Alper Çiftci

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.

Prevalence and Characterization of Coagulase Positive Staphylococci Isolated from Salted Anchovy

A total of 100 salted anchovy samples were used to investigate the prevalence of S. aureus and other coagulase positive Staphylococci (CPS) as well as to determine the methicillin (MR) and antibiotic resistance (AR) profile, the presence of Panton-Valentine leukocidine (PVL) toxin gene (lukS/F-PV), slime factor properties (SFP), and the genotypic relatedness of the isolates. Agar disc diffusion assay (ADDA) and microdilution broth susceptibility test (MDBST) were applied to compare the specificity and sensitivity of the MR detection methods. A total of 41 CPS isolates were detected at the 102 and 103 CFU/g levels in contrast to S. aureus. The 16S rRNA (genus specific) was detected in all the isolates in contrast to nuc (species-specific) and lukS/F-PV genes. A total of 16/41 isolates were found to be MR by using the three methods. Polymerase chain reaction (PCR) assay was a more sensitive and reliable method for the detection of MR isolates. The antibiotic resistance rates were 75.60, 73.17, 51.21, 31.70, 12.19, and 4.87% to penicillin, ampicillin, tetracycline, erythromycin, ciprofloxacin, and clindamycin, respectively. All the isolates were sensitive to gentamicin and vancomycin. The SFP were determined in all the isolates by using Congo Red agar, and 20 different genotypes were determined by using randomly amplified polymorphic DNA (RAPD)-PCR assay.

Evaluation of PCR methods for detection of Brucella strains from culture and tissues

The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

Evaluation of microbial contamination of resilon and gutta-percha cones and their antimicrobial activities

The aims of this study were to determine the microbial contamination of gutta-percha and resilon cones and to evaluate their antimicrobial activities against five different microorganisms (Staphylococcus aureus ATCC 29213, Pseudomonas auroginosa ATCC 27853, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212 and Candida albicans ATCC 10231). Resilon and gutta-percha cones from seven manufacturers were used in this study. Antimicrobial activities of the cones against five different microorganisms were tested by agar disc diffusion method. To detect the bacterial contamination of cones during clinical use, the cones taken from the previously opened packages were placed into tripticase soy agar. Bacterial growth was detected in 3 out of 28 packages of cones. Clinical use of all the cones led to increase in bacterial contamination. ProTaper® and Mtwo® gutta-percha cones were determined to have lowest antibacterial activities against S. aureus and P. aeruginosa strains. The cones may be contaminated right after opening their packages and/or during clinical use. It is recommended that they should be disinfected before placing into the canals.