samples, has been the subject of numerous research studies all over the globe (Dos Santos et al., 2013;Schaufler et al., 2015;Chen et al., 2019;Kristianingtyas et al., 2020;Gruel et al., 2021., Ullah et al., 2023).In our country, cattle, sheep, and goats have been the subjects of the most exhaustive studies of E. coli infection (Johura et al., 2017).However, dogs and cats have recently resided near humans; consequently, the likelihood of pathogenic microorganism transmission to humans is extremely high.Therefore, some research must determine the abundance of AMR genes in E. coli isolates from domesticated pets.However, to our best knowledge, no published data are available in Bangladesh on identifying pathogenic E. coli strains and finding antibiotic resistance genes in cats and their owners.Considering the paucity, the research addressed pathogenic E. coli prevalence in domestic pet cats, their AMR pattern, and molecular detection of resistance genes.

Ethic al approval
Current research proceeded in accordance with the guidelines of the Animal Welfare and Experimental Ethics Committee, Bangladesh Agricultural University.The samples (rectal swabs from cats) were obtained after getting the appropriate consent from cat owners and explaining the study's objective.Approval No: AWEEC/ BAU/2019(51).

Sample collection and processing
In total, 210 rectal swabs were obtained from pet cats enrolling in veterinary clinics in the communities of Dhaka and Mymensingh.The target population for this study is client-owned cats with access to a bed that visited veterinary clinics for routine exams, vaccinations, or other health complications with or without diarrhea.In order to circumvent self-contamination, we utilized sterile cotton buds and submerged the swabs in the sterile nutritional broth after collecting the swabs.Immediate transport to the lab facilitated overnight incubation at 37 °C of the obtained samples.

Isolation and identification
The enriched broth was diluted and inoculated onto Eosine Methylene Blue (EMB) (H.I. media, India) agar overnight at 37 °C.The colonies exhibiting classic E. coli cultural features were subcultured on selective media (EMB) to obtain purified isolates.Gram staining was used to confirm the morphology.Observing the cultural and Gram's straining attributes, selected colonies were determined to perform several biochemical tests such as primary sugar (Sucrose, Maltose, Lactose, Mannitol, Dextrose) fermentation test, Coagulase test, Methyl-red test, Catalase test, Indole test, and Voges-Proskauers test (Farhad et al., 2021).

Molecular detection E.coli and virulence genes
Each E. coli strain's genomic DNA was retrieved utilizing a boiling protocol (Hossain et al., 2013).Molecular detection was performed using genus specific primers listed in Table 1.Total 25 ll of PCR mixture was prepared where 12.5 ll of master mix (Promega, USA), 1 ll of forward and reverse primer (20 pmol/L) (Table 1), and 1 ll of DNA template was mixed in 9.5 ll of nuclease-free water were used.The results of PCR were filtered on a 1.2% agarose gel, tainted with Ethidium bromide, visualized with UV-transilluminator, and photographed.Confirmed E. coli isolates were then subjected to PCR for the confirmation of virulence genes including rfbO157, stx-1, and stx-2 using specific primers listed in Table -1.

Antimicrobial susceptibility testing and detection of ESBLproducing E. coli
In order to ascertain the antibiotic susceptibility profile, twelve commonly used antibiotics for cats from seven antimicrobial classes were preferred.Antibiotic susceptibility testing was done among all genotypically confirmed E. coli isolates utilizing the standard agar disc diffusion method (Bauer et al., 1966).The antimicrobial assay used the commercially available Mueller Hinton Agar (H.I. media, India).Used antimicrobial agents were Erythromycin (E, 15 lg); Ampicillin (AMP, 10 lg); Cefuroxime (CXM, 30 lg); Cefotaxime (CTX, 30 lg); Cefixime (CFM, 5 lg); Norfloxacin (NOR, 10 lg); Chloramphenicol (C, 30 lg); Ciprofloxacin (CIP, 5 lg); Azithromycin (AZM, 15 lg); Gentamicin (CN, 10 lg); Imipenem (IMP, 10 lg); Florfenicol (FFC, 30 lg).Antimicrobial test findings are categorized according to the zone diameter interpretive criteria mentioned in the guideline of the Clinical Laboratory and Standards Institute (CLSI) (Clinical and Institute, 2017).MDR isolates have developed resistance to three or more different types of antimicrobial classes.Double-disk synergy assay was performed to determine the presence of ESBL-producing E. coli isolates according to previous study (Cui et al., 2022).

