STUDIES ON ANTIBIOTIC RESISTANCE AMONG MAJOR BOVINE MASTITIS PATHOGENS IN ANDHRA PRADESH
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Date
2016-12
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT:
Mastitis remains as a major problem to the dairy industry worldwide, as it
affects the quality and quantity of milk production. In the present study a total of
130 milk samples were collected from clinical (101) and subclinical cases (29) of
bovine mastitis from different regions of Andhra Pradesh. Isolation of causative
bacteria was carried out and a total of 105 bacterial isolates were obtained. The
incidence of Staphylococcus spp. (70/105, 66.66%) was found to be high, followed
by Enterobacter spp. (16/105, 15.23%), E. coli (11/105, 10.47%) and Streptococcus
spp. (8/105, 7.61%). Among the 70 isolates of Staphylococcus spp., 37 were
identified as coagulase positive Staphylococci (CPS) and 33 were identified as
coagulase negative Staphylococci (CoNS) on tube coagulase test and in coagulase
gene PCR. Multiplex PCR carried out for species identification of Staphylococcal
and Streptococcal isolates, confirmed all the 37 CPS isolates as S. aureus, a CoNS
isolate as S. epidermidis and six isolates of Streptococci as St. agalactiae.
The in vitro antibiotic sensitivity test of Staphylococcal isolates revealed
high frequency of resistance to pencilin G (48 isolates, 68.57%) followed by
cefoxitin (35, 50%), oxacillin (24, 34.28%), gentamicin (6, 8.57%), ciprofloxacin (5,
7.14) and ceftriaxone (2, 2.85%). Interestingly, all the isolates were found
susceptible to chloramphenicol. As cefoxitin is used as surrogate for mecA mediated
oxacillin / methicillin resistance, 35 (50%) isolates that showed resistance to
cefoxitin were phenotypically identified as methicillin resistant, out of which 18
were MRSA and 17 were CoNS. In PCR for mecA and mecC genes that confer methicillin resistance in Staphylococci, only ten (10/70, 14.28%) Staphylococcal isolates were found to carry mecA gene. Three of them were S. aureus and the remaining seven were coagulase
negative Staphylococci. The relative frequencies of MRSA and MR-CoNS were
8.1% (3/37) and 21.2% (7/33) respectively. All these mecA positive isolates were
found resistant to cefoxitin which is a surrogate for mecA mediated oxacillin/
methicillin resistance. However, six of these mecA positive isolates were found
susceptible to oxacillin. None of the 70 Staphylococcal isolates carried mecC gene.
Phenotypic resistance was observed in three isolates of St. agalactiae (3/8,
37.5 %), but none was found to carry resistance genes tetO and ermB in PCR.
Out of 27 (11 E. coli and 16 Enterobacter spp.) isolates of coliforms, 14
(14/27, 51.85%) isolates were suspected as ESBL producers as they showed
resistance to any of the 3rd generation non combination cephalosporins tested in
phenotypic screening test. Among these fourteen isolates, only four (4/14, 28.57%)
have shown increased diameter of inhibition zones (≥ 5 mm) with the drugs in
combination with ß- lactamase inhibitors over the individual drugs and hence these
four isolates were phenotypically confirmed as ESBL producers. All the 27 isolates
were susceptible to ertapenem and combination drugs of 3rd generation
cephalosporins with ß-lactamase inhibitors i.e. ceftriaxone + tazobactem,
ceftazidime + clavulanic acid and cefotaxime + clavulanic acid.
Out of 27 isolates of coliforms tested for ESBL genes, six isolates (3 E. coli
and 3 Enterobacter spp.) were found carry SHV gene in m PCR-I. In m PCR-II, an
isolate each of E. coli and Enterobacter spp. were found to carry CTX-M-1 gene and
another isolate of E. coli was found to carry both CTX-M-1 and CTX-M-2 gene.
Hence these three isolates were confirmed as ESBL producers genotypically.
Among the 4 phenotypically confirmed ESBL producers, ESBL genotype
was confirmed only in 3 of them (2 E. coli and 1 Enterobacter spp.) with the
presence of CTX-M genes. The other ESBL isolate didnot carry any of the ESBL
genes.
Results of the present study indicate considerably high levels of antibiotic
resistance among the major bacterial species causing mastitis in cattle and buffaloes.
Hence, it is imperative to go for antibiotic susceptibility testing prior to choosing an
appropriate antibiotic for treatment.
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