Indian Journal of Animal Research

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Full Research Article

Detection of Antimicrobial Resistance by Phenotypic and Genotypic Methods in E. coli Isolated from Diarrhea of Cattle Calves in Mhow, Indore

Ravi Sikrodia1,*, Daljeet Chhabra1, Supriya Shukla2, S.D. Audarya3, Joycee Jogi1, Rakhi Gangil1, G.P. Jatav2
1Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Dr. Ambedkar Nagar- Mhow-453 466, Madhya Pradesh, India.
2Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Dr. Ambedkar Nagar - Mhow-453 466, Madhya Pradesh, India.
3Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Kuthuliya-486 001, Rewa, Madhya Pradesh, India.

ABSTRACT

Background: Neonatal calf diarrhoea, a complex illness, continues to be the leading cause of neonatal calf deaths. The complex pathophysiology of infectious calf diarrhoea results in severe losses to the dairy sector, either directly via mortality or indirectly through the costs of prophylaxis and slowed growth in infected calves. Neonatal calf diarrhoea during the first month of life causes between 80 to 85 per cent of all deaths in India. 

Methods: A total 137 cattle calve diarrhea samples were collected from calves that show clinical signs of diarrhea. Swabs of the rectum were taken directly from diarrhoeic calves and collected in sterile test tubes. They were then immediately transported to the laboratory for the isolation of E. coli. 

Result: A total 64 E. coli isolates were detected and identified from samples via biochemical and molecular assays employing PCR targeting the E. coli specific 16s rRNA gene. The result of antibiotic sensitivity of 64 isolated strains of E. coli to 15 antimicrobial drugs shows highest sensitivity towards antibiotic gentamycin (68.75%) followed by sulfadiazine and co-trimoxazole (39.06), ciprofloxacin (32.81%) and highest resistance against ampicillin (87.5%) followed by ceftriaxone (82.813%), ceftazidime plus clav (81.25%). There is the variation in the expression of the AMR genes in the isolated E. coli strains. The detection rate of AMR gene blaTEM was highest 90% and that of aadA was the lowest (0%). The other AMR genes blaCTX-M, blaSHV, sul1 and tetA showing 50%, 20%, 50% and 30%.

INTRODUCTION

Around the world, newborn calves are a significant source of animal production for either meat or breeding. In various nations, diarrhoea is one of the extremely prevalent disease syndromes in newborn calves and this can have detrimental effects on both the economy and animal welfare (Africa Union, 2008). Despite decades of research, neonatal calf diarrhoea, a complex illness, continues to be the leading cause of neonatal calf deaths (Uhde et al., 2008). The complex pathophysiology of infectious calf diarrhoea results in severe losses to the dairy sector, either directly via mortality or indirectly through the costs of prophylaxis and slowed growth in infected calves (Radostits, 2007). The non-infectious causes (predisposing variables) (flaws or gaps in management-inadequate nourishment, exposure to a harsh environment, neglect of the newborn calf, or a combination of these) and infectious causes interact in a variety of ways (Blanchard, 2012; Cho and Yoon, 2014). Neonatal calf diarrhoea during the first month of life causes between 80-85 per cent of all deaths in India (Singh et al., 2009). According to several studies, E. coli is the main cause of diarrhoea, hemorrhagic colitis and dysentery in weak, undernourished, unhealthy and immunosuppressed calves (Ellaithi, 2004 and Mohamed et al., 2007).

MATERIALS AND METHODS

Study area
 
This study was conducted from July 2020 to the end of June 2021. Neonatal cattle calves of different ages (1 day-4 months old) examined clinically for diarrhea on dairy farms in and around Mhow, Indore, Madhya Pradesh, India. On the old Mumbai-Agra Street, 23 kilometers south of Indore city, Mhow is toward Mumbai. In 2003, the Madhya Pradesh government changed the town’s name to Dr. Ambedkar Nagar, Mhow, Madhya Pradesh, India, has a latitude of 22.552437 degrees and a longitude of 75.756531 degrees. In Mhow, the average annual temperature is 25.1°C. The rainfall here is around 1126 mm per year. This region is home to a large number of organized and unorganized dairy farms that provide the town and its neighbours’ residents with milk and milk-related products. Depending on the scale of production, these dairy farms either have local or crossbred animals.
 
