Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 57 issue 1 (january 2023) : 66-73

​Detection of Carbapenemase Genes (bla-NDM, bla-KPC, bla-OXA-48) in Escherichia coli and Klebsiella species, Isolated from Milk Samples of Bovine in Eastern Plain Zone of Uttar Pradesh

Vibha Yadav1,*, Rajesh Kumar Joshi1, Namita Joshi2, Amit Kumar3, Satyavrat Singh4
1Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj-224 229, Ayodhya, 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-224 229, Ayodhya, Uttar Pradesh, India.
3Department of Immunology and Defense Mechanism, College of Biotechnology, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut-250 110, Uttar Pradesh, India.
4Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj-224 229, Ayodhya, Uttar Pradesh, India.
Cite article:- Yadav Vibha, Joshi Kumar Rajesh, Joshi Namita, Kumar Amit, Singh Satyavrat (2023). ​Detection of Carbapenemase Genes (bla-NDM, bla-KPC, bla-OXA-48) in Escherichia coli and Klebsiella species, Isolated from Milk Samples of Bovine in Eastern Plain Zone of Uttar Pradesh . Indian Journal of Animal Research. 57(1): 66-73. doi: 10.18805/IJAR.B-4543.
Background: Among enterobacteria E. coli and Klebsiella spp. are of great concern in health care settings, as these bacteria sometimes may contaminate the milk due to unhygienic practices and poor udder condition which have been associated with various illnesses. Therefore, this study aimed to detect the carbapenem resistant E. coli and Klebsiella spp. of bovine milk origin with regard to the risk of human transfer via the food chain in community.

Methods: Total 240 samples were collected from Ayodhya and Sultanpur districts of Eastern Plain Zone of Uttar Pradesh (India). Confirmation of E. coli and Klebsiella spp. isolates was done by using species specific uidA and 16S rRNA gene, respectively. Then, carbapenemase positive E. coli and Klebsiella spp. were confirmend by DDST, MBL E-strip test and PCR analysis by targeting (bla-NDM, bla-OXA-48 and bla-KPC). Antibiogram of all carbapenemase positive isolates was performed against 20 antibiotics of 12 different classes.

Result: In the present study, total 74(30.83%) isolates were identified including 55(22.92%) E. coli and 19(7.92%) Klebsiella spp. by PCR, out of which 12(16.21%) isolates were confirmed as carbapenemase producers comprising 7(12.72%) E. coli and 5(26.31%) Klebsiella spp by DDST and E-strip. All carbapenemase positive E. coli were found 100% sensitive to polymyxin-B and chloramphenicol, while all Klebsiella spp. were 100% sensitive to amikacin and polymyxin-B. Resistance against imipenem, meropenem, cefotaxime, cefpodoxime, ceftazidime, ceftriazone, aztreonam and ampicillin ranged between 80.0%-100%. All carbapenemase positive isolates were found multidrug resistant. Carbapenemase genes bla-NDM and bla-KPC were detected in E. coli while bla-OXA-48 and bla-KPC were detected in Klebsiella spp.
Carbapenems are one of the most important groups of antimicrobials which are used as a last resort of antibiotics to combat the infection of multidrug resistant (MDR) pathogens. Unfortunately, the use of carbapenems has increased worldwide in past few years (Wang et al., 2015), hence MDR and carbapenemase producing Enterobacteriaceae (CPE) have been increasing globally at a alarming rate.  Resistance to carbapenem is generally mediated by various mechanisms such as loss of outer membrane porins, production of carbapenemase enzymes and over-expressed efflux pump. Majority of bacterial isolates that harbour these enzymes belong to Enterobacteriacae, because Gram negative bacteria producing ESBL enzyme led to higher carbepenem usage in human being which has resulted in the wider occurrence and spread of CPE (Muller et al., 2018) in environment. The spread of these bacteria into many species may occur though sewage and waste water. Among Enterobacteriacae, E. coli and Klebsiella spp. are main environmental pathogens, associated with various illnesses and acute bovine mastitis (Koovapra, 2015). Resistant genes of these antibiotics are frequently located on mobile genetic elements which can be horizontally transferred (Ansari et al., 2018) between the bacteria. CPE among livestock are of great public health concern because as these can be transferred to human and animal via contaminated food and water. Although very few works have been done on CPE in bovine in India and abroad, their occurrence in animal raises concern in particular to risk of transfer to human via food chain, through contact or environment.

Keeping these facts in view, the present study was undertaken to detect the carbapenem resistant genes in E. coli and Klebsiella spp., since, no in-depth study has been done on carbapenemase producing enterobacteria in cattle and buffaloes in Eastern Zone of Uttar Pradesh. It will help the researchers, field veterinarians and policy makers to develop appropriate control strategy for farmers of this region.
Study area
 
The present study was carried out in the Department of Veterinary Microbiology, C.V.Sc. and A.H. Kumarganj, Ayodhya. The samples were collected from Ayodhya and Sultanpur district of Eastern Plain Zone of Uttar Pradesh, India. The study was conducted between August 2019 and June 2020.
 
Sample collection
 
In present study, total 240 milk samples (160 normal and 80 mastitic milk) of cattle and buffaloes were collected from 5 tehsils of Ayodhya and 3 tehsils of Sultanpur district. Sampling consisted of 10 normal and 5 mastitic milk samples from each of the animal from total eight tehsils. California Mastitis Test was used for screening of mastitis. Approximately 5 ml normal and mastitic milk was collected into sterilized test tubes following strict aseptic conditions. All collected samples were transported to bacteriology laboratory of the department under a cold chain for further processing.
 
Isolation and Identification
 
Samples were enriched with 2ml nutrient broth and incubated for 24hrs at 37oC. A loopful of inoculum was taken and directly streaked on MacConkey agar (MLA) plates added with 1 mg/L imipenem and incubated at 37oC for 24 hr. Colonies showing lactose fermenting characteristics were picked up and transferred to nutrient agar slant and incubated at 37oC for 24 hrs. Thereafter, cultures were streaked on Eosine Methylene Blue (EMB) agar plates and colonies showing specific characteristics were identified by the method of Cruickshank et al., (1975). Further identification of the isolates was done by various biochemical and sugar fermentation reaction as per the method of Edward and Ewing (1972).
 
Extraction of genomic DNA
 
The DNA templates were prepared by using snap-chill method as described by Franco et al., (2008).
 
Molecular identification of E. coli and Klebsiella spp
 
All presumptively positive E. coli isolates were confirmed by PCR amplification using species specific uidA and Klebsiella spp. by bacteria specific 16S rRNA gene as per method described by Anbazhagan et al., (2010) and Andersson et al., (2008), respectively (Table 1). PCR reaction was carried out in 25 µl volume that constituted 12.5 µl of 2X EmeraldAmp GT Master Mix, 8.5 µl nuclease free water, 1 µl mixture of the forward and reverse primers (0.5 µl each primer) and 3.0 µl of template DNA. Amplification was performed using thermal cycler (Bio-Rad, USA). The cycling conditions of PCR are mentioned in (Table 1). PCR amplicons were stored at 4oC until electrophoresis.

Table 1: Oligonucleotide primer sequences used for amplification of uidA and 16S rRNA genes and PCR cycling conditions used.


 
Sequencing of PCR Amplicons
 
The E. coli and Klebsiella spp. PCR products (Fig 1, 2) were submitted to Sanger sequencing Biokart India Pvt Ltd, Bengaluru, India and sequences were subjected to Blast Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) in order to confirm the identity of the isolates.

Fig 1: PCR amplification of uidA gene (556 bp). M: 1Kb ladder, Lane 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 positive for uidA gene (556 bp), Lane 1 negative for uidA gene.



Fig 2: PCR amplification of 16S rRNA gene (265 bp). M: 1 Kb ladder, Lane 1, 2, 3, 4, 5, 6 and 7 positive for 16S rRNA (265 bp).


 
Screening of carbapenemase producing isolates
 
All confirmed isolates were subjected to carbapenemase screening using imipenem and meropenem having 10 μg conc. of disc (Hi-Media, India) by disk diffusion method (Bauer et al., 1966). The results were interpreted as per CLSI (2019) guidelines. The isolates showing reduced susceptibility to any one of these agents were further subjected to confirmatory phenotypic tests.
 
Confirmation of carbapenemase positive isolates
 
Two phenotypic methods were used for confirmation of carbapenemase production.
 
