Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Prevalence of Extended-spectrum β-lactamase Producing E. coli and Klebsiella spp. Isolated from Buffaloes in Eastern Plain Zone of Uttar Pradesh

V. Yadav1,*, R.K. Joshi1, N. Joshi2, S.V. Singh3, R.K. Gupta4, D. Niyogi4, D.K. Yadav3
1Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya-224 229, Uttar Pradesh, India.
2Department of Veterinary Public Health and Epidemiology, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya-224 229, Uttar Pradesh, India.
3Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya-224 229, Uttar Pradesh, India.
4Department of Veterinary Pathology, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya-224 229, Uttar Pradesh, India.
Background: Antimicrobial resistance (AMR) due to emergence and spread of Extended-spectrum β-lactamase (ESBL) producing bacteria are point of discussion in both human as well as in animals across the world as it is one of the latest challenges faced by scientific community. The present study highlighted the prevalence of ESBL producing E. coli and Klebsiella spp. isolated from buffaloes in Uttar Pradesh.

Methods: Total 240 samples were collected from two district of eastern plain zone of Uttar Pradesh during January 2020 to March 2021. E. coli and Klebsiella spp. isolates were confirmed by cultural characteristics in selective media and biochemical tests. ESBL producing isolates were confirmed by DDST, ESBL E-strip and PCR analysis using specific primer for (bla-CTX-M-1, bla-CTX-M-2, bla-CTX-M-9, bla-TEM and bla-SHV). Antibiogram of all confirmed ESBL producing isolates was performed against 20 antimicrobials under 12 different classes.

Result: In the present study, a total of 147 (61.25%) E. coli and 19 (7.91%) Klebsiella spp. were isolated and identified based on cultural and biochemical characteristics. A total of 104 (62.65%) and 99 (59.63%) isolates were confirmed as ESBL producers using DDST and ESBL E-strip tests respectively. PCR analysis, revealed the presence of selected ESBL genes in 92 (55.42%) isolates, among them bla-CTX-M-1 was found most dominant gene (69.41%) in E. coli and (71.42%) in Klebsiella spp. All the ESBL positive E. coli and Klebsiella spp. isolates were found to be 99-100% and 86-100% sensitive to chloramphenicol, polypeptides and aminoglycosides classes respectively. Both isolates of E. coli and Klebsiella spp. showed 100% resistance to cefotaxime, cefpodoxime ceftriazone and ampicillin, 76.47% and 71.42% to ceftazidime respectively and 81.52% isolates were found to be MDR.
Extended-spectrum β-lactamase (ESBL) producing organisms are expanding rapidly throughout the world and have become a major problem in both human and veterinary medicine. Among Enterobacteriaceae, E. coli and Klebsiella spp. are major ESBL producers and have been identified as an emerging global threat due to their increasing prevalence in livestock during last few years (Reuland et al., 2013) and are main environmental pathogens, associated with various illnesses. ESBL are enzymes that hydrolyse most of β-lactam antibiotics and mediate resistance against penicillins, 3rd and 4th generation cephalosporins (Saravanan et al., 2018). The genes encoding for these enzymes are commonly located on mobile genetic elements, among the species belonging to family Enterobacteriaceae, which are horizontally transferred between a close families of bacteria and also help to spread the AMR gene in the environment (Ansari et al., 2018). As a result, ESBLs have emerged as a cause of resistance in Enteribacteriaceae, particularly E. coli and Klebsiella spp. Extensive and indiscriminate use of antibiotics leads to emergence of resistance against a variety of antibiotics called MDR organisms, which poses significant challenges for both scientists and clinicians (Kappeli et al., 2019). MDRs in Enterobacteriaceae is increasing day by day, leads to limited antimicrobial therapeutic options and major cause of morbidity and mortality worldwide (WHO, 2016).
 
Since no in depth study has been done on prevalence and distribution of ESBL genes (bla-CTX-M, bla-TEM and bla-SHV) among E. coli and Klebsiella spp isolated from buffaloes in this area of study and their importance in development of resistance to other species or pathogens. Molecular characteristics and AMR pattern, will help the researchers, field veterinarians and policy makers to developed a guideline to scrutinize the use of antibiotics for therapeutic purpose and also helpful in controlling the spread of these bacteria in community setting.
Study area
 
The study was carried out in the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya. The samples were collected from Ayodhya and Sultanpur districts of eastern plain zone of Uttar Pradesh, India. The study was conducted between January 2020 and March 2021.
 