Study population
 
Animals included in this investigation were cattle calves under 4 months of age with clinical diarrhoea, showing symptoms of systemic sickness (such as impaired suckling reflex, poor appetite, fever and dehydration) and pasty-watery feces. A total 137 diarrhea samples were collected from calves that show clinical signs of diarrhea. Swabs of the rectum were taken directly from diarrhoeic calves and stored in sterile test tubes and transported to the laboratory of the department of Veterinary Microbiology in the college of Veterinary Science and Animal Husbandry in Mhow for processing.
 
Isolation and identification of E. coli
 
The sample was inoculated in BHI broth and incubated aerobically at 37°C for 24 hours, followed by streaking on MacConkey agar plate and EMB for selective isolation of E. coli. The plates were incubated aerobically at 37°C for 24 hours. Bacteria producing pink colony on MacConkey agar and characteristic purple-black colonies with dark centre with metallic sheen on EMB agar suggestive of E. coli (Fig 1) and (Fig 2). Following the guidelines outlined in Quinn et al., (2002), conventional bacteriological methods were used to identify probable E. coli colonies. As a result, IMViC assays, such as indole, methyl red, Voges Proskauer and citrate utilization, were used to preliminary characterize  E. coli isolates.
 

Fig 1: Pink colonies of E. coli on MacConkey agar.


 

Fig 2: E. coli on EMB agar showing metallic sheen.


 
DNA extraction
 
The isolates were inoculated in Brain heart infusion broth (Hi Media, Mumbai) and incubating at 37°C for 12-18 hours. Extraction of DNA was done using readymade kit (GenEluteTM Bacterial Genomic DNA Kit, Sigma) as per the manufacturer’s instructions. The extracted DNA was stored at -20°C till further use.
 
Molecular identification of E. coli isolates using polymerase chain reaction (PCR)
 
16S rRNA is a highly conserved gene that was chosen for the molecular detection for E. coli isolates according to Sobur et al., (2019) (Table 1). DNA amplification was accomplished in a total 25 μL mixture volume, including 2 μL of DNA template, 12.5 μL of Master mix (SIGMA life science), 1 μL of 16S rRNA forward and reverse primers (20 pmol/μL) and 8.5 μL PCR-grade nuclease free water (SIGMA life science). Samples were subjected to initial denaturation for 5 min at 94°C followed by 35 cycles of denaturation (94°C, 30 s), annealing (58°C, 30 s) and elongation (72°C, 2 min). These cycles were followed by a final elongation step (7 min at 72°C).
 
Antimicrobial susceptibility tests
 
Isolates were screened for resistance to antibiotics by a disk diffusion method. Bacteria were suspended in saline to the density of a McFarland No. 2 standard, diluted 1:20 and streaked by the method of Bauer et al., (1966) on Mueller-Hinton agar and incubated for 24 hours at 37°C. Characterization of strains as sensitive, intermediate or resistant was based on the manufacturer’s instructions (Hi-media). Fifteen antibiotics like Ampicillin (AMS) 10 µg, Cefotaxime (CTX) 30 µg, Enrofloxacin (Ex) 10 µg, Gentamycin (G) 10 µg, Azethromycin (AZM) 15 µg, Ceftrixone (Ci) 10 µg, Ciprofloxacin (CIP) 5 µg, Colistin (CL) 10 µg, Co-trimoxazole (COT) 25 µg, Ceftazidime plus Clav (CAC) 30 µg, Aztreonam (AT) 30 µg, Nitrofurantoin (NF) 100 µg, Streptomycin (S) 10 µg, Sulphadiazine (SZ) 100 µg, Tetracycline (TE) 10 µg were used in this investigation.
 