Double disc synergy test (DDST)
 
The test was performed by placing the commercially available imipenem disc (10µg) and combination of imipenem + EDTA (10/750 µg) discs (HiMedia, India) at 25 mm apart on Muller Hinton agar (MHA) (HiMedia) plate inoculated with 1.5x108 organisms/ml and incubated at 37oC for 24 hrs (Fig 3). The results were interpreted as per CLSI guidelines (2019).

Fig 3: Double disc synergy test for confirmation of carbapenemase producing E. coli and Klebsiella spp.


 
Minimum inhibitory concentration (MIC) MBL E-test
 
MBL E-strip test was done by placing strip on MHA plate inoculated with 1.5x108 organisms /ml and incubated at 37oC for 24 hrs. Results were interpreted as per CLSI guideline (2019) (Fig 4).

Fig 4: MIC MBL E- strip test for confirmation of carbapenemase producing E. coli. and Klebsiella spp.


 
Detection of carbapenemase genes by PCR assay
 
Isolation of plasmid DNA
 
A single pure colony of each isolates was inoculated into 10 ml Luria-Bertani (LB) broth medium (HiMedia, India) and incubated at 37oC for 12 -15 hrs in a shaking incubator. The pellets were prepared by centrifuging the bacterial suspension at 10,000 rpm for 3 min. Plasmid DNA was isolated using the GeneJet plasmid Miniprep kit (Cat No. #K0503,Thermo Scientific) as per the instruction of the manufacturers.
 
Detection of bla-NDM, bla-KPC and bla-OXA-48 genes
 
Carbapenemase gene detection was carried out in a total reaction volume of 25 µl as per method described by Mushi et al., (2014) for bla-NDM and Dallenne et al., (2010) for bla-KPC and bla-OXA-48 genes. The primer sequence of targeted genes and amplicon sizes are listed in Table 2. 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 ethidium bromide 1 µl (5 μg/ml) in 60 ml and run slowly at 80-100V, 60-70 mA for 1 hrs and the gels were visualized using the UV illuminator (GeNei Bangalore, India). Ladder DNA of 1 kb (Thermo Scientific # SM 0311) was used as a marker for band interpretation.

Table 2: Detail of primers used for molecular characterization of carbapenemase genes in isolates of E. coli and Klebsiella spp.


 
Study of Multi-drug resistance (MDR) pattern
 
All phenotypically confirmed carbapenemase isolates of E. coli and Klebsiella spp. were checked for their multidrug resistance pattern using 20 antibiotics of 12 different classes. It was performed by agar disk diffusion test on MHA using following discs of HiMedia: Aminoglycosides viz. gentamicin (10 µg), amikacine (30 µg), polymyxin (300 unit), 3rd generations cephalosporins viz. cefotaxime (30 µg), cefpodoxime (30 µg), monobactams viz. aztreonam (30 µg), 2nd generation cephalosporins viz. cefoxitin (30 µg), fluoroquinolones viz. enrofloxacin (10 µg), ofloxacine  (2 µg), nalidixic acid (30 µg), penicillins viz. ampicillin (25 µg), tetracyclines (30 µg), carbapenem viz. imipenem (10 µg), meropenem (10 µg), sulphonamide viz. trimethoprim (30 µg), co-tromoxazole (30µg), amoxyclav (20/10 µg), chloramphenicol (30 µg). Isolates were classified as susceptible and resistant based upon interpretation criteria of CLSI (2019) and those showing resistance to at least one antibiotic in three or more classes were defined as MDR.
In this study, total of 240 samples comprising 160 normal and 80 mastitic milk samples were processed for isolation and identification. On the basis of morphological, growth and biochemical characteristics, 29.58% and 9.16% isolates were presumed as E. coli and Klebsiella spp., respectively (Table 3). Thus, larger proportion of these was confirmed as (22.92%) E. coli while 7.92% as Klebsiella spp. through PCR amplification (Fig 2, 3 and Table 3). These findings were found in agreement with the reports of previous worker (Ibrahim et al., 2018; Geser et al., 2012). Comparatively higher isolation rate of E coli in this study can be attributed to high prevalence of E. coli in the GIT flora.

Table 3: Isolation rate of E. coli and Klebsiella spp. in normal and mastitic milk samples of cattle and buffaloes.