Molecular detection of antimicrobial resistant genes
The E. coli isolates underwent further testing for the presence of beta-lactamase producing genes including bla TEM , bla CTX-M , bla SHV2 .The primers that were utilized in order to accomplish this PCR are shown in Table 1.After PCR, the product was illustrated as the procedure described before (Hossain et al., 2013).

Antibiotic susceptibility profile and ESBL producing E. coli
Erythromycin and imipenem resistance was 100% in stx-1 and rfbO157-positive E. coli.The pathogenic E. coli isolates containing stx-1 gene were resistant to azithromycin and ampicillin (83.3%) but sensitive to chloramphenicol (100%), florfenicol (83.3%), and intermediate in gentamicin.Subsequently, the rfbO157 gene containing E. coli isolates exhibited 100% resistance to cefixime and azithromycin, whereas it was 75 % sensitive to florfenicol and chloramphenicol (Fig. 4).Among ten virulence gene containing E. coli four were producing ESBL enzyme listed in Table-3.

Molecular detection of beta-lactamase genes in E. coli
Each pathogenic E. coli containing stx-1 and rfbO157 genes had at least a single antibiotic resistance gene (bla TEM ), and four of them possessed three resistance genes (bla TEM , bla CTX-M , bla SHV2 ) (Table 3).Molecular detection of these genes is depicted in Fig. 5, Fig. 6 & Fig. 7.

Discussion
The expansion of antimicrobial resistance in microbes from different animals has triggered a significant due to the risk of resistant infections and commensal bacteria being transmitted to humans.In recent years, there has been a rise in the number of  people who keep pets as companions, which has increased the potential of zoonotic bacteria being passed from pets to humans (Cui et al., 2022).E. coli is an essential zoonotic agent linked to human and animal infectious diseases (Allocati et al., 2013;Bentancor et al., 2007).This study confirmed multidrug-resistant pathogenic stx-1 and rfbO157 E. coli strains in pet cats, for the first time in Bangladesh.This study revealed a 2.86% prevalence of STEC in pet cats.This stx-1 gene incidence could be attributed to the habitat of cats living in an unhealthier environment and cats having outdoor assess roaming around in areas where pathogenic microorganisms are abundant and eating raw carcasses (Egberink et al., 2013).Cats can also be exposed to pathogenic microorganisms by consuming contaminated water (Srikullabutr et al., 2021).Moreover, wastes from households and the environment, like raw fish, meat, milk, vegetables, fruits, soil, water, etc., work as an essential medium for transmitting pathogenic enterobacteria to cats (WHO, Cairncross 1990;Bach et al., 2002;Radi et al., 2014;Navab-Daneshmand et al., 2018).
On the other hand, the rfbO157 genes were screened to detect the serotype and found a 1.90% prevalence among the cats..The trend of offering companion animals raw meat-based diets (RMBDs) risks the transmission of these zoonotic pathogens (van Bree et al., 2018).Furthermore, these pathogenic strains of E. coli were predominantly detected in rectal swabs of cats, which could result from bacteria being shed via their feces (Ercumen et al., 2018;Navab-Daneshmand et al., 2018;Srikullabutr et al., 2021).Moreover, cats can serve as a repository of enteropathogenic E. coli even in the absence of diarrhea, which could better explain the availability of E. coli strains rectally (Morato et al., 2009).
Furthermore, E. coli, which produces Vero-toxin (Shiga-like toxin), was found in abundance in the feces of healthy domestic cats (Bélanger et al., 2011).Even so, animal-derived Extraintestinal Pathogenic E. coli (ExPEC) could be considered zoonotic, meaning they can be transferred to people directly or indirectly (Bélanger et al., 2011).Similarly, a kid who lived near a pet cat was a carrier of STEC strains affected with the hemolytic urinary syndrome (HUS) that was previously reported (Rumi et al., 2012).As a result, the chance of contamination between humans and cats remained.However, none this study's STEC strains belonged to O157:H7 serotypes.They might belong to other enterohemorrhagic E. coli (EHEC) serogroups noticed in prior research (Abdulrazzaq et al., 2021).
In the antibiotic sensitivity test by disc diffusion method, isolates containing rfbO157 and stx1 genes were entirely resistant to erythromycin and imipenem.Additionally, complete resistance of Azithromycin and Cefixime was found in rfbO157 isolates.Higher resistance of ciprofloxacin, ampicillin, cefuroxime, and cefotaxime was observed in the rest of the isolates, whereas Chloramphenicol and Florfenicol were found sensitive to all the isolates.The current study's inferences coincide with those of prior studies (Madiha et al.,     Metwally 2015; Liu et al., 2015;Saputra et 2017).This study shows that intermediate-resistance organisms can develop resistance at any time, which is concerning.On the other hand, chloramphenicol and florfenicol have proven to be the most efficient antibiotics against E. coli infection in cats (Carattoli et al., 2005;Johnson et al., 2009).This research observed MDR in all the patho-genic E. coli isolates.In total, three patterns of MDR were observed in which Erythromycin, Imipenem, Azithromycin, Ciprofloxacin, Ampicillin (E-IMP-AZM-CIP -AMP) (66.67%), and Erythromycin, Imipenem, Azithromycin, Ciprofloxacin, Ampicillin, Cefuroxime (E-IMP-AZM-CIP -AMP-CFM) (50%) were observed in stx1 gene containing E.coli and erythromycin, imipenem, azithromycin, and   ciprofloxacin (E-IMP-AZM-CIP) (100%) observed in rfbO157 gene containing E. coli.This investigation discovered the MDR isolates of E. coli, which is concerning.Similar findings on the antimicrobialresistant patterns of MDR-producing E. coli isolates have also been noticed earlier (Shaheen et al., 2010;Rzewuska et al., 2015;Chen et al., 2019;Piccolo et al., 2020).This study used bla TEM , bla CTX-M , and bla SHV2 genes to detect E. coli isolates that produce ESBL enzymes.Similarly to our findings, earlier research has documented the presence of multidrugresistant ESBL-producing E. coli in cats and dogs (Zogg et al., 2018;Carvalho et al., 2021;Sfaciotte et al., 2021).Our pathogenic isolates were all bla TEM gene positive; four were bla TEM , bla CTX-M , and bla SHV genes positive.In prior research, these resistance genes were also found in cat feces (Toro et al., 2005;Weese et al., 2022).This is because cats have a close relationship with environmental elements, particularly water, and contaminated water is a significant factor in the spread of E. coli, which produces betalactamase among humans.Because of the negligent application of broad-spectrum antibiotics in animal feeds for treatment and prevention, these products may also pose a risk to pets (Wegener, 2003).Moreover, the sub-therapeutic usage of antibiotics in animals could also disseminate resistant organisms surrounding the environment, endangering animal and human health (Ejaz et al., 2021).The presence of ESBL-producing E. coli isolates in cats is alarming, and there remains a high chance of transferring these ESBL genes of E. coli to the owners and other people who come closer to them (Carvalho et al., 2016;Abbas et al., 2019).

Conclusions
The study accentuates the possible role of domestic cats in Bangladesh as reservoirs of ESBL-producing E. coli and multidrugresistant Shiga-toxin-generating E. coli strains.The existence of multidrug-resistant stx-1 and rfbO157 E. coli strains in pet cats is an enormous public health issue as these animals can act as carriers for antibiotic-resistant strains to spread into humans.In order to combat multidrug-resistant E. coli infections and Bangladesh's rising antimicrobial resistance, governments, stakeholders, and the human and animal health sectors must work collaboratively.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Table 1
List of primers with sequences used in this study.
Pathogenic E. coli No of total samples No. of malB gene positive E. coli samples No. of virulence gene positive E. coli Occurrence of pathogenic E. coli in the total sample (%) Occurrence of pathogenic E. coli in malB gene positive samples (%)

Table 3
Distribution of antibiotics resistant phenotypes and resistance genes among isolated E. coli.