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Subject: Veterinary Microbiology × Author: BHASKARA RAMARAJU SAGI, S × End date: 2018 × Start date: 2010 × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    DETECTION OF GENETIC DETERMINANTS OF ANTIBIOTIC RESISTANCE AND BIOFILM FORMATION IN Staphylococcus aureusISOLATES FROM BUBALINE MASTITIS
    (SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI – 517 502. (A.P) INDIA, 2014-10) BHASKARA RAMARAJU SAGI, S; Anand Kumar, P (Major); SRINIVASA RAO, T; SRINIVASA RAO, G
    ABSTRACT : Mastitis in buffaloes is the most important cause of economic loss to Indian dairy industry due to its adverse impact on milk production of the milch animals. Different bacterial pathogens are responsible for mastitis. However, Staphylococcus species are widely implicated. Among the Staphylococcus species, Staphylococcus aureus is recognized as the most frequent cause of intramammary infections like mastitis in cows and buffaloes. Rapid and accurate detection of S. aureus is important for controlling mastitis in a herd. Mastitis is the most frequent reason for antimicrobial drug use in dairy herds and as antimicrobial resistance is associated with the improper use of antimicrobial agents, it is important to monitor antimicrobial susceptibility of mastitic pathogens. Detection of antibiotic resistance genes in S. aureus isolates by polymerase chain reaction (PCR) test and also performing phenotypic tests such as antibiotic sensitivity test (ABST), minimal inhitory concentration (MIC) assay and starch-iodide agar test for β-lactamase production with the S. aureus isolates throws light on the antibiotic resistance pattern in S. aureus isolates from bubaline mastitis milk samples. Biofilm formation in S. aureus is correlated to antibiotic resistance. Therefore detection of genes that control biofilm formation (a marker of virulance) and also performing phenotypic tests like modified Congo red agar test and microtitre plate assay for detecting biofilm formation help to identify the strains of S. aureus with biofilm formation ability circulating in the given area. Certain strains of S. aureus secrete Luk M/F’-PV or PVL (Panton Valentin Leukocidin) toxin, a highly active bicomponentleukotoxin pair that causes intensive damage of bovine mammary tissues. These strains have the potential to epidemiologically spread in the community. Therefore detection of pvlgene in S. aureus isolates from bubaline mastitis is useful to identify the strains of S. aureus with pvl gene circulating in the given area. Out of the 71 mastitic milk samples of buffaloes collected during the present study, 64.78% (n=46) of the samples wereprovisionally confirmed for Staphylococcus species based on conventional tests, which were later subjected to confirmation by PCR test. In California Mastitis Test (CMT) out of the 46 mastitic milk samples tested, majority (47.82%, n=22) scored CMT score of 3 indicating severe clinical form of mastitis. With the Staphylococcus genus specific primers targeting 16S rRNA all the 46 samples were confirmed as Staphylococcus species in polymerase chain reaction (PCR) test by yielding specific product of size 228 bp, Whereas 52.17% (n=24) of them were confirmed as S. aureus with species specific primers (Staur 4 and Staur 6) yielding specific PCR product of size 1250 bp. With regard to antibiotic sensitivity of S. aureus isolates in in vitro antibiotic sensitivity test (ABST) with antibiotic discs, 100% of S. aureus isolates (n=24) were found to be resistant to penicillin, 95.8% (n=23) were resistant to methicillin, 66.6% (n=16) were resistant to amoxycillin, 58.3% (n=14) were resistant to ceftriaxone, 29.1% (n=7) were resistant to amoxycillin+clavulanic acid and 4.1% (n=1) was resistant to ceftriaxone+tazobactum. In MIC assays all the 24 S. aureus isolates isolated in the present study were considered as resistant to amoxycillin, 11 isolates (UNG 142, UNG 155, GDV 156, GDV 162, GDV 167, GDV 168, GDV 169, GDV 170, GDV 171, GDV 173 and GDV 174) were considered as resistant to amoxyillin+clavulanic acid and the remaining 13 isolates of S. aureus were considered as susceptible to amoxyillin+clavulanic acid. With the antibiotic ceftriaxone eight S. aureus isolates (UNG 146, UNG 155, GDV 150, GDV 155, GDV 176, GDV 177, GDV 178 and GDV 179) were regarded as susceptible to ceftriaxone and the remaining 16 isolates were considered as moderately resistant as per the performance standards for antimicrobial susceptibility testing of Clinical and Laboratory Standards Institute, (2007). All the 24 isolates of S. aureus were tested for the antibiotic