Identification of antibiotic resistance genes
 
All E. coli strains were analysed for six antibiotic resistance genes blaTEM (betalactams), blaCTX-M (Cephalosporin), blaSHV (betalactams), tetA (tetracycline), sul1 (sulfonamides) and aadA (streptomycin) by PCR. PCR were used to investigate the target genes of the E. coli isolates. Lists of primers used and the amplified products are presented in Table 1. All reactions were carried out on a PCR thermocycler (Applied Biosystems). Amplification of DNA was conducted in a total volume of 25 µl, including 2 μL of DNA template, 12.5 μL of Master mix (SIGMA life science), 1 μL of forward and reverse primers (20 pmol/μL) each and 8.5 μL PCR-grade nuclease free water (SIGMA life science). The PCR were carried out as per specific cyclic conditions with different genes as per the Table 2. The amplified products were separated by electrophoresis through 1.5% agarose (wt/vol), stained with 0.5 μg/mL ethidium bromide, visualized under UV illumination, imaged with a Gel Doc 1000 fluorescent imaging system (Uvitech).
 

Table 1: Details of primers used for PCR reactions.


 

Table 2: Thermocyclic conditions for different AMR genes.

RESULTS AND DISCUSSION

When diseased calves were examined clinically, it was discovered that they had varying degrees of profuse watery diarrhoea as well as varying degrees of dehydration, which could be seen in their decreased skin elasticity, sunken eyes and inability to stand. The E. coli strains were diverse and varied, with several different virulence factors. The presence of a large molecular weight plasmid harbouring several genes involved in disease was the cause of the heterogeneity. This plasmid contributes to the pathotypes heterogeneity by facilitating the acquisition or deletion of virulence genes. Variation in resistance and sensitivity per cent has been found in various reports. Following a bacterial analysis, A total 64 E. coli isolates were detected and identified from 137 diarrheic samples via biochemical and molecular assays employing PCR targeting the E. coli specific 16s rRNA gene (Plate 1). Walid et al., (2020) and Mousa and Abo Shama (2021) also used 16S rRNA to confirm the E. coli genotypically. Over all, Escherichia coli (E. coli) appeared to be an important causative agent either alone or in combination with other bacteria in the etiology of calf diarrhea and were isolated with the highest frequency from diarrhoeic calves in our study. In accordance with our findings, numerous researchers have also reported a significant prevalence of E. coli strains in cases of infectious diarrhea in calves. (Nguyen et al., 2011 and Shahrani et al., 2014).
 

Plate 1: Results of PCR Showing 16 S rRNA gene of E.coli.


       
The results of Antibiotic sensitivity test of E. coli isolates showed variable percentage of sensitivity and resistance to the different antibiotics Fig 3. The result of antibiotic sensitivity of 64 isolated strains of E. coli to 15 antimicrobial drugs are presented in Fig 4. The highest sensitivity was attributed towards antibiotic gentamycin (68.75%) followed by sulfadiazine and co-trimoxazole (39.06), ciprofloxacin (32.81%), Azithromycin (31.25%), Tetracycline (25%), Nitrofurantoin (21.87%), Aztreonam and streptomycin (20.31), cefotaxime (12.5%), Ceftizidime plus clav (7.81%), ceftriaxone (6.25%), enrofloxacin (4.687%), Ampicillin (3.125%) and colistin (0%). In present study gentamycin showed the highest sensitivity. In accordance with our findings, numerous scientists have also reported a sensitivity to gentamycin The Hemashenpagam et al., (2008) recorded 75% and Kirkan et al., (2018) recorded 89% which is very close to our percent. Whereas lower sensitivity viz. 62.5%, 60.2% was recorded by Abdeen et al., (2019) and Cengiz and Adiguzel (2020), respectively.
 

Fig 3: Results of antibiotic sensitivity test.


 

Fig 4: Sensitivity of E. coli isolates to different antimicrobial agents.


       
The highest resistance was recorded against ampicillin (87.5%), ceftriaxone (82.813%), ceftazidime plus clav (81.25%), colistin (76.56%), enrofloxacin (75%), Azethromycin (68.75%), cefotaxime (67.18%), tetracycline (62.5%), aztreonam (57.81%), sulphadiazine (48.438%), Nitrofurantoin (46.875%), co-trimoxazole (43.75%), streptomycin (37.5%), ciprofloxacin (29.688%) and gentamycin (12.5%).
       
E. coli showed variable percent of resistance against Ampicillin by many researchers viz. Hemashenpagam et al., (2008) (66.66%), Abdeen et al., (2019) (81.25%), Manjushree et al., (2019) (84%), Adeladlew et al., (2020) (100%) and Sobhy et al., (2020) (54.5%). In contrast of present finding Kadam (2018) (60%), Sumedha et al., (2019) (66%), Srivani et al., (2020) (100%), Algammal et al., (2020) (78.5%) and Merera et al., (2020) (73.85%) reported higher sensitivity against Ampicillin. To compare the phenotypic resistance the isolates were subjected to detection of six different antibiotic resistance gene belonging to 4 different antibiotic groups presented in Table 2.  There is the variation in the expression of the AMR genes in the E. coli. The detection rate of AMR gene blaTEM was highest 90% and that of aadA was the lowest (0%). The other AMR genes blaCTX-M, blaSHV, sul1and tetA showing 50%, 20%, 50% and 30% (Plate 2-6). Similarly, many scientists studied the AMR gens in isolated E. coli. Jiang and Zhang (2013), El Bably et al., (2016) and Abdeen et al., (2019) detected blaTEM gene in 84.6%, 80% and 100%, isolates respectively, which is in close agreement with the present research and, however lower percent of blaTEM gene was detected by Algammal et al., (2020) and Khawaskar et al., (2022) reported 21.5% and 8.3% respectively. El Bably et al., (2016) and Yue et al., (2021) reported the blaSHV gene in 33.3% and 18.5%, respectively which is close to the present detection level. Abdeen et al., (2019), Khawaskar et al., (2022) reported lower per cent of blaCTX-M 31.25% and 28.3%, respectively. However, Esmaeel and Naseer (2017) reported gene in 80%, higher as compared to the present findings. Abdulgayeid et al., (2015) and El Bably et al., (2016) reported 60% and 50% isolates to be positive for sul1 gene, respectively which is very similar to the present findings but higher percent of sul1 gene 74%, 81.25% and 100%, was recorded by Jiang and Zhang (2013), Abdeen et al., (2019) and El sayed et al., (2020), respectively. In contrast to present finding very higher percentage was tetA gene reported by Abdeen et al., (2019), Abdulgayeid et al., (2015), El Bably et al., (2016), Yue et al., (2021) and El sayed et al., (2020) reported 93.75%, 100%, 50%, 96.3% and 100%, respectively. In contrast to findings of present study Jiang and Zhang (2013) and Yue et al., (2021) reported 73.3% and 70.4% aadA gene in isolated E. coli. A great variation in gene detection percentage in E. coli was reported by different scientists. These differences might be partly explained by differences in geographic location, the complexity of the healthcare institutions involved, the exposure to healthcare environments, the usage of antibiotics and antibiotic stewardship practices.
 

Plate 2: Results of PCR showing bla CTX-M gene of E.coli.


 

Plate 3: Results of PCR sowing blaSHV gene of E.coli.


 

Plate 4: Results of PCR showing blaTEM gene of E.coli.


 

Plate 5: Results of PCR showing Sul1 gene of E.coli.


 

Plate 6: Results of PCR Showing tetA gene of E.coli.

CONCLUSION

The intensity and incidence of the disease are regulated by several variables that interact dynamically over time since diarrhoea is a complicated condition that includes various bacteria, among these members of Enterobacteriaceae family specially E. coli and Salmonella are major pathogens. Antimicrobial resistance in bacteria is frequently linked to various resistant genes, transmission mechanisms and reservoirs of antibiotic resistance among various species.  In present study high level of antibiotic resistance is reported which is one of the major issue globally.

ACKNOWLEDGEMENT

The authors are thankful to College and NDVSU University officials for providing necessary facilities for research.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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