Sample collection
 
Total 240 samples (120 faecal and 120 milk) were collected from 5 tehsils of Ayodhya district and 3 tehsils of Sultanpur district. Sampling was done randomly and consisting of 10 apparently normal healthy and 5 diarrhoeic animals from each of the tehsil. Likewise, 10 apparently healthy and 5 clinical mastitic milk samples from same regions. California Mastitis Test was used for screening of mastitic milk samples. Faecal samples were collected by swab technique and approximately 5 ml of milk samples were collected in sterilized test tubes. All collected samples were transported immediately to Bacteriology Laboratory under cold chain.
 
Isolation and identification
 
All samples were enriched with 2ml nutrient broth and incubated at 37°C for 24 hrs. A loopful inoculum was taken and directly streaked on MacConkey agar plates added with 2 mg/L cefotaxime and incubated at 37°C for 24 hrs. Colonies with rose pink colouration were picked up and streaked on Eosine Methylene Blue agar plates. Colonies showing greenish metallic sheen (Fig 1) were tentatively considered as E. coli while light purple colonies with dark centred and mucoid appearance (Fig 2) were suspected as Klebsiella spp. After this, pure colonies were taken onto sterilized nutrient agar slant and further identification was done by various biochemical tests viz. IMViC pattern, catalase test, (Fig 3) nitrate reduction, urease test, triple sugar iron agar and sugar fermentation reaction as per the method of Edward and Ewing (1972).

Fig 1: Metallic sheen of E. coli on EMB agar.



Fig 2: Purple dark mucoid colony of Klebsiella spp. on EMB agar.



Fig 3: Catalase positive test.


 
Screening of ESBL producing isolates
 
All confirmed isolates of E. coli and Klebsiella spp. were subjected to screening of ESBLs, using 3rd, 4th generation cephalosporins, monobactam and carbapenems as per Kirby-Bauer’s. The results were interpreted as per CLSI guidelines (2019). Each isolates which showed resistant to one or more of these antibiotics, were screened for ESBL production.
 
Confirmation of ESBL producing isolates by phenotypic methods
 
Double disc synergy test (DDST)
 
All screened isolates were further confirmed by using ESBL kit 1 and kit 3 (Hi-media) (Fig 4). These commercially available discs were placed on MHA plates, infused with 1.5´108 organism/ml and incubated at 37°C for 24 hrs. The results were interpreted as per CLSI guidelines (2019).

Fig 4: DDST for confirmation of ESBL producing E. coli and Klebsiella spp.


 
Minimum inhibitory concentration (MIC) ESBL E-test
 
This test was performed by placing E-strip on MHA plates infused with 1.5x108 organism/ml and incubated at 37°C for 24 hrs. The results were interpreted as per CLSI guidelines (2019) (Fig 5).

Fig 5: ESBL E-strip test for confirmation of ESBL producing E. coli and Klebsiella spp.


 
Molecular characterization of ESBL producing E. coli and Klebsiella spp.
 
Extraction of plasmid DNA
 
Plasmid DNA was extracted from phenotypically confirmed isolates using GeneJet plasmid Miniprep kit (Thermo Scientific) as per the protocol of the manufacturers.
 
Molecular characterization of CTX-M genes (bla-CTX-M-1, bla-CTX-M-2, bla-CTX-M-2), bla-TEM and bla-SHV gene
 
ESBL gene detection was carried out in a total reaction volume of 25 µl for CTX-M and bla-TEM as per method described by Dallenne et al., (2010) and bla-SHV by Bhattacharjee et al., (2007). The primer sequence of targeted genes and amplicon size are listed in Table 1. Visualization of PCR product was done by mixing 5 µl of amplified products with 3 µl of bromophenol blue dye (6X) and electrophorased in 0.8% agarose gel in 1X TAE buffer mixed with 1 µl (5µg/ml) ethidium bromide, using 1kb DNA ladder (Thermo Scientific # SM 0311) at 60-70 mA for 40 min and gel was visualized using the UV illuminator (GeNei Bangalore, India).

