Characterization of Extended Spectrum Beta Lactamase Producing Escherichia coli from Calves

Neonatal calf diarrhea is multifactorial symptom which despite of continuous research is major cause of poor growth in young calves. Gram negative bacteria are a major therapeutic challenge in both livestock and human beings. The rapid global rise of Escherichia coli infections that are resistant to therapeutically important antimicrobials, including first line drugs such as cephalosporins and fluoroquinolones, is of serious concern, as it hampers treatment of infections leading to significant morbidity, mortality, treatment costs as well as production losses in livestock (de Been et al., 2014).
       
Beta lactam antimicrobial drugs exhibit the most common treatment for bacterial infections. Extended spectrum beta lactamases (ESBL) are classified into several groups, the prominent among them are TEM, SHV and CTXM types (Singh et al., 2017). Members of the family Enterobacteriaceae commonly produce ESBLs that confer resistance to the 3^rd and 4^th generation of cephalosporins, penicillins and monobactams. ESBLs are of great concern because they are plasmid associated. ESBL genes are situated on plasmids that can be easily transferred between and within bacterial species. Some ESBL genes are mutant derivatives of established plasmid mediated beta lactamases (e.g. bla TEM/SHV) and others are mobilized from environmental bacteria (e.g. bla CTXM). E. coli has a very wide natural distribution and a propensity of plasmid carriage (Sherley et al., 2003). Detection of ESBL producing E. coli in food producing animals and edible animal products has become a serious cause of concern for the consumers (Kar et al., 2015). The increase, emergence and spread of antimicrobial resistance among E. coli are the most important health problems worldwide. This increased antibiotic resistance is mainly due to increased prevalence of ESBL producing Enterobacteriaceae (Canton et al., 2008).

The present study was carried out in the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Jabalpur (M.P.). A total 52 rectal swabs were collected from Instructional Livestock Farm Complex, Adhartal, Private dairy farms and Gaushala of Jabalpur district betweenthe month of October 2019 to February 2020, in transport media and incubated at 37°C for 18 hrs. All samples were processed for isolation of E. coli. Bacterial isolation was carried out as per Markey et al., (2013). The inoculum was inoculated in to selective MacConkey agar and incubated at 37°C for 18 hrs. All the isolates were lactose fermenters as indicated by small bright pink colonies on MacConkey agar medium.The pink colonies were further inoculated on Eosin Methylene Blue agar and incubated at 37°C for 18 hrs resulted in metallic green sheen colonies. Identification of E. coli was done by Gram’s staining, catalase, oxidase, indole, methyl red, Voges Proskauer and citrate utilization tests.
       
Extended spectrum beta lactamase production was studied as per the recommendations of CLSI (2013). The antibiotics used for the study were cefpodoxime, ceftazidime, cefepime and aztreonam (30 mcg) for initial screening.  The isolates exhibiting resistance to the extended spectrum cephalosporin and monobactam group of antibiotics were selected for confirmation of ESBLs production by ESBL identification kit and double disc diffusion test (DDDT). HX096 disc containing Cefpodoxime 10 mcg and Cefpodoxime/Clavulanic acid 10/5 mcg, Ceftazidime 30 mcg and Ceftazidime/Clavulanic acid 30/10 mcg, Cefotaxime 30 mcg and Cefotaxime/Clavulanic acid 30/10 mcg disc was placed on the inoculated Mueller Hinton agar plate. It was incubated overnight and difference in zone of inhibition of ≥5 mm of cephalosporin and their cephalosporin plus clavulanic acid containing disc indicate production of ESBL. ESBL production was analyzed by the DDDT. The central disc was Amoxicillin/Clavulanic acid 20/10 mcg. Four other discs were placed within a 20 mm radius of the first one: Ceftazidime 30 mcg, Ceftriaxone 30 mcg, Cefepime 30 mcg and Aztreonam 30 mcg. Samples were considered positive for ESBL when the inhibition zone around any cephalosporin increased toward the central disc with AMX/AC and when the inhibition zone around at least one of the cephalosporins were smaller than 19 mm.
       