To confirm the identity of isolates, gene sequencing was done and uidA gene sequence data indicated that E. coli isolates with Accession No: MW353603 and Accession No: MW353604 possessed great similarities (99.82%) to E. coli strain K-12 (LR881938.1), E. coli ST18 strain (CP060709.1), E. coli O68:H12 strain (CP061758.1) and E. coli strain EC93 (CP061329.1). Klebsiella isolates with Accession No: MW346043 and Accession No: MW346044 were identified as Klebsiella pneumoniae strain K1and Klebsiella pneumoniae strain K3, respectively. These isolates possessed 100% similarities to Klebsiella pneumoniae strain NU-CRE047 (CP025037.1), Klebsiella pneumoniae strain G17SC 16S (KX610833.1) and Klebsiella pneumoniae strain WP5-S18-ESBL-06 (AP022157.1) on gene sequencing of 16S rRNA.

To study the occurrence of carbapenemase producers in milk, total 74 confirmed isolates were selected. On preliminary screening, 21.62% isolates were presumed as carbapenemase producers, out of which 17.56% isolates were found positive using DDST and 16.21% using MBL-E-strip test (Fig 5). There was little difference in the sensitivity of both of the test used for detection of carbapenemase producers and this observation corroborated with the findings of Gupta et al., (2013) and Bora et al., (2014). The finding of present study revealed higher frequency of carbapenemase producers in Klebsiella spp. (26.31%) than in E. coli (12.72%), which has been reported as major carbapenemase producer in previous studies also (Gupta et al., 2013 and Bora et al., 2014). The occurrence of carbapenemase producers was 10.0% (5.0% E. coli and 5.0% Klebsiella spp.) in cattle’s mastitic milk, while 20.0% (12.5% E. coli and 7.5% Klebsiella spp.) in buffaloes’ mastitic milk. The overall prevalence of carbapenemase producers was 5.0% with 2.91% E. coli and 2.08% Klebsiella spp. None of the carbapenemase producers was detected in normal milk (Table 4). There is scanty information on isolation of carbapenemase producers from milk both in India and abroad. In India, Ghatak et al. (2013) have reported very low percentage of carbapenemase positive E. coli in mastitic milk from NEH region while Diab et al., (2017) reported 1.6% Klebsiellae in raw milk samples from Lebanon. Similar to our finding, low percentage of carbapenemase positive E. coli has also been reported by Braun et al., (2016), Webb et al., (2016) and Nirupama et al., (2018) from faecal sample of different species. However, some of the workers have reported higher prevalence of carbapenemase producers among faecal samples ranging between 21.64%- 29.03% from different parts of India (Gupta et al., 2019; Murugan et al., 2019; Pruthvishree et al., 2017). These differences in findings of various co-workers may be due to variations in source, type of samples, animal husbandry practices in geographical locations.

Fig 5: Distribution of carbapenem positive E. coli and Klebsiella spp. isolates according to screening and phenotypic confirmation tests (DDST and MBL-E strip).



Table 4: Occurrence of carbapenemase producing of E. coli and Klebsiella spp. among various sources.



Genotypic analysis of 12 phenotypically confirmed carbapenem resistant isolates (07 E. coli and 05 Klebsiella spp.) was done by targeting carbapenem genes viz. bla-NDM, bla-KPC and bla-OXA-48 (Fig 7, 8, 9). The overall gene distribution study showed 42.85% occurrence of bla-NDM and bla-KPC in E. coli isolates while 20.0% of bla-OXA-48 and bla-KPC in Klebsiella spp. E. coli isolates of cattle harboured both bla-NDM and bla-KPC gene (14.28% each) whereas Klebsiella spp. harboured only bla-OXA-48 gene (20.0%) in mastitic milk. In case of Buffalo, E. coli isolates exhibited bla-NDM and bla-KPC gene (28.57% each), while Klebsiella spp. isolates revealed only bla-KPC gene (20.0%) in mastitic milk (Table 5). It was notable in this study that bla-KPC gene was present in both E. coli and Klebsiella spp. isolates and bla-OXA-48 gene was present only in one isolates of Klebsiella spp. Although occurrence of these genes in animals is very low but it is very significant from public health point of view, as these are increasingly being reported from nosocomial infection in which Klebsiella spp. is most commonly implicated. To the best of our knowledge, this is the first report of bla-KPC and bla-OXA-48 gene detection in mastitic milk of bovine from India. Earlier to our study, only Ghatak et al., (2013) have reported bla-NDM gene in mastitic milk. The occurrence of such genes in milk samples of bovine may be presumed due to horizontal transfer by unclean hands of workers, water supplied for washing of udder and utensils or poor environmental condition. These carbapenemase genes have also been reported in previous studies like bla-NDM-1 and bla-OXA-48 genes in piglets (Pruthvishree et al., 2017; Nirupama et al., 2018), bla-VIM in calves (Murugan et al., 2019), bla-OXA-48 in cattle faeces (Braun et al., 2016) and in milk (Diab et al., 2017) from India and abroad. The detection of these genes in milk sample isolates is a matter of serious concern, because it is very easier for such isolates to enter the food chain leading to serious food-borne illnesses.