Table 1: Detail of primers used for molecular characterization of ESBLs genes in isolates of E. coli and Klebsiella spp.


 
In vitro antibiotic sensitivity test of ESBL producing isolates
 
Antibiogram of all phenotypically confirmed E. coli and Klebsiella spp. was performed using 20 antibiotics of 12 classes (Hi-Media), mentioned in Table 6. It was done by disc diffusion method on Muller Hinton Agar (MHA, Hi-Media) plates inoculated with 1.5x108 organism/ml and incubated at 37°C for 24 hrs and isolates were classified as susceptible and resistant as per interpretation criteria of Clsi (2019) and those organisms showing resistance to at least one antibiotic of three or more classes, were called Multi-drug resistant (MDR) bacteria.
A total 240 milk and faecal samples were collected from different tehsils of Ayodhya and Sultanpur districts. Out of them 147 (61.25%) isolates were identified as E. coli and 19 (7.91%) as Klebsiella spp. on the basis of their morphology, cultural characteristics in selective media and biochemical tests (Table 2, Fig 1, Fig 2 and Fig 3). In this study, isolates of E. coli was found to be predominate among isolates recovered from both milk and faecal samples. This finding was also supported by findings of various workers (Ibrahim et al., 2018; Kotsoana et al., 2019). Higher isolation rate of E. coli in faecal samples may be attributed to high prevalence of E. coli in GIT flora of ruminants.

Table 1: Detail of primers used for molecular characterization of ESBLs genes in isolates of E. coli and Klebsiella spp.



To study the prevalence of ESBL producing isolates among clinical and apparently healthy isolates, total 166 (147 E. coli and 19 Klebsiella spp.) were subjected to screening, confirmatory phenotypic tests and PCR analysis. On preliminary screening, 79.5% isolates presumed as ESBL producer. Using phenotypic confirmatory testing, 62.65% isolates were confirmed as ESBL by DDST and 59.63% by ESBL-E strip test and final confirmation was done by PCR analysis, which revealed 55.42% ESBL positive isolates (Table 3). Total 92 (38.33%) were ESBL positive comprising 05 (1.25%), 16 (40.0%), 51 (63.75%) and 20 (50.0%) from normal milk, mastitic milk, normal faecal and diarrhoeic faecal samples, respectively (Table 2). These results were in conformity with findings of various workers (Kotsoana et al., 2019; Yadav et al., 2019). The prevalence of ESBL producers was found much higher in mastitic milk than normal milk irrespective of pathogen, which may be attributed to indiscriminate and irrational use of antibiotics for treating mastitis in Eastern Plain Zone of Uttar Pradesh.

Table 3: Distribution of ESBL strains, according to screening, phenotypic and genotypic confirmatory tests.



Genotypic analysis of confirmed ESBL producing isolates (85 E. coli and 07 Klebsiella spp.) was done by targeting bla-CTX-M (bla-CTX-M-1, bla-CTX-M-2, bla-CTX-M-9,) bla-TEM and bla-SHV genes (Fig 6, 7, 8, 9, 10). The present study showed the overall predominance of bla-CTX-M-1 (69.56%) followed by bla-CTX-M-9, bla-SHV, bla-TEM and bla-CTX-M-2 with 40.21%, 33.69%, 32.60% and 4.34% respectively (Table 4). This study discloses the predominance of bla-CTX-M-1 gene in this area. Likewise various co-workers across the world have also reported the high frequency of this gene in different sample sources (Ibrahim et al., 2018; Paghdar et al., 2020; Yadav et al., 2019). Overall distribution of genes according to organisms, E. coli revealed highest (69.41%) bla-CTX-M-1 followed by bla-CTX-M-9 (42.35%), bla-TEM (32.94%) bla-SHV (32.94%) and bla-CTX-M-2 (4.71%) where as Klebsiella spp. revealed highest (71.42%) bla-CTX-M-1 followed by bla-SHV (42.85%), bla-TEM (28.57%) and bla-CTX-M-9 (14.28%) (Table 5). It was notable in this study that bla-CTX-M-1 and bla-CTX-M-9 was present in both isolates but bla-CTX-M-2 gene was only present in E. coli isolates. Multiple co-existence of bla genes were also reported which has been mentioned in Table 4. Similar to this finding, multiple co-existence of bla genes were also noticed by various workers (Yadav et al., 2019; Tekinar and Ozpinar, 2016)

Fig 6: PCR amplification of bla-CTX-M-1 gene (688bp). M: 1kb ladder, Lane 1, 3, 4, 5, 6 and 7 positive for bla-CTX-M-1 (688bp).



