Detection of carbapenem resistant Escherichia coli in cattle and pigs

The aim of this study was to detect the presence of carbapenem resistant Escherichia coli (E. coli) in cattle and pigs. Altogether, 100 samples were collected, 50 each from cattle and pigs including mastitic milk and faecal samples from healthy as well as diarrhoeic cattle and faecal samples and slaughter specimens from pigs. Bacteria were isolated from the samples and antibiotic sensitivity test was carried out. Eight isolates of E. coli were obtained from cattle and 15 isolates from pigs. The phenotypic resistance patterns against carbapenems (ertapenem, imipenem and meropenem) were analysed. In cattle, the four isolates from milk were observed to be sensitive to all the three carbapenems whereas, all the isolates obtained from faecal samples were resistant to all the three carbapenems. The four faecal and three intestinal isolates of E. coli from pigs showed sensitivity to all the three carbapenems. From a total of 10 faecal samples from pigs, four, three and one isolates showed resistance to ertapenem, imipenem and meropenem respectively. Among five isolates from intestine, one isolate showed resistance to ertapenem and imipenem and no isolate showed resistance to meropenem. Polymerase chain reaction (PCR) was carried out to find the presence of carbapenem resistance genes (bla SHV, bla CTX, bla TEM and bla NDM ) in all the 23 isolates and the results revealed that seven (58.3 per cent) E. coli isolates carried bla SHV gene and four (33.3 per cent) contained bla CTX gene which encodes carbapenem resistant enzymes. Antibiogram using 14 commonly used antimicrobial agents revealed varied multidrug resistant patterns among the E. coli isolates.

as the last resort in treatment of multi-drug resistant (MDR) infections in human beings. The bacterial infections associated with carbapenem resistant Escherichia coli (E. coli) strains are emerging as a major public health concern globally (Baran and Aksu, 2016). Production of β-lactamases or β-lactam hydrolysing agents is the most common mechanism of drug resistance; especially in E. coli against the β-lactam antibiotics like carbapenems. The spread of these carbapenem hydrolysing enzymes among enteric bacteria in hospital settings is of grave concern. Intestinal microflora of animals carry genes namely, bla CTX-M , bla TEM, bla SHV and integrons, which can act as a possible cause for drug resistance in humans and animals (Tshitshi et al., 2020;Ejaz et al., 2021). Various ESBL genes viz., bla SHV (33.1 per cent), bla TEM (22.5 per cent), bla CTX-M (20.5 per cent) and bla OXA (11.3 per cent) were detected in CRE. It was reported that 76.3 per cent of Gram negative bacteria isolated from dogs were positive for atleast one carbapenemase gene (Sankar et al., 2021).
Carbapenem resistant Enterobacteriaceae (CRE) isolates were detected in food-producing animals and companion animals (Taggar et al., 2020). There are reports of identification of carbapenem resistant E. coli in cattle and pigs in different parts of the world. But studies carried out in India are rare and no studies have been carried out so far in Kerala. Hence, the present study was undertaken to detect the presence of carbapenem resistant E. coli in cattle and pigs. The study also aims at detecting the susceptibility of these isolates to other commonly used antibiotics. The collected samples were immediately plated onto brain heart infusion agar (BHIA) under aseptic conditions and were incubated at 37°C for 24 h. The isolates were analysed for its colony morphology and subjected to Gram's staining. Those colonies, which revealed Gram negative rods were subcultured onto Mac Conkey Agar (MCA) and eosin methylene blue (EMB) agar and further subjected to various biochemical tests for the confirmation of the presence of E. coli. All the procedures are followed as per Quinn et al. (1994).

Antibiogram
Disc diffusion method (CLSI, 2018) was performed on Mueller Hinton Agar (MHA) plates to determine susceptibilities of different β-lactam and non-β-lactam antibiotics. Three carbapenem antibiotics (ertapenem, meropenem, imipenam) and 14 other commonly used antibiotics belonging to various classes with known concentration were analysed.