Fig 7: PCR amplification of bla-NDM gene (521 bp). M: 1 Kb ladder, Lane 2, 4 and 5 positive for bla-NDM gene (521 bp), Lane 1 and 6 negative for bla-NDM gene.



Fig 8: PCR amplification of bla-KPC gene (538 bp). M: 1 Kb ladder, Lane 3, 4, 5 and 6 positive for bla-KPC gene (538 bp), Lane 1, 2,7 and 8 negative for bla-KPC gene.



Fig 9: PCR amplification of bla-OXA-48 gene (281 bp). M: 1 Kb ladder, Lane 4 positive for bla-OXA-48 gene (281 bp), Lane 1, 2, 3 4, 5, 6 and 7 negative for bla-OXA-48 gene.



Table 5: Distribution of carbapenemase genes according to various sources and organisms.



Antimicrobial resistance is currently a serious global problem that has received the attention of larger scientific community. Antimicrobial susceptibility testing (AST) of carbapenemase positive isolates revealed that all isolates of E. coli and Klebsiella spp. were (80%-100%) resistant to imipenem, meropenem cefotaxime, cefpodoxime, ceftazidime, ceftriazone, aztreonam and ampicillin. The plausible factors for high degree of resistance to these antibiotics may be due to persistent antibiotic pressure or acquired from environmental sources or farm workers. Susceptibility pattern of these isolates differed with different classes of non-β-lactam antibiotics except polymyxin-B for which both isolates were found 100% susceptible (Fig 6). E. coli isolates were found 85.0% to 100% sensitive against gentamicin and chloramphenicol, respectively while Klebsiella spp. was found 80% to 100% sensitive against chloramphenicol and amikacin. There is abundant evidence to corroborate the emergence of resistance against 3rd generation cephalosporins and ampicillin in India and abroad for both E. coli and Klebsiella spp. isolated from milk of bovine origin (Batabyal et al., 2018; Ghatak et al., 2019; Badri et al., 2017; Ibrahim et al., 2018). In this study all carbapenemase producing isolates were found to be multi-drug resistant (MDR) i.e., resistant to three or more classes of antimicrobials. This finding was in concordance with the finding of Bora et al., (2014). The occurrence of MDR in this study highlighted a potential threat by limiting the therapeutic options.

Fig 6: AMR pattern of carbapenemase positive E. coli and Klebsiella spp. isolates of bovine origin.

It is noteworthy from the present findings that carbapenems are not used in animal husbandry practices in this area; even then resistance in animal isolates has been observed which may be attributed to horizontal transfer of resistance gene between human and animal in community setting. Horizontal transfer of such pathogen or genes from one species to another may easily occur in highly populous country like India. Most of the isolates were resistant to 3rd and 4th generation cephalosporins and showed MDR which is cause of concern about Enterobacteriaceae. Therefore, a specific study on rational use of antibiotics and continuous monitoring for resistance gene against these antibiotics in livestock is warranted. Recovery of carbapenemase producers is very worrisome to human health in this area.
The research work was conducted in Veterinary Bacteriology Laboratory of Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Kumarganj and Centre of Excellence in Agriculture Biotechnology, College of Biotechnology, S.V.P.U.A.T., Meerut, U.P. The author is thankful to Dean, College of Veterinary Sciences and Animal Husbandry, Kumarganj and livestock owners of the Ayodhya and Sultanpur districts for their kind support during collection of samples.

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