All the E. coli isolates obtained from different samples were investigated for presence of ESBL genes namely bla CTX-M, bla TEM and bla AmpC. The chromosomal DNA was extracted as per the method of Wilson (1987) with a slight modification. The extracted DNA samples were stored at -20°C for further molecular analyses. Plasmid from each tested ESBL producing isolates were extracted as per the method described by Sambrook and Russel (2001).
       
All the 51 isolates of E. coli were genotypically characterized for 16S rRNA by PCR assay using specific primer as mentioned in Table 1.  PCR was carried out in a thermal cycler and the cycling condition for 16SrRNA gene was: initial denaturation at 95°C for 5 min followed by 30 cycles of amplification with denaturation at 94°C for 30 s, annealing at 69°C for 30 s and extension at 72°C for 2 min, ending with a final extension at 72°C for 10 min. All the isolates found tobe positive for ESBLs production phenotypically were tested for the presence of bla CTX-M, blaTEM and bla AmpC genes by PCR assay using specific primers (Table 1). Total DNA (3 μl) was used in a 25 μl reaction mixture that contained 12.5 μl of Dream Taq Green PCR master mix(2X) (Thermo Fisher Scientific, UK) (containing DreamTaqTM DNA polymerase, optimized DreamTaq Green buffer, 0.4mMof each of the dNTPs, 4 mM MgCl₂), 7.5 μl of nuclease free water and 1 μl of each primer. PCR was carriedout in a thermal cycler and the cycling condition forbla CTX-M was: initial denaturation at 94°C for 1 min followed by 35 cycles of amplification with denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min, ending with a final extension at 72°C for 10 min. For blaTEM gene, initial denaturation at 95°C for 5 min followed by 35 cycles of amplification with denaturation at 95°C for 30 s, annealing at 45°C for 1 min and extension at 72°C for 1 min, ending with a final extension at 72°C for 10 min. For bla AmpC gene, initial denaturation at 94°C for 3 min followed by 25 cycles of amplification with denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 1 min, ending with a final extension at 72°C for 7 min.

Isolation and Identification of E. coli
 
The emergence of multidrug resistant bacteria is a global health concern. ESBL are rapidly spreading worldwide and are frequently isolated from animals and humans Enterobacteriaceae isolates. In the present study 52 rectal swabs of healthy as well as diarrheic cattle calves in and around Jabalpur were collected for characterization of ESBL producing Escherichia coli. A total 51 (98.07%) samples yielded E. coli which include 27(100%) from non diarrheic cattle and 24 (96%) from diarrheic cattle calves (Table 2). In this study, singlex PCR was performed for targeting the highly conserved gene 16S rRNA for confirmation and rapid diagnosis of E. coli. Total 39 (76.47%) isolates produced 1476 bp of amplicon and were confirmed as E. coli (Fig 3a).



        
The present study shows much higher isolation rate than Masud et al., (2012) (30.71%) and Gebregiorgis and Tessema (2016) (36.8%). The reason why the result of the current study varies from the other reports might be due to variations in farm management conditions. The present study findings corroborate with the result of Pereira et al., (2014); Awosile et al., (2018) and Putra et al., (2020), they isolated and identified E. coli from calf and confirmed E. coli in 96%, 88.1% and 100%, respectively. E. coli are the normal commensal bacteria of intestine so they were present in all the non diarrheic calves as stated by Saidani et al., (2017). Prevalence of E. coli was lower in diarrheic calves (88.46%) compared to other studies. The differences in the prevalence rates of E. coli among diarrheic calves may be attributed to the geographical locations of the farm, management practice and hygienic measures (El-Seedy  et al., 2016).
 
ESBL production
 
Out of 51 E. coli isolates, 07 (13.72%) E. coli isolates showed reduced susceptibility to one or more antimicrobials of initial screening (Fig 1). Total 05 (9.8%) isolates were phenotypically confirmed as ESBL producing E. coli by using ESBL identification kit and double disc diffusion test (Table 3, Fig 2). Out of the 05 phenotypically positive isolates screened for the presence of bla genes by PCR, 01 (20%) isolate was found to be positive for bla CTX-M gene (Fig 3b), 04 (80%) isolate was found to be positive for blaTEM gene (Fig 3c) and 03 (60%) isolates were positive for bla AmpC gene (Fig 3d). Coexpression of bla TEM and bla AmpC was recorded in 03 (60%) isolates (Table 4).

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Findings of present study are supported by observations of Liu et al., (2018) from China using double disc diffusion test reported 9.60% E. coli isolates from pigs as ESBL producer. In France, Haennia et al., (2014) reported prevalence of ESBL E. coli to be 29.40% in calves fecal flora. Hiroi et al., (2011) screened 16 E. coli isolates, out of which two isolates (12.50%) were phenotypically confirmed as ESBL producers. Similar findings of lower prevalence of ESBL producer E. coli were observed in the present study it might be due to differences in the detection methods. As documented in the Schmid et al., (2013) using enrichment and selective media (MacConkey agar containing cefotaxime) for isolation of ESBL producing E. coli. Olowe et al., (2015) performed PCR in E. coli isolates obtained from animal fecal samples in Nigeria and detected blaTEM and bla CTX gene in 48 (42.10%) and 51 (44.70%) isolates, respectively. Liu et al., (2018) from China reported 9.60% E. coli isolates from pigs as ESBL producer harbored at least one type of beta lactamase, with bla CTX-M, bla TEM, being detected in 90.90% and 68.18%, respectively.  The present study revealed bla TEM shows higher prevalence which is similar to the finding of Montso et al., (2019) from South Africa screened 53.1% E. coli isolates as ESBL producers. The bla TEM and bla CTX-M genes were detected in 85.5% and 58.00%, respectively. Dewangan et al., (2017) from Chhattisgarh detected 39.4% isolates were found to harbour the blaTEM gene on their plasmid DNA indicating the presence of multidrug resistant ESBL producing E. coli in foods of animal origin and human samples. Tekiner and Ozpýnar (2016) from Turkey detected blaTEM and blaCTX-M 96.40% and 53.70%, respectively in foods of animal origin.
 
Plasmid profiling
 
The plasmid profile of 05, ESBL producing E. coli isolates from healthy and diarrheic cattle calves are presented in Table 5. Plasmid profiling of ESBL producing E. coli isolates from healthy and diarrheic cattle calves were observed by agarose gel electrophoresis which showed plasmid bands in different combinations (Fig 4). The average plasmid number among the isolates was 2.4 and ranging from 0-4 plasmids per isolate. Three isolates (60%) were harboring 04 plasmids which were highest number of plasmid and no plasmid was observed in two isolates (40%). The plasmid size was observed in different combinations which ranged from 900 bp to>10 k bp in all isolates. pQE-30 Xa vector was used as positive control in our study.

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In E. coli, the antimicrobial resistance genes reside in plasmids which are responsible for resistance to numerous antimicrobial agents. We recorded smaller plasmids of <20 kb in size. Existence of common plasmid among the isolates implies the spread of resistant plasmid in the community. Gupta et al., (2014) reported smaller size plasmid ranging from 3 kbp to 8 kbp and their number also varied from one to four. Whereas Gohar et al., (2015) reported plasmid size from 100 bp to 12 kbp. Furthermore, the similarity in plasmids among different isolates suggested plasmid movement between bacteria. From ESBL resistant isolates, 40% of the isolates we couldn’t recover plasmid although the concentration of plasmid was visible in spectrophotometer but on electrophoresis, we were not able to amplifying it. It may be due to low copy number or loss of plasmid during curing in laboratory procedure. 

In the present study detection of antibiotic resistance genes showed prevalence of ESBL producing E. coli in cattle calves in and around Jabalpur. Existence of common plasmids among isolates implies the spread of resistant plasmid in the community.