Polymerase Chain Reaction
The DNA was extracted from E. coli isolates using the Genomic DNA Purification Kit (Origin, Kerala) as per manufacturer's instructions. The concentration and purity of DNA were measured spectrophotometrically. The DNA extracted from E. coli isolates, showing phenotypic resistance to carbapenems, were subjected to PCR using primers targeting bla SHV , bla CTX (Bora et al., 2014), bla TEM and bla NDM genes (Sankar et al., 2021).
A total volume of 12.5 µL reaction was prepared. The composition of single reaction mix for amplification was 6.25 μL of EmeraldAmp® GT PCR Master Mix, 1 μL each of forward and reverse primer (10 pm/μL), 3 μL of template DNA and 1.25 μL of nuclease free water. To each reaction tube, 9.5 µl of reaction mix and 3 µL of template DNA were added and subjected to initial denaturation (94ºC for 5min) followed by denaturation (94ºC for 1 min), annealing (56.9ºC, 63.2ºC, 65.6ºC, 66.7ºC for 1min for bla SHV , bla CTX , bla TEM , bla NDM , respectively), extension (72ºC for 2 min for bla SHV , 72ºC for 1 min for bla CTX , bla TEM , bla NDM , respectively), final extension (72ºC for 10 min for bla SHV , bla CTX , bla NDM , 72ºC for 5 min for bla TEM ) and hold (4ºC for 10 min).

Results and discussion
The presence of E. coli was detected in eight samples from cattle (four isolates from mastitis milk and four isolates from diarrhoeic samples). Major pathogen associated with clinical mastitis in dairy cattle was reported to be Staphylococcus aureus followed by E. coli (Sumathi et al., 2008). In a study conducted by Ranjan et al. (2011) on aetiological agents associated with bovine mastitis in Jharkhand, E. coli could be isolated from only 8.95 per cent. Lakshmi and Jayavardhanan (2016) reported that 27 per cent of isolates from mastitic milk samples of cattle in organised farms in Kerala were E. coli. Fifteen E. coli isolates were obtained from pigs (10 isolates from faecal samples and 5 isolates from intestine). There were reports of isolation of E. coli from more than 50 per cent of faecal samples collected from pigs (Kagambega et al., 2012;Lalzampuia et al., 2013). Individual animals shed E. coli in different concentrations due to several factors such as pathogen, host and environment and E. coli might not be isolated from all the faecal samples (Stein et al., 2017).
The carbapenem resistance pattern of E. coli isolates obtained from cattle and pigs were analysed against three carbapenem discs using disk diffusion method (CLSI, 2018) on MHA plates. Antibiotic susceptibility of the isolates to 14 commonly used antibiotic discs were also analysed. The varying degrees of resistance and susceptibility were observed (Table 1).  All the E. coli isolates obtained from faecal samples collected from cattle were found to be resistant to all the three carbapenems (ertapenem, meropenem and imipenem) and all the four isolates found from milk were observed to be sensitive to all the three (Fig. 1). Similar results were made by Papp-Wallace et al. (2011) and Chika et al. (2017) who reported the presence of carbapenem resistant E. coli in cattle. However, in the present study, no difference in the resistance could be observed among the three carbapenems.
All the ten E. coli isolates from faecal sample from pigs showed different resistance patterns. Four isolates showed resistance to ertapenem and three were resistant to imipenem; only one isolate showed resistance to meropenem. Among five isolates obtained from intestine, only one isolate was resistant to ertapenem and imipenem where no isolate showed resistance to meropenem (Fig. 2).
The study revealed the presence of carbapenem resistant E. coli in cattle and pigs. Out of 23 E. coli isolates obtained from cattle and pigs, 12 isolates were found to be resistant to atleast one of the carbapenem antibiotics.
316 Detection of carbapenem resistant Escherichia coli _____________________________________________ These 12 isolates were subjected to PCR, targeting resistance genes such as bla SHV , bla CTX, bla NDM and bla TEM using specific primers. Seven were positive for bla SHV , four were positive for bla CTX (Fig. 3 and 4) and all were negative for bla TEM and bla NDM . Rita et al. (2021) confirmed the presence of bla CTX gene in one E. coli isolate out of 27 isolates obtained from cattle. However, there are various reports on detection of bla SHV and bla CTX from E coli isolates from humans (Gondal et al., 2022). The transmission of these resistant genes from humans to animals and vice-versa pose a major threat to control of infection using these antimicrobials. However, further detailed surveillance of the resistant genes in personnels in contact with these animals is needed. Carbapenem resistant E. coli were reported from pigs . In the present study, among the ten isolates from faecal samples from pigs, six (60 per cent) were resistant to at least one of the carbapenem drugs. This indicates that the personnel working in these pens with animals harbouring resistant microbes may have a chance to get horizontal transmission. From the five isolates from slaughter house specimens, two (40 per cent) were carbapenem resistant. None of the isolates were resistant to all the three as against Pruthvishree et al. (2016), who reported that 30 per cent of the carbapenem resistant E. coli were resistant to all the three carbapenems.
Antibiotic susceptibility testing using commonly used antibiotics revealed that all the eight isolates from cattle were found to be multidrug resistant. The four isolates from milk were resistant to cefotaxime, ceftriaxone/ sulbactam, cefotaxime/clavulanic acid and cefpodoxime. Among them, three were resistant to enrofloxacin (C2, C3 and C4), ceftriaxone/ tazobactam (CI, C2 and C3) and piperacillin/ tazobactam (C2, C3 and C4). Two isolates showed resistant to amoxicillin (CI and C4) and tetracycline (C3 and C4). Only one isolate showed resistance to ceftriaxone (C2), cefalexin (C3) and gentamicin (C4). All the four isolates were found to be sensitive to ceftazidime and co-trimoxazole (Fig. 5).
There are several reports on resistance of E. coli isolated from cattle to a variety of antimicrobials including penicillin, amoxicillin, tetracycline, oxytetracycline, methicillin, amoxyclav, cloxacillin, ceftriaxone, gentamicin and enrofloxacin (Chandrasekaran  Das et al., 2017, Verma et al., 2017Revathi, 2018;Xavier et al., 2020). However, Sumathi et al. (2008) observed that gentamicin was found to be the most effective drug. All the eight isolates were found to be sensitive to co-trimoxazole. Revathi (2018) also obtained similar result.
E. coli in pigs were reported to be resistant to antibiotics like amoxicillin, oxytetracycline, trimethoprim, sulphonamide, streptomycin, cefixime, cefazolin, cephalexin, ceftriaxone, cefotaxime, enrofloxacin, ampicillin, chlortetracycline, erythromycin and lincomycin (van den Bogaard et al., 2000;Enne et al., 2007;Lalzampuia et al., 2013). The present study revealed varying degrees of resistance and susceptibility of the isolates to the antimicrobial agents in the study.

conclusion
The reports on detection of carbapenem resistant bacteria are rare in cattle and pigs. The occurrence of these bacteria in livestock has serious consequences and may affect therapeutic options in human medicine, since carbapenems are the last resort of antibiotic therapy. A key finding of this study was that 100 per cent of E. coli isolates recovered from faecal samples from cattle were resistant to all three carbapenems, ertapenem, meropenem and imipenem, which are not used in the livestock in Kerala. The high rate of resistance may reflect the increased clinical use of these antimicrobials among humans, which needs a thorough investigation. Hence, constant monitoring of carbapenem susceptibility among enteric bacteria of livestock is warranted. The bacteria were found to have various mechanisms conferring decreased sensitivity. The necessity for the study of antibiotic resistance and the associated genetic mechanisms is highly essential. Since a dependable substitute to carbapenems is not there, rationalisation of use of these antibiotics in both humans and animals is needed. The infection control measures should be meticulous whenever carbapenem resistance is detected and the active surveillance of carbapenemaseencoding genes are of the extreme importance. The tracing of the source of infection in livestock is important and all the steps should be taken to prevent the spreading of bacteria.

acknowledgement
The authors thank Kerala Veterinary and Animal Sciences University, Pookode for providing the facilities for the conduct of the study