Genetic diversity of Ichthyophthirius multifiliis (Fouquet, 1876) infecting farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) in Turkey.

We assessed genetic diversities among Ichthyophthirius multifiliis (Ich) field isolates collected from farmed rainbow trout (Oncorhynchus mykiss) in Turkey. The overall prevalence of Ich was 35.3% (634/1798). Five novel Ich genotypes (ImulTR1 and ImulTR3-ImulTR6) were described based on mitochondrial cox-1 and nad1_b genes. The remaining genotype ImulTR2 was identical to the previously reported NY3 (or Ark9 and TW7) genotype from the United States and South Asia. Phylogenetic analysis indicated Turkish Ich isolates separated genetically into at least four distinct groups. Our study presents the first data on the genotypes of Ich in Turkey. We also provide evidence for the wide distribution of the NY3 genotype (or Ark9 and TW7) from the United States and South Asia to Turkey. Genetic diversities within the mitochondrial genes provided adequate resolution for describing novel genotypes and identifying the known genotype within Turkish Ich isolates. Description of the Ich genotypes allows for tracking of pathogen genotypes worldwide. Thus, we can better understand the connections between Ich outbreaks in the fisheries aquaculture.

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.

[Detection of the first QnrS gene positivity in aquatic Aeromonas spp. isolates in Turkey]. / Türkiye'de su kaynakli Aeromonas spp. izolatlarinda saptanan ilk QnrS gen pozitifligi.

Aeromonas spp. are oxidase positive, gram-negative, facultative anaerobic bacilli that are widely distributed in aquatic environments. A.hydrophila, A.sobria and A.bestiarum may cause severe infections in both human and cold-blooded animals. Environmental persistance of quinolones that are widely used in both human and veterinary medicine plays an important role in the selection of resistant mutants. Plasmid-mediated resistance is one of the main mechanisms involved in quinolone resistance, and qnr, qepA, aac(6')-Ib-cr, oqxAB genes are identified as resistance determinants. Determination of various types of qnr gene in different bacteria mainly in Enterobacteriaceae, suggests that they are widely distributed in nature. Recently, plasmid-mediated quinolone resistance was defined among Aeromonas species isolated from water. The aim of this study was to investigate the presence of qnr genes among aquatic Aeromonas spp. in Turkey. A total of 45 Aeromonas strains isolated from water and fishes collected from three different geographical regions (Aegean, Mediterranean and Blacksea) in Turkey, were included in the study. The isolates were identified at species level by the use of 16S rDNA-RFLP (Restriction fragment length polymorphism) analysis and multiplex polymerase chain reaction (M-PCR). Among the isolates, 20 were identified as A.sobria, 10 as A.hydrophila, nine as A.salmonicida, four as A.bestiarum and two as A.veronii. The plasmid-mediated quinolone resistance determinants, qnrA, qnrB, qnrC and qnrS genes, were investigated by M-PCR, and sequence analysis was performed for nine qnr-positive isolates. According to the sequence analysis of the genes, qnr genes were characterized in six A.sobria, in two A.bestiarum and in one A.hydrophila isolate (9/45; 20%). When the sequence was compared with GenBank database, this gene was found as qnrS2. All qnrS-positive Aeromonas spp. isolates were ciprofloxacin-susceptible, while five of them were resistant to nalidixic acid. This study is the first research about the plasmid-mediated quinolone resistance and the presence of qnrS2 genes among Aeromonas spp. isolated from fishes and water in Turkey. In conclusion, various resistance genes of aquatic bacteria may constitute a potential risk for the transmission of those genes to other bacteria as well as clinical isolates.

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 Turkey's 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 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.

Occurrence and molecular characterization of Hysterothylacium aduncum (Nematoda: Anisakidae) from Merlangius merlangus euxinus and Trachurus trachurus off the Turkish coast of Black Sea.

A total of 286 larval forms of Hysterothylacium aduncum were collected from Merlangius merlangus euxinus and Trachurus trachurus captured at different sites of the Black Sea coast of Turkey. Prevalence of H. aduncum in M. merlangus euxinus and T. trachurus was 37.4 and 29.3 %, respectively. The fourth-stage larvae from M. merlangus euxinus and T. trachurus of H. aduncum were characterized genetically using a molecular approach. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region (ITS-1, 5.8S subunit, ITS-2) was amplified and sequenced. Two isolates of H. aduncum obtained from M. merlangus euxinus and T. trachurus in Black Sea showed a 100 % nucleotide similarity. Pairwise comparison between the entire ITS fragment including ITS-1, 5.8S, ITS-2 sequences of the H. adumcum isolates of M. merlangus euxinus and T. trachurus from Black Sea (Turkey, JX413596-JX413597) and other H. adumcum isolates from Baltic Sea (Poland, AJ937672), North Sea (Denmark, HM598666), Mediterranean Sea (Tunisia, HQ270427), Japan Sea (Japan, AB277826), Adriatic Sea (Croatia, JQ934878), East Greenland Sea, English Channel, Bay of Biscay, Adriatic Sea, and North Sea showed differences ranging from 0.1 to 0.7 %. With the present study, larvae of H. aduncum infecting M. merlangus euxinus and T. trachurus caught off the Black Sea, Turkey were characterized for the first time by sequencing of the ITS rDNA.

Phenotypic and molecular characterization of Yersinia ruckeri isolates from rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) in Turkey.

