Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 56 issue 12 (december 2022) : 1557-1561

Molecular Detection of Carbapenem Resistant Gram Negative Bacterial Isolates from Dogs 

Surya Sankar1,*, Thresia1, Anu Bosewell1, M. Mini1
1Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy-680 651, Kerala, India.
Cite article:- Sankar Surya, Thresia, Bosewell Anu, Mini M. (2022). Molecular Detection of Carbapenem Resistant Gram Negative Bacterial Isolates from Dogs . Indian Journal of Animal Research. 56(12): 1557-1561. doi: 10.18805/IJAR.B-4297.
Background: Carbapenems are beta-lactam antibiotics that are considered as the last line of therapy against multidrug resistant extended spectrum beta-lactamase. The resistance to carbapenems predominantly through carbapenemase is one of the most important emerging health problems worldwide in the therapy of clinical infections. The objective of the present study is to determine the presence of carbapenemase encoding genes among Gram- negative bacterial spp. associated with clinical infections in dogs. 

Methods: 30 Escherichia coli, 11 Klebsiella pneumoniae and three Pseudomonas aeruginosa isolated from urine, swabs from lesional skin and anterior vagina of dogs presented with different clinical ailments formed the samples for the study. Polymerase chain reaction was carried out to detect the presence of carbapenemase encoding genes viz., KPC, NDM, OXA, VIM and IMP among the isolates.

Result: Out of the 44 Gram- negative isolates tested, 28 (76.3%) were positive for at least one tested carbapenemase gene. The highest frequency of carbapenemase recorded was for NDM followed by OXA-181, KPC, OXA-48 and VIM. Our study identified a high prevalence of carbapenemases among companion animals like dogs which could act as potential source of transmission of these resistance bacteria or their genomes to humans.
Antibiotic resistance is an emerging threat globally. Multidrug resistant (MDR) extended spectrum beta-lactmase (ESBL) producing Gram-negative bacterial spp. are becoming a major concern in the therapy of clinical infections in both human and Veterinary sector. Carbapenem group of antibiotics are considered as the last choice against ESBL producing Enterobacteriaceae. This group of drug is known to be stable to hydrolysis by beta-lactamase and the nature of its chemical structure permits the drug for easy entrance through porin channel into bacteria (Mulla et al., 2011). Resistance to carbapenem occur due to the presence of carbapenem hydrolyzing beta-lactamase called carbapenemase or combination of some mutations along with the production of other beta lactamase such as the ESBL/AmpCs with the loss of porins and/or active efflux pumps. Most widespread mechanism of resistance is due to the presence of carabapenemases which may be chromosomal or plasmid encoded or associated with various mobile genetic structures (insertion sequences, integrons, transposons), enhancing their spread (Mohanty et al., 2017). They includes Klebsiella pnuemoniae carbapenemase (KPC), New Delhi metallobeta-lactase-1(NDM-1), Oxacillinase (OXA), Verona integron encoded metallobeta-lactamase (VIM) and Imipenemase (IMP). The emergence of carbapenem resistant Enterobacteriaceae is a rapidly evolving global public health dilemma and calls for urgent action within the international scientific community. Production of KPC and NDM-1 is currently the most common resistance mechanism encountered (Little et al., 2012; Ahmed-Bentley et al., 2013; Rolain and Cornaglia, 2014; Codjoe and Donkor, 2018). Various studies in different parts of the world have now established the increasing prevalence of carbapenemase in animals (Abraham et al., 2014; Gentilin et al., 2018; Bourafa et al., 2018). The reports on the detection of carbapenemase among animals or products of animal origin in India in scanty (Singh et al., 2012; Ghatak et al., 2013; Bandyopadhyay et al., 2015; Naik et al., 2015; Pruthvishree et al., 2017). There were no reports on the prevalence of these resistance genes among companion animals like dogs in Kerala. Since the number of companion animals in veterinary practice is on rise now and they could act as potential source for the transfer of multidrug resistant ESBL producing bacteria or their resistance genes to humans (Queenan and Bush, 2007; Johnson et al., 2008; Kock. 2018; Hong et al., 2020), the present study was envisaged to detect their presence among dogs in Kerala employing polymerase chain reaction (PCR) assays.
Bacterial isolates
A total of 100 clinical samples obtained over a period of six months formed the materials for the study. The samples included in the study were urine, swabs from lesional skin and anterior vagina of various dogs. Gram-negative bacilli isolated from these samples were subjected to cultural, morphological and biochemical characterisation as per Quinn et al., (1994). The isolates obtained included 30 Escherichia coli, 11 Klebsiella pneumoniae and three Pseudomonas aeruginosa and were subjected to DNA extraction using HiPurA Multi-Sample DNA Purification Kit.
Standardisation of PCR conditions
The standardised conditions of PCR for different genes was initial denaturation at 95°C for 3 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing temperature at 65°C for NDM, 61°C for KPC, 60°C for IMP, 52°C for VIM and 55°C for OXA for 45 sec followed by extension at 72°C for 45 sec and final extension at 72°C for 5 min. One negative control without template DNA was included to monitor any contamination. Two similar reactions using Staphylococcus aureus genomic DNA as templates was set up to determine the specificity of the primers. Corresponding positive controls were also set up for each set of PCR reactions. The sequences of the primers used for amplification were given in Table 1. The PCR amplicons were further subjected to agarose gel electrophoresis.

Table 1: Sequences of the primers used for polymerase chain reaction.

Carbapenems are beta-lactam antimicrobial agents with a broad in vitro spectrum against Gram-negative, many Gram-positive and anaerobic bacteria (Zhanel et al., 2007). They are considered as one of the most critically important antimicrobials for human treatment. Worldwide, the emergence and global spread of microorganisms with acquired carbapenemases is of great concern. The reservoirs for such organisms are increasing, not only in hospitals, but also in the community and environment. Recent studies have highlighted the presence of such organisms in livestock, companion animals and wildlife (Abraham et al., 2014). The detection of carbapenemase producing Escherichia coli, Salmonella spp. (VIM-1 producers) and Acinetobacter spp. (producing OXA-23 and NDM-1) in livestock animals (poultry, cattle and swine) and their environment have been reported (Fischer et al., 2012, 2013; Poirel et al., 2012; Zhang et al., 2013). In addition, the isolation of NDM-1 producing E. coli, OXA-48 in E. coli and K. pneumoniae or OXA-23 in Acinetobacter spp. from companion animals (cats, dogs or horses) has also been observed (Smet et al., 2012; Shaheen et al., 2013; Stolle et al., 2013).
Carbapenems are the last sort drugs against MDR, ESBL producing Gram-negative bacterial spp. and their prescription is increasing in humans due to the increased prevalence of ESBL and other multi resistant organisms (Papp-Wallace et al., 2011; Patel and Bonomo, 2013; Woodford et al., 2014). Considering the close contact between humans and their pets and the potential for cross-species transmission, the emergence of carbapenem resistance in companion animals is a public health concern. The discovery of carbapenem resistance in companion animals could possibly be linked to their widespread use in humans or may be through environment. it may also be attributed to their use in veterinary practice and even though carbapenems are not registered for use in animals in any major jurisdiction, off-label veterinary use of this critical ‘last-line’ antimicrobial class has been reported in dogs for the treatment of urinary tract infection (UTI) and postoperative infection caused by multidrug-resistant E. coli (Gibson et al., 2008). Since these drugs are not currently licensed, for use in Veterinary sector, resistance to carbapenems is not routinely evaluated in animal isolates, so it seems likely that its prevalence is underestimated. The great increase and spread of carbapenemase producing enterobacteriacea from human sources and its recent isolation from animals may reflect an emerging problem in human and veterinary medicine, as interspecies transmission may occur between humans and companion animals within the same household (Johnson et al., 2008; Livermore, 2012; Meletis, 2016).
Previous research works conducted in our institute have identified a high prevalence of MDR, ESBL producing Gram-negative bacteria among the companion animals like dogs (Paulson et al., 2019). Since no studies have been conducted so far in Kerala in this regard, we have envisaged a pilot study to detect the prevalence of major carbapenemase encoding genes among the companion animals in Kerala. In the present study, out of 100 samples collected from dogs with different clinical ailments, 44 Gram-negative isolates could be obtained which includes 30 E. coli, 11 K. pneumoniae and 3 P. aeruginosa based on cultural, morphological and biochemical characteristics.
Polymerase chain reaction assays were standardised to detect the presence of common carbapenemase in animals which includes NDM-1, KPC, OXA-181, OXA-48, VIM and IMP.  The highest prevalence obtained was that for NDM in 23 isolates (52.27%; 18 E. coli, 3 K. pneumoniae, 2 P. aeruginosa) followed by OXA-181 in 10 (22.73%; 7 E. coli, 2 K. pneumoniae, 1 P. aeruginosa), KPC in 8 (18.18%; 7 E. coli, 1 K. pneumoniae) OXA-48 in 6 (13.64%; 1 E. coli, 4 K. pneumoniae, 1 P. aeruginosa) and VIM in 2 (4.55%; 2 E. coli) (Fig 1,2,3,4 and 5). None of the isolates revealed positive amplicons for IMP genes. As per Bartolini et al., (2014),  NDM and KPC were the predominant carbapenemase conferring resistance among Gram negative bacterial spp. isolated from human and reported now worldwide, emerging as a health crisis in treatment (Diene and Rolain, 2014). In the present study we recorded a high prevalence of NDM followed by OXA-181 in dogs. This is in accordance with other studies in companion animals where the most detected carbapenemase were NDM and OXA (Smet et al., 2012; Shaheen et al., 2013; Stolle et al., 2013; Gonzalez-Torralba et al., 2016).The results are pointing to an impending threat in the therapy of clinical infections in both human and veterinary sector. Hence, there is an urgent need to curb the irrational and excessive use of antibiotics in both human and veterinary sector. The already established national policies and guidelines might be the beginning to combat this problem. Regular and systematic screening using modern molecular and culture-based methods on the prevalence of carbapenem resistance is required because of the variety of bacterial species and the different genetic elements involved. Transmission of resistance bacteria/genetic elements between animals and humans in either direction to be investigated thoroughly for an evidence-based public health risk assessment (Daniel-Haardt et al., 2018). The present study could be a pilot one in the state in this regard.

Fig 1: Agarose gel electrophoresed image of the amplicons of polymerase chain reaction targeting NDM-1.


Fig 2: Agarose gel electrophoresed image of the amplicons of polymerase chain reaction targeting OXA-48.


Fig 3: Agarose gel electrophoresed image of the amplicons of polymerase chain reaction targeting KPC.


Fig 4: Agarose gel electrophoresed image of the amplicons of polymerase chain reaction targeting OXA-48.


Fig 5: Agarose gel electrophoresed image of the amplicons of polymerase chain reaction targeting VIM.

Carbapenems are the last resort drugs against MDR extended spectrum beta-lactamase producing Gram-negative bacterial organisms. The present study identified a high prevalence of carbapenem resistance among companion animals, which indicated an impending crisis in therapy of clinical infections, because they could act as potential source of transmission of resistance to humans. Therefore, firm control on infection and surveillance measures combined with prudent use of antibiotics in both human and veterinary practice is essential to control the spread of resistance against carbapenems.
We are thankful to the Kerala Veterinary and Animal Sciences University for providing the facilities for the conduct of research.

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