STUDIES ON BETA-LACTAMASE ANTIMICROBIAL RESISTANCE IN CANINE MICROBIOTA AND SCREENING OF LACTIC ACID BACTERIA FOR PROBIOTIC ACTION
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Date
2016-12
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
Microbiota plays a central role in host health and disease. Alterations in gut
microbiota can have major consequences, both beneficial and harmful, for host health. In
view of this, rectal swab samples from healthy (92) and diarrhoeic (44) dogs as well as
pus swabs from pyometra (15) and otitis (15) clinical cases were analyzed. Isolation and
identification of canine microbiota was carried out by conventional cultural methods. Gut
microbiota isolated include E. coli (65.2% incidence in healthy versus 75% in diarrhoeic
dogs), Proteus spp. (61.9% vs. 65.9%), Enterobacter spp. (26% vs. 11.3%), Klebsiella
spp. (26% vs. 20.4%) and Pseudomonas spp. (28.2% vs. 45.4%). Microbiota isolated
from pus swabs include Staphylococcus spp. (73.3% incidence in pyometra vs. 100% in
otitis), Pseudomonas spp. (80% vs. 53.3%), Proteus spp. (0% vs. 60%) and E. coli
(46.6% vs. 0%). E. coli isolates were further confirmed by PCR targeting E16S gene and
also sent for serotyping based on ‘O’ antigen. Extensive usage of antibiotics in canine practice may lead to development of antimicrobial resistance in dogs. In view of this, all the isolates obtained in the present study were screened for β-lactamase resistance both phenotypically and genotypically. Overall incidence of β-lactamase antimicrobial resistance in phenotypic screening test was found to be 35.8% (125/349), which includes 54 E. coli, 40 Pseudomonas, 22
Klebsiella and 9 Enterobacter species. Of these 125 isolates, resistance to cefotaxime was
observed in 80.8%, ceftriaxone in 57.6%, ceftazidime in 52% and aztreonam in 26.4% of
isolates. β-lactamase resistance was detected in 34.5 and 42.7% of gut microbiota isolated
from healthy and diarrhoeic dogs, respectively; 46.6 and 12.5% of microbiota isolated
from pyometra and otitis pus samples, respectively. All the Proteus and Staphylococcus
spp. were found to be highly sensitive or intermediately sensitive to β-lactam antibiotics.
Overall incidence of ESBL phenotype in phenotypic confirmatory test was found
to be 14.6% (51/349), with highest incidence detected in E. coli (31%, 31/100) followed
by Klebsiella (21.2%, 7/33) and Pseudomonas (19.6%, 13/66) species. ESBL phenotype
was detected in 12.5 and 17.7% of gut microbiota of healthy and diarrhoeic dogs,
respectively and 33.3% of microbiota of pyometra pus samples.
Detection of β-lactamase genes in canine microbiota was carried out using a set of
three multiplex PCR assays and a single uniplex PCR assay. The overall incidence of β-
lactamase genes in canine microbiota was found to be 57.3% (200/349). Predominant β-
lactamase genes detected in canine microbiota include blaAmpC in E. coli (87%), blaSHV in
Klebsiella (84.8%) and Enterobacter (48.2%), blaOXA in Pseudomonas (66.6%) species.
Majority of the isolates with confirmed ESBL phenotype carried blaCTX-M G1 gene
(72.5%). The blaACC and blaMOX genes were not detected in the canine microbiota.
The efficacy of antibiotics against bacterial infections is decreasing with rise in
antimicrobial resistance, thus, there is a need to search for novel probiotic strains as
potential alternatives to antibiotics. In India, there are no probiotics available for canine
usage, as they are host specific. In view of this, rectal swabs (67) from healthy pups were
analyzed and a total of 49 (73.1%) Lactobacillus isolates were identified based on
morphological, biochemical characteristics and genus specific PCR. Of these 49 isolates,
23 were found to be positive for Group IV; six for Group I, four for Group II and 16 were
found to be negative for Lactobacillus group specific PCR. In vitro antibacterial activity
of canine Lactobacillus isolates on test pathogens like E. coli, Klebsiella and
Enterobacter species have been studied using agar well diffusion assay. Out of 49
Lactobacillus isolates, the supernatants of 20 isolates showed inhibition against majority
of the test pathogens examined. The inhibition zones were large and clear against E. coli
and Klebsiella spp., but limited and hazy zones were observed against Enterobacter spp.
Reduction in antibacterial activity was noticed after neutralization, proteinase K and heat
treatment of supernatants, suggesting that the antibacterial activity might be partly due to
organic acid production and partly due to heat labile antimicrobial proteins.
Nucleotide sequence analysis of genus specific PCR products of 10 selected
Lactobacillus isolates that showed consistently high antibacterial activity revealed
maximum sequence homology with Lactobacillus fermentum strain RCM 14 (for six
isolates), Lactobacillus agilis strain 76CL (for one isolate), Pediococcus acidilactici
strain G4 (for 2 isolates) and Weissella confusa strain 3W (for one isolate).
In conclusion, the present study revealed alarming β-lactamase resistance in
microbiota of dogs in Andhra Pradesh. Therapeutic failures may likely to occur as
resistance to commonly prescribed third generation cephalosporins was observed.
Lactobacillus strains of dog faecal origin were found to have potent in vitro antimicrobial
action. Furthermore, the present study highlighted need for in vivo studies in India to
establish probiotic potential of dog faecal Lactobacillus species in the near future.
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