Fig 7: PCR amplification of bla-CTX-M-2 gene (404bp). M: 1Kb ladder, Lane 5 positive for bla-CTX-M-2 gene (404bp), Lane 1, 2, 3 and 4 negative for bla-CTX-M-2 gene, Lane 6 negative control.



Fig 8: PCR amplification of bla-CTX-M-9 gene (561bp). M: 1 Kb ladder, Lane 1, 2, 3, 5, 7, 8, 9, 10 and 11 positive for bla-CTX-M-9 gene (561bp), Lane 4 and 6 negative, Lane 12 negative control.



Fig 9: PCR amplification of bla-TEM gene (800bp). M: 1kb ladder, Lane 1, 2, 3, 4, and 5 positive for bla-TEM gene, Lane 6 and 7 negative for bla-TEM gene.



Fig 10: PCR amplification of bla-SHV gene (392bp). M: 1kb ladder, ladder 2, 4 and 5 positive for bla-SHV gene (392), 1 and 2 negative for bla-SHV gene, Lane 6 negative control.



Table 4: Prevalence of ESBL genes among ESBL positive isolates.



Table 5: Distribution of ESBL genes according to organisms.



Antimicrobial resistance is currently a most serious problem that received the attention of larger scientific community across the world. In present study, antimicrobial susceptibility test (AST) of all ESBL positive isolates was performed against 20 antibiotics of 12 different classes. All isolates of E. coli and Klebsiella spp. were found (70-100%) resistant to 3rd, 4th generation cephalosporins and ampicillin (Table 6). The plausible factors for high degree of resistance against these antibiotics might be due to persistent antibiotic pressure or acquired from horizontal transmission. Susceptibility pattern of these isolates varied with different classes of non-β-lactam antibiotics. E. coli isolates were found 98.83% to 100% sensitive against chloramphenicol, polypeptides and aminoglycosides classes respectively while Klebsiella spp. was found 85.7% to 100% against chloramphenicol, polypeptides and aminoglycosides (Table 6). There are abundant evidences that corroborate with this finding in India and abroad for both E. coli and Klebsiella spp. isolated from bovine (Batabyal et al.,2018 ; Gupta et al., 2019; Ghatak et al., 2013; Ibrahim et al., 2018). In this study resistance to carbapenem antibiotics also reported, even though these antibiotics are not used in animal husbandry practices across the country, this may be attributed as a result of clinical use in human medicine and transfer of these resistant genes to zoonotic pathogens (Bhardwaj et al., 2015).

Table 6: AMR pattern of ESBL positive E. coli and Klebsiella spp. Isolates.



Presently, Multi-drug resistant (MDR) isolates is a cause of concern, they may possess severe health complications by limiting the treatment options. In this study 81.52% isolates were found to be MDR i.e. resistant to at least one antibiotic of three or more classes of antimicrobials, which highlighted the potential threat by limiting the therapeutic options.
Present study highlighted the prevalence of ESBL producing bacteria in eastern plain zone of Uttar Pradesh, India. Larger proportion of E. coli and Klebsiella spp. (81.52%) was found to be MDR. Despite this some isolates of E. coli and Klebsiella spp. also exhibited resistance against carbapenems, even without its use in animal husbandry practices, which is not a good sign from public health point of view. Taking into consideration, this emerging drug resistance, the practice of routine ESBL testing along with conventional antibiogram would be useful for all cases, which will help in appropriate selection of antibiotic and also prevent further development of AMR. This study also reveals high prevalence of bla-CTX-M gene in this area which will help in reliable epidemiological investigation of AMR. These enzymes may be chromosomal or plasmid mediated which play important role in the dissemination of antimicrobial drug resistance in health care settings. Therefore, continuous monitoring of resistance genes against these antibiotics in livestock is warranted. 
The author is thankful to Dean, College of Veterinary Science and Animal Husbandry, Kumarganj and livestock owners of the Ayodhya and Sultanpur districts for their kind support during collection of samples.
None

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