resistance genes blaZ and mecA in PCR test with specific oligonucleotide primers. Out of 24 isolates of S. aureus tested 58.33% isolates (n=14) were positive for blaZ gene and 41.66% isolates (n=10) were positive for mecA. However 41.66% isolates (n=10) were positive for both blaZ and mecAgenes, and 16.66% (n=4) isolates (UNG 142, UNG 151, UNG 152 and GDV 177) were positive for only blaZ. The S. aureus isolates that were positive for mecA gene were also positive for blaZ gene. It is interesting to note that in GDV 171 S. aureus isolate an additional PCR product between 800 to 900 bp was observed in addition to the specific PCR product of 517 bp for blaZ gene, which needs further investigation. Although in phenotypic test (ABST), all the S. aureus isolates (100%) were found to be resistant to penicillin and 95.8% isolates were resistant to methicillin, either blaZ gene or mecA gene was not detected in 10 isolates (UNG 143, UNG 144, UNG 145, UNG 146, GDV 150, GDV155, GDV 170, GDV 176, GDV 178 and GDV 179). Staphylococcus species (other than S. aureus) isolates GDV 152, GDV 158 and GDV 166 also showed the presence of blaZ gene in PCR test with oligonucleotide primers specific to blaZ gene. It is not uncommon to detect the blaZ gene in Staphylococcus species other than S. aureus. The starch iodide agar test employed for detection of β-lactamase produced by S. aureus isolates revealed that except one S. aureus isolate (UNG 146), all other isolates were positive for β-lactamase production. The isolate UNG 146 was also found to be negative for blaZ gene in PCR test. This may be due to failure of phenotypic expression of β-lactamase controlled by other β-lactamase producing genes like blaIand blaR, which needs further investigation.Although 95.8% isolates of S. aureus (n=23) were found resistant to methicillin in ABST, only 41.66% of S. aureus isolates (n=10) viz. UNG 153, UNG 155, GDV 156, GDV 162, GDV167, GDV 168, GDV 169, GDV 171, GDV 173 and GDV 174 were found to be positive for mecA gene in PCR test. However the same S. aureus isolates were positive for both blaZ and mecAgenes. Further investigation is required for the remaining isolates to detect the presence of mecA gene analogue mecC gene, which also confers resistance against methicillin. Results of the biofilm formation by S. aureus isolates indicate that 4 isolates of S. aureus (UNG 146, GDV 156, GDV 174 and GDV 176) were negative for biofilm formation on modified Congo red agar, including by spot inoculation test. Eight isolates of S. aureusviz. (UNG 153, UNG 155, GDV 155, GDV 162, GDV 167, GDV 170, GDV 171 and GDV 178) yielded absorbance value of higher than 1.0 in microtitre plate assay, thus indicating that they are strong in biofilm formation. Six isolates of S. aureus (UNG 144, UNG 152, GDV 168, GDV 169 GDV 177 and GDV 179) yielded absorbance value of 1.0 in microtitre plate assay and also considered as strong for biofilm formation. In their reactivity with oligonucleotide primers specific to icaA gene, none of the S. aureus isolates in the present study showed specific PCR product of 188 bp in PCR test, however two isolates of S. aureus (GDV 171 and GDV 173) yielded PCR product of about 550 bp with the primers specific to icaAgene. In their reactivity with oligonucleotide primers specific to icaD gene, 37.5% (n=9) of isolates of S. aureus viz. UNG 151, UNG 155, GDV 155, GDV 162, GDV 167, GDV 168, GDV 169, GDV 170 and GDV 171 were positive with specific PCR product of 198 bp. Among them, four isolates (GDV 167, GDV 168, GDV 169 and GDV 171) yielded a different PCR product in addition to specific product of 198 bp. However, two S. aureus isolates (GDV 177 & GDV 178) yielded a different size of PCR product (very faint band, other than specific PCR product of 198 bp) on reactivity with icaDspecific primers. The findings of the present study also confirms the correlation between antibiotic resistance and biofilm formation in S. aureus. On reactivity with oligonucleotide primers specific to pvl gene of S. aureus, in the present study 29.16% of S. aureus isolates (n=7) were positive for pvlgene with PCR product of 83 bp. Out of the seven isolates of S. aureus that are positive for pvl gene, the isolates UNG 151 and UNG 152 and GDV 170 had a score of 2 in CMT, whereas the isolates GDV 155, GDV 156, GDV 169 and GDV 171 had a score of 3 in CMT. In conclusion further investigation is required to study the correlation among antibiotic resistance, biofilm formation and virulence factors like pvlleucotoxin of S. aureus that damage mammary tissue so that appropriate therapeutic and control measures may be taken up.