The aim of the study was the phenotypic and molecular characterization of Yersinia (Y) ruckeri strains, the causative agent of Enteric Redmouth Disease (ERM), by antibiotyping, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of whole cell proteins. For this aim, a total of 97 Y ruckeri isolates were analyzed. The isolates were distinguished into ten antibiotypes and six phenotypes according to their resistance properties and whole cell protein profiles, respectively. Also, a glycoprotein band of approximately 25.5 kDa was observed in all Y ruckeri strains tested. In all strains, six different RAPD types were observed. In conclusion, Y ruckeri strains isolated from rainbow trout of fish farms in Turkey showed variation according to their phenotypic and genotypic characteristics, and the use of these three typing techniques in double and triple combinations could be more useful for discriminating the 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.

Slime production and antibiotic resistance of Enterococcus faecalis isolated from arthritis in chickens.

Slime factor production and antibiotic resistance of 67 Enterococcus faecalis strains isolated from chicken arthritis were investigated in this study. Slime factor productions of enterococci were found as 59.7%. The antibiotic resistances were investigated by testing gentamycin, penicillin, streptomycin, vancomycin, danofloxacin, and enrofloxacin. The resistance rates were found as 62.68%, 76.11%, 67.16%, 13.43%, 47.76%, 43.28%, respectively. For slime factor positive enterococci, the antibiotic resistance rates were found as follows respectively; 82.50%, 87.50%, 92.50%, 17.50%, 72.50%, and 60.00%. In conclusion; the slime factor might play a role as a colonization factor for chicken arthritis and slime factor positive enterococci were found to be more resistant to these antibiotics. The resistance rates between slime factor positive and negative enterococci against the tested antibiotics except for vancomycin were found statistically significant (p<0.05).

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis / Detecção de resistência a meticilina e produção do fator slime por Staphylococcus aureus em mastite bovina

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 methicillinresistant and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slimeproducing 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.

Este estudo objetivou a detecção de Staphylococcus aureus resistente a meticilina e produtor do fator slime em casos de mastite bovina. Um PCR triplex foi otimizado, com alvo no genes 16SrRNA, nuc e mecA para detecção de Staphylococcus spp, S. aureus e resistencia a meticilina, respectivamente. Para detecção das cepas produtoras do fator slime, empregou-se um PCR com alvo nos genes icaA e icaD. No estudo, 59 cepas foram identificadas como S. aureus por testes convencionais e PCR, sendo 13 resistentes a meticilina e quatro positivas para o gene mecA. Embora 22 das 59 cepas tenham sido produtoras do fator slime em Agar Vermelho Congo, no teste PCR somente 15 foram positivas para os genes icaA e icaD. Dezesseis e 38 das 59 cepas foram positivas para os genes icaA e icaD, respectivamente. Somente duas das 59 cepas foram positivas simultaneamente para resistência a meticilina e produção do fator slime, sugerindo falta de correlação entre estas características. Em conclusão, o PCR triplex otimizado neste trabalho mostrou-se ser um método rápido e confiável para detecção de S.aureus meticilina resistente. Por outro lado, somente PCR para os genes icaA e icaD pode não ser suficiente para detectar produção de fator slime e outros estudos com alvo em outros genes ica são necessários para um avaliação correta da produção do fator slime por S. aureus.

[Investigation of biofilm formation and relationship with genotype and antibiotic susceptibility of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis]. / Kistik fibrozlu hastalardan izole edilen Pseudomonas aeruginosa izolatlarinin biyofilm olusturma yeteneklerinin arastirilmasi ve bu özelligin genotip ve antibiyotik duyarliligi ile iliskisinin belirlenmesi.

Pseudomonas aeruginosa is a frequent cause of respiratory infections in cystic fibrosis (CF) patients. P. aeruginosa strains isolated from these patients have often a mucoid phenotype at advanced disease. This mucoid structure contains a dense amount of alginate type polysaccharide which facilitates bacterial attachment to lung epithelia and provides protection from the immune system due to biofilm formation. The aims of this study were to investigate the biofilm formation and the relation of this property with genotype and antibiotic susceptibilities of P. aeruginosa strains isolated from CF patients. The biofilm formation was determined by using the Congo Red agar and Christensen methods. RAPD-PCR (Random amplification of polymorphic DNA polymerase chain reaction) and disc diffusion methods were used for genotyping and antibiotic susceptibility testing, respectively. Biofilm production was found positive in 33.3% (20/60) of P. aeruginosa tested. While 9 of these 20 isolates were of mucoid colony morphotype, among the 40 biofilm negative isolates mucoid colony was detected in 16 of them. RAPD genotyping based on 70% similarity yielded 19 (A-S) clusters and subtypes related to five of these clusters (K1, K2, N1, N2, Q1, Q2, R1, R2, S1, S2) making up a total of 24 genotypes. Nine of these genotypes composed of biofilm positive isolates and 15 were biofilm negative ones. Most of the biofilm positive strains belonged to K1 (n = 5) and K2 (n = 6) genotypes while biofilm negative isolates were in the L (n = 8) and O (n = 7) genotypes. The comparison of antibiotic susceptibilities in both groups revealed no statistically significant difference (p > 0.0%). However, highest rate of resistance was detected for tobramycin and lowest rate for piperacillin/tazobactam. The data obtained from this study indicated that biofilm negative and positive P. aeruginosa isolates clustered in different groups. These results should be supported with larger scale multi-center studies which may provide information about P. aeruginosa dynamics in CF lungs.

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.