Ertan Emek Onuk

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 identification of Anisakis species (Nematoda: Anisakidae) from marine fishes collected in Turkish waters.

Anisakid nematodes are important etiological agents for zoonotic human anisakiasis (or anisakidosis). These parasites in the Turkish waters still remain unexplored. This study aims the molecular identification of Anisakis species in Turkeys coast from Black, Aegean and Mediterranean Sea and specifically to screen for zoonotic species in commonly commercialized a total of 1145 fish belonging to 31 different species using both polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) and sequencing of the ribosomal internal transcribed spacer (ITS) regions and the mitochondrial cytochrome C oxidase subunit II (cox2) gene. A total of 776 Anisakis type I larvae were isolated in 56/1145 (4.8%) fish of 7 species from Turkish waters. The combining all of our results, e.g., morphology, PCR-RFLP, ITS region, and the cox2 gene, conclusively supported the identification of 3 Anisakis spp. taken from marine fish hosts, namely Anisakis pegreffii, Anisakis typica and Anisakis simplex sensu stricto (s.str.)/A. pegreffii hybrid genotype. No Anisakis larvae were isolated from the Black Sea whereas A. pegreffii, A. typica and A. simplex s.str./A. pegreffii hybrid genotype was found in the Aegean Sea and A. pegreffii was only isolated from the Mediterranean Sea. This study represents the first identification of A. typica and A. simplex s.str./A. pegreffii hybrid genotypes from Turkish waters. Moreover, in the present study first record of the presence of A. pegreffii is also reported from Turkish coasts of Aegean and Mediterranean Sea. No zoonotic Anisakis species were found in commonly commercialized 1025 fish belonging to 16 different species from the Black Sea, thus Turkish populations who consume captured fish from the Black Sea may have a less risk of human anisakiasis or allergies. However, the prevalence of larvae were 47.1% and 46% and recognized zoonotic A. pegreffii were identified from the Aegean and Mediterranean Sea coast, suggesting a high threat of anisakiasis or allergies for Turkish populations who consume fish originating in these regions.

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 characterization of Hysterothylacium fabri (Nematoda: Anisakidae) from Zeus faber (Pisces: Zeidae) caught off the Mediterranean coasts of Turkey based on nuclear ribosomal and mitochondrial DNA sequences.

In the present study, Hysterothylacium fabri was found in the coasts of the Mediterranean Sea, Turkey and characterized by sequencing of nuclear (internal transcribed spacer, ITS) and mitochondrial (cytochrome c oxidase subunit 2, cox2) markers. Pairwise comparison between the entire ITS fragment including ITS-1, 5.8S, ITS-2 sequences of the H. fabri isolates from the Mediterranean Sea (Turkey, KC852206) and other H. fabri isolates from the South China Sea (JQ520158), the South Korea waters (JX974558) showed differences ranged from 0.1 and 1.1%. With the present study, H. fabri from the Mediterranean Sea was characterized for the first time by sequencing of the cox2 gene.

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.

Pyrosequencing Analysis of Cryogenically Ground Samples from Primary and Secondary/Persistent Endodontic Infections

Introduction This study aimed to characterize the microbial communities of primary and secondary/persistent endodontic infections using high‐throughput pyrosequencing from the pulverized samples. Methods The roots of 20 extracted human teeth with primary endodontic infection and 20 teeth with secondary/persistent endodontic infection were collected. The outer surfaces of the roots were disinfected, and whole roots were cryopulverized. 16S amplicon pyrosequencing data from the DNA extracted from the pulverized root powders were obtained, and microorganism abundance and diversity were calculated. Data were analyzed using statistical and bioinformatic methods. Results Pyrosequencing analysis resulted a total of 2,606,128 sequences from 40 samples. A total of 15 phyla, 160 genera, and 368 species were detected. No significant difference between primary and secondary/persistent endodontic infections was found regarding the diversity and richness of operational taxonomic units at the phyla, genera, and species levels (P > .005). Conclusions The present study revealed that the microbial diversity of secondary/persistent endodontic infections did not differ than those of primary endodontic infections. A new archaeal species, Candidatus Nitrosoarchaeum limnia, was detected in root canals of 1 patient with primary endodontic infection for the first time. HighlightsMicrobial community profiles of endodontic infections were investigated.Cryopulverized samples were used to represent microbial community structure.Microbial diversity was similar in primary and secondary/persistent infections.Candidatus Nitrosoarchaeum limnia was reported for the first time in a root canal sample.

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.