STUDIES ON BIOFILM FORMING BACTERIAL PATHOGENS OF BOVINE MASTITIS
Loading...
Date
2016-12
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA
Abstract
ABSTRACT:
Bovine mastitis is the most common infectious diseases of dairy cattle which is
economically important in dairy industry all over the world. The main etiological agents
causing bovine mastitis are Staphylococcus aureus, Streptococcus agalactiae and
Escherichia coli. Antimicrobial therapy is commonly implemented for mastitis prevention
and control. Restriction of antimicrobial agents gaining access to the pathogens due to the
impervious nature of the biofilm encased extracellular matrix, plays a pivotal role in
biofilm antimicrobial resistance. Hence, the present study was undertaken to study the
prevalence of biofilm forming bacteria involved in bovine mastitis and evaluation of their
antibiogram profile. A total of 81 mastitis milk samples were investigated in the present study. Out of
these, 67 (82.7%) samples were positive. No pathogens were isolated from 14 (17.3%) milk
samples. A total of 95 organisms were recovered. Staphylococcus species (46.9%) was the
predominant bacteria isolated followed by E. coli (33.3%), Streptococcus species (29.6%),
Pseudomonas species (6.17%) and Listeria monocytogenes (1.23%).All 38 isolates of Staphylococcus spp. and 24 isolates of Streptococcus species were confirmed using genus specific primers. Whereas, 27 isolates of E. coli, 17 isolates of
S. aureus, 12 isolates of Streptococcus uberis, 6 isolates of S. agalactiae, 4 isolates of
Streptococcus dysgalactiae and one isolate of Listeria monocytogenes were confirmed
using species specific oligonucleotide primers.The antibiogram profile indicated that Staphylococcus species, E. coli and
Streptococcus species showed highest resistance to ampicillin, penicillin-G and
ceftriaxone+ sulbactum followed by enrofloxacin, tetracycline and oxacillin and had least
resistance to amoxicillin+ clavulanic acid and gentamicin. Whereas, the antibiogram of
biofilm producing bacterial pathogens revealed more resistance to antibiotics used in the
present study when compared to nonbiofilm producers.The MIC of Staphylococcus isolates ranged from 7.81- 0.49 μg/ml for enrofloxacin,
0.49- 7.81 μg/ml for gentamicin, 0.49- 31.25 μg/ml for amoxicillin, 0.49- 125 μg/ml
penicillin-G, 3.9- 1000 μg/ml for oxacillin and 0.49- 31.25 μg/ml for ceftriaxone. The
observed MIC of E. coli isolates ranged from 0.49- 31.25 μg/ml for gentamicin, 0.49- 125
μg/ml for amoxicillin, 3.9- 1000 μg/ml for penicillin-G, 0.49- 250 μg/ml for nalidixic acid
and 0.49- 1.95 μg/ml for ceftriaxone. All isolates of E. coli were inhibited at a
concentration of 0.49 μg/ml for enrofloxacin.The genotypic antibiotic profile of E. coli and Staphylococcus species was
investigated. Among the 38 isolates of Staphylococcus species, 33 (86.8%) and 28 (73.7%)
were carrying mecA and blaZ genes respectively. Both mecA and blaZ genes were found in
25 (65.8%) isolates of Staphylococcus species. Out of 17 isolates of S. aureus, mecA and
blaZ genes were detected in 13 (76.5%) isolates each respectively. Both mecA and blaZ
genes were found in 10 (58.8%) isolates of S. aureus. Tetracycline resistance genes were
found in 74.07% of E. coli isolates in the present study. Among tetracycline resistance
genes, tetB was found in higher frequency of 51.8%, and tetA was found in 48.1%. Whereas
25.9% isolates carried both tetA and tetB genes. None of the E. coli isolate was positive for
quinolones gene.The biofilm formation of bacterial isolates was detected by using congo red agar
method (CRA), microtitre plate assay (MTPA) and polymerase chain reaction (PCR). In
E. coli, the frequency of biofilm producers was 66.7% when identified by CRA method,
while it was 77.7% using MTPA method and by PCR, 88.8% of isolates were positive for
biofilm gene (luxS). Out of 38 isolates of Staphylococcus species, 17 (44.7%) were
producing biofilm by CRA method, 35 (92.1%) by MTPA. icaA and icaD genes
responsible for biofilm formation in S. aureus were detected in three (17.6%) and seven
(41.2%) of S. aureus isolates respectively. Two (11.76%) strains of S. aureus carried both
icaA and icaD genes. None of the Staphylococcus isolates were positive to icaBC and bap
genes. Out of 24 Streptococcus isolates, 12 (50%) were positive for biofilm production by
CRA method, whereas 19 (79.2%) by MTPA method. Among 12 S. uberis isolates, biofilm
formation was detected in 10 (83.3%) by CRA method, all 12 isolates were biofilm
producers by MTPA method and the luxS gene responsible for biofilm production was
detected in five (41.7%) isolates.The results of nucleotide sequences of biofilm genes of S. aureus (icaD), E. coli
(luxS) and S. uberis (luxS) when blasted with reference strains in NCBI database revealed
98%, 100% and 100% homology respectively.
Description
THESES
Keywords
null