Arzu Findik

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis

This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.

Molecular typing and cdt genes prevalence of Campylobacter jejuni isolates from various sources

The genetic diversity of 168 Campylobacter jejuni isolates originating from human (n = 30), cattle (n = 36), sheep (n = 44), dog (n = 35), and poultry (n = 21) and cdt genes prevalence of the isolates were investigated. To determine the genetic diversity of these strains, random amplified polymorphic DNA–polymerase chain reaction (PCR) using a random primer (M13) was performed. The numbers of genotypes determined in human, cattle, sheep, dog, and poultry isolates were 19, 18, 17, 18, and 6, respectively. To find out the prevalence of cdt genes in C. jejuni isolates simultaneously, a multiplex PCR was performed. The prevalence of the separate cdt genes was found to vary from 69% to 100% for cdtA, 92% to 100% for cdtB, and 39% to 98% for cdtC. These rates without host discriminating were 95%, 98%, and 93% for cdtA, cdtB, and cdtC, respectively. The prevalence of all three cdt genes in strains originating from human, cattle, sheep, dog, and poultry were 87%, 67%, 84%, 89%, and 39%, respectively. These results showed the relatively high genetic heterogeneity and variation of cdt genes among C. jejuni isolates from various sources except for poultry isolates. This study gives baseline data on molecular characterization of C. jejuni strains from different sources.

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.

Molecular typing of Staphylococcus aureus strains from ovine mastitis by pulsed-field gel electrophoresis and polymerase chain reaction based on coagulase and protein A gene polymorphisms

Staphylococcus aureus is one of the most important etiologic agents of ovine mastitis. To develop effective control measures for mastitis, it is important to type S. aureus strains that have considerable genetic heterogeneity. In the current study, 47 S. aureus strains isolated from ovine mastitis were typed by polymerase chain reaction (PCR) based on coagulase (coa) and protein A (spa) polymorphisms and by pulsed-field gel electrophoresis (PFGE). Eight different coa types and 4 spa types were identified by PCR. While the most prevalent coa type was CG2 (42.56%), the spa types S4 and S1 were the most commonly observed (44.68% and 38.29%, respectively). Nineteen different pulsotypes were identified, and 12 of these were represented by a single isolate. Pulsotypes J and K were predominant and each represented 9 isolates (19.14%). All isolates belonging to J and K pulsotypes were CG2. Although all 9 isolates belonging to the J pulsotype were S4, all isolates in the K pulsotype were S1. While PFGE was found to be the best discriminatory technique for distinguishing strains, coa and spa types were found to be in correlation with PFGE types and can be used for quick, preliminary epidemiologic studies for detecting strains that may cause mastitis.