Genotypic Evidence of Extended-spectrum β-lactamase and Carbapenemase-producing Shigatoxigenic and Intimin-producing Shigatoxigenic Multidrug-resistant Escherichia coli from Diarrheic Calves: Hidden Zoonotic and Public Health Risk

Shiga toxin-producing Escherichia coli (STEC) is a pathotype of E . coli associated with a wide variety of diarrhoea in neonatal calves, causing a global economic loss in the dairy industry with significant zoonotic risks via STEC and intimin-producing STEC, resulting in enteric and systemic illness, including diarrhoea, haemorrhagic colitis (HC), and haemolytic uremic syndrome (HUS) in humans. The prominence of multidrug-resistant (MDR) STEC from neonatal diarrhoeic calves is increasing public health risks and restricted treatment alternatives. The prevalence of STEC was investigated in 75 rectal swabs from diarrhoeic calves aged ≤ 12 weeks, collected before initiating antibiotic treatment. The E. coli isolation rate among diarrhoeic calves was 80% (60/75; 95% CI: 69.17–88.35). The presence of stx genes ( stx1 , stx2 ), intimin-producing eae gene, carbapenemase-producing genes ( bla _NDM group and bla _KPC group), and extended-spectrum β-lactamase genes ( bla _TEM group, bla _CTX−M group, and bla _SHV group) was screened by polymerase chain reaction (PCR). The prevalence of pathogenic E. coli was 13.33% (10/75; 95% CI: 6.5–23.1). The STEC isolates were detected in 12% (9/75, 95% CI: 5.64–21.56). Of these pathogenic E. coli isolates, the STEC with both stx genes, stx1 only, and stx2 only genotypes were present in 40% (4/10) and 10% (1/10), respectively. Intimin-producing STEC isolates ( eae -STEC) were identified in 40% (4/10) among the pathogenic isolates, with the following genotypes: stx1 + eae (2/10, 20%), stx2 + eae (1/10, 10%), and stx1 + stx2 + eae (1/10, 10%). Only one (1/10, 10%) eae- encoded non-STEC isolate was found, called enteropathogenic E. coli (EPEC). All STEC (100%) isolates were tested positive for either ESBL or carbapenemase or both ESBL and carbapenemase in the phenotypic assays. ESBL-producing STEC isolates were genetically identified in 6.67% (5/75) with the following combinations: three isolates coharbored stx1 - bla _TEM , two had stx1 - bla _SHV , and stx2 - bla _TEM , respectively. All intimin-producing STEC ( eae- STEC) isolates were both ESBL and carbapenemase producers, which were identified in 5.33% (4/75), with followings genotypic expression: stx1 + stx2 + eae / bla _TEM + bla _CTX−M + bla _NDM + bla _KPC in 1.33% (1/75), stx1 + eae / bla _TEM + bla _KPC in 1.33% (1/75), stx1 + eae / bla _TEM + bla _CTX−M +bla _KPC in 1.33% (1/75), and stx1 + eae / bla _TEM + bla _CTX−M +bla _KPC in 1.33% (1/75). ESBL and carbapenemase-producing eae -STEC isolates were more likely to be multidrug-resistant (MDR) than ESBL-STEC and EPEC isolates. The highest antimicrobial resistance rates were observed in ciprofloxacin and meropenem (100%), nitrofurantoin and cefoxitin (90%), ampicillin, streptomycin, and trimethoprim-sulfamethoxazole (80%), chloramphenicol (70%), and doxycycline, tetracycline, and cefotaxime (60%). In contrast, the lowest resistance rates were found in gentamycin (20%) and amoxicillin-clavulanate (40%). In terms of pathogenicity, only STEC isolates induced mild to moderate non-bloody diarrhoea, whereas intimin-producing STEC caused severe bloody diarrhoea with 100% mortality (4/4) ( p  < 0.05) in neonatal calves. To the best of our knowledge, this is the first report on ESBL and carbapenemase-producing MDR STEC from diarrhoeic calves in Bangladesh.