Bacterial metagenome analysis of Mytilus galloprovincialis collected from Istanbul and Izmir coastal stations of Turkey.

Mytilus galloprovincialis is a marine mollusk belonging to the Bivalvia class. It has been distributed largely in Turkish shores and worldwide aquatic environments. Besides being known as an environmental pollution indicator, it is highly consumed as a food and has a high economic value. Due to their nutritional mechanisms by filtering water, they are affected by pollution in seawater and mussels can host-microbial diversity of environmental origin as well as pathogenic bacteria. Therefore, in this study, bacterial species found in Mediterranean mussels collected from the coastal stations of Istanbul [Rumeli Kavagi (RK), Kucukcekmece (KC)], and Izmir [(Foca (MF), Urla (MU)] were investigated and compared with microbiological and metagenomic analyses. According to microbiological analysis results, 34 mussel-associated Enterobacteriaceae and Vibrionaceae family members were identified. As a result of the culture-independent metagenomic analysis, taxonomic groups for each station were identified and compared based on Operational Taxonomic Unit data. For all stations, the most abundant bacterial genera were the unclassified bacterial genera. The total number of mussel-related total richness identified in all groups was 4889 (RK = 1605; KC = 1930; MF = 1508; and MU = 1125). According to the metagenomic data obtained in this study, different relative amounts of Lachnospiraceae and Bacteroidetes taxa groups were reported for all stations. The pathogenic bacterial genera identified by metagenomic analyses which may be significant for the public health are Arcobacter, Clostridium, Aeromonas, Vibrio, Escherichia_Shigella, Klebsiella, Campylobacter, Helicobacter, Pseudomonas, Morganella, Serratia, Corynebacterium, Enterococcus, Staphylococcus, Yersinia, Mycoplasma, Brucellaceae_unclassified, Pantoea, and Proteus.

The relationship between phylogenetic classification, virulence and antibiotic resistance of extraintestinal pathogenic Escherichia coli in Izmir province, Turkey.

BACKGROUND: Extraintestinal pathogenic Escherichia coli (ExPEC) is an important bacterium and responsible for many bloodstream infections, including urinary tract infections and even fatal bacteremia. The aim of this research was to investigate whether ExPEC strains isolated from Turkish blood cultures have a relationship between 16S rRNA based phylogenetic clusters and antibiotic resistance profiles, virulence factors or clonal lineages. METHODS: Phenotypically identified ExPEC blood culture isolates (n = 104) were included in this study. The 16S rRNA partial sequence analysis was performed for genotypic identification of ExPEC isolates. Antibiotic susceptibility and Extended-Spectrum ß-Lactamase testing of isolates were performed. Phylogenetic classification (A, B1, B2 and D), Multi Locus Sequence Typing analysis and virulence-associated genes were investigated. RESULTS: Based on 16S rRNA partial sequence analysis, 97 out of 104 (93.26%) ExPEC isolates were confirmed as E. coli. Ampicillin (74.22%) and cