Isolation of E. coli from rectal swabs of diarrhoeic piglets
In the present study a total of 102 samples of rectal swab from diarrhoeic piglets were examined for isolation of
E. coli of which, 92 (90.2%) samples yielded
E.
coli showing characteristic morphology (Fig 1 and 2). In respect to source of samples, 41 (91.12%) out of 45 samples were collected from ICAR-AICRP/MSP on Pig, College of Veterinary Science, Assam Agricultural University, Khanapara and 32 (88.89%) out of 36 samples were collected from ICAR-National Research Centre on Pig, Rani, Guwahati, Assam while 19 (90.48%) out of 21 samples were collected from different unorganised farms (Table 3).
Biochemical characterization of E. coli
The isolates were subjected to biochemical tests like IMViC tests, catalase test and motility tests (Table 4). 92 isolates were positive for indole and methyl red tests, in which the isolates have produced red/pink colour ring (Fig 3) and red colour respectively (Fig 4). None of the samples were positive for Voges-Proskauer (Fig 5) and citrate utilization tests (Fig 6). Catalase (Fig 7) and motility tests (Fig 8) showed positive reaction for 92 isolates. All the biochemical reactions confirmed the presence of
E. coli.
Pathotyping of E. coli isolates
Nucleic acid amplification by Polymerase Chain Reaction is recognised as one of the most important scientific developments to come along during the last two centuries for detection of genes encoding virulence factors of pathogenic organisms (
Rahman, 2002;
Moon et al., 2003). Multiplex PCR is a useful adoption in which several PCR primers are combined to detect virulent genes in a single reaction. In the present study, a oligonucleotide primer set was used which encodes the genes typically found in pathotypes of
E. coli prevalent in piglet diarrhoea viz. Enterotoxigenic or ETEC (
est1, elt1), Shigatoxigenic or STEC
(stx1) and Enteropathogenic or EPEC
(eaeA) E. coli. Primers were selected on the basis of published nucleotide sequence of the 127 bp for
stx1, 222 bp for
est1, 308 bp for
elt1 and 535 bp for
eaeA genes
(Rajkhowa et al., 2015).
A total of 92 isolates of
E. coli were examined for the presence of
stx1,
est1,
elt1 and
eaeA gene by multiplex PCR using standard primers. The study revealed that 25 isolates were positive for
stx1 gene (27.17%), 18 for
est1 gene (19.56%), 6 for
elt1 genes (6.52%), 3 for presence of both genes
est1 and
elt1 (3.26%) and 12 for
eaeA genes (13.04%), which corroborates with the findings of
Bessone et al., (2017) and
Fratamico et al., (2008).
This suggests that the
E. coli isolates were capable of producing heat stable (ST) and heat labile (LT) toxins. Prevalence of ETEC in
E. coli isolates were also reported by some workers (
Garcia-Menino et al., 2018,
Yang et al., 2019, Li et al., 2020).
stx1 gene encodes for Shiga toxin 1 which is a characteristic cytotoxin released by verocytotoxigenic
E. coli (VTEC) or Shigatoxigenic
E. coli (STEC). Shiga toxin-producing
E. coli (STEC) are a group of highly pathogenic foodborne zoonotic pathogens, associated with diarrhoea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans. As a matter of fact, STEC have been isolated from pigs and pork products, in some cases associated with episodes of HC and HUS in humans
(Baranzoni et al., 2016; Honish et al., 2017). This is an important finding from public health concern as these strains can be cross transferred to human via food chain. Overall, pathotyping of the
E. coli isolates by detection of the virulent genes revealed that 25 (27.17%) isolates belonged to STEC, 27(29.34%) to ETEC and 12(13.04%) to EPEC as depicted in Table 5, 6 and Fig 9, 10.
Antimicrobial resistance pattern of the isolates
The widespread use of antibiotics in livestock has led to the emergence of multidrug-resistant (MDR)
E. coli, posing a threat to animal and human health. In the present study,
E. coli isolates resistant to more than 3 drugs were considered MDR
Sahm et al., (2001) and 50% of
E. coli isolates were found to be MDR, exhibiting resistance to three or more antimicrobials. In all, 46 (50%) of the 92
E. coli isolates were found to be MDR. Table 7 and Fig 11 shows that the
E. coli isolates tested for antimicrobial susceptibility showed maximum resistance towards tetracycline (71.74%), nalidixic acid (70.65%), framycetin (59.78%), cefazolin (54.35%), ofloxacin (51.09%), erythromycin (50%), gatifloxacin (48.91%) and ampicillin (47.83%), while moderate resistance was observed against nitrofurantoin (43.48%), co-trimoxazole (41.30%), cefixime (41.30%), neomycin (41.30%) ciprofloxacin (40.22%) and streptomycin (35.87%). Moderate susceptibility was recorded to piperacillin/ tazobactum (33.69%), cefuroxime (33.69%) cephalothin (32.61%), chloramphenicol (29.35%) and levofloxacin (29.34%), while maximum susceptibility was observed for meropenem (21.74%), norfloxacin (21.74%), cefoxitin (21.74%), cefepime (16.30%), gentamicin (11.95%), ceftazidime (10.87%), amikacin (8.69%), polymixin- B (6.52%) and imipenem (4.34%). Fig 12-15 depicts the antimicrobial sensitivity and resistance patters of
E. coli isolates on Mueller Hinton agar.
High resistance rates were observed for tetracycline (71.74%), nalidixic acid (70.65%), framycetin (59.78%), cefazolin (54.35%) and several other commonly used drugs. Moderate resistance was noted for nitrofurantoin, co-trimoxazole and ciprofloxacin, among others. The frequent use of these antibiotics likely drives resistance development. The occurrence of MDR
E. coli with resistance to up to 20 antimicrobials suggests plasmid-mediated resistance and highlights the urgent need for prudent antibiotic use in livestock to mitigate the spread of resistance
(Chakravarti et al., 1990). Conversely, higher susceptibility to carbapenems (meropenem, imipenem), polymyxin B, and certain cephalosporins indicates limited use of these drugs in veterinary practice, preserving their effectiveness.
