ASSESSMENT OF ANTIBIOTIC RESISTANCE PATTERN AND BIOFILM FORMATION IN Pseudomonas aeruginosa ISOLATES FROM BUFFALOES AND DOGS
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Date
2022-03
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
The current research is conducted to assess the antibiotic resistance pattern and
biofilm formation ability in Pseudomonas aeruginosa isolates from the clinical samples
of buffaloes and dogs. Samples were inoculated in Brain Heart Infusion (BHI) broth and
cultured on Pseudomonas isolation agar. Based on pigment production and biochemical
tests (catalase, oxidase, IMViC), 52 samples (34.8%) out of 149 collected in the present
study were provisionally confirmed for Pseudomonas species. The isolates were further
confirmed by Polymerase Chain Reaction (PCR) assay with Pseudomonas aeruginosa
species-specific primers (16S rRNA) and all the 52 isolates yielded specific PCR
product of 956 bp.
One of the main objectives of the present study, antibiotic resistance pattern was
determined by subjecting the PCR confirmed 52 P. aeruginosa isolates to 12 different
antibiotic discs in vitro by Kirby Bauer method. The results of the test revealed that
co-trimoxazole was highly resistant (98.07%), followed by ampicillin (96.15%),
clindamycin (92.30%), oxytetracycline (88.46%) and amoxycillin (84.61%).
Streptomycin (75%), ceftriaxone (50%) and norfloxacin (36.53%) were moderately
resistant. Ciprofloxacin (5.76%), enrofloxacin (9.61%), amikacin (11.53%) and
gentamicin (21.15%) were least resistant than others. The Multiple Antibiotic resistance
(MAR) indices of P. aeruginosa isolates ranged from 0.33 to 0.91 in the current study,
51 isolates were confirmed as Multi Drug Resistance (MDR) and 1 isolate was found to
be Extensively Drug Resistance (XDR).
Phenotypic screening for extended spectrum β-lactamases production was
carried by employing phenotypic screening test (PST). Isolates were tested with four
cephalosporin antibiotics (cefotaxime, ceftazidime, ceftriaxone, and aztreonam). Forty
seven out of the 52 PCR confirmed Pseudomonas aeruginosa isolates were found to be
resistant against one or more cephalosporin discs, 44 isolates were resistant against
cefotaxime, 26 were resistant against ceftriaxone, 9 were resistant to ceftazidime and 5
against aztreonam. Hence they were considered as ‘suspect ESBL producers’ as defined
by the CLSI (2018) guidelines. Thereafter, the suspect ESBL producers were confirmed
for Extended Spectrum β Lactamases (ESBL) presence by phenotypic confirmatory
tests (PCTs). ESBL production was confirmed in 42 isolates by combination disc
method (CDM) and 37 isolates exhibited double disc synergy in double disk synergy
test (DDST).
Further, all the 52 isolates were tested for genetic determinants of antibiotic
resistance with oligonucleotide primers specific for extended spectrum genes (blaOXA,
blaSHV, blaTEM). A PCR product of 564 bp, 713 bp and 800 bp size was yielded for
blaSHV, blaOXA and blaTEM genes, respectively.
Out of 52 isolates of Pseudomonas aeruginosa, 10 isolates were positive only
for blaOXA, 9 isolates were positive only for blaSHV, 3 isolates were positive only for
blaTEM. Both blaSHV and bla OXA were observed in 5 isolates (9.61 %), both blaSHV and
blaTEM were observed in 3 isolates (5.76 %), whereas blaOXA and blaTEM together were
observed in 6 isolates (11.50 %). All the three genes blaSHV, blaOXA, blaTEM were
observed in 3 isolates (5.67 %). No ESBL genes were observed in 13 isolates.
The biofilm formation ability was assessed by processing the P. aeruginosa
isolates through congo red agar method (CRA) and tube method. CRA method detected
that 7 isolates (13.46%) were weak, 26 (50%) as moderate and 19 (36.53%) isolates to
be strong biofilm producers, whereas, in tube method 11 (21.15%) isolates were
detected weak, 18 (34.61%) moderate, 23 (44.23%) as strong biofilm producers.
In conclusion, the current study investigated the presence of genetic
determinants of multidrug resistant P. aeruginosa isolates from clinical samples of
buffaloes and dogs in Andhra Pradesh. There is a great necessity to pursue further
research on multi-drug and extended drug resistance in
Pseudomonas aeruginosa isolates from dairy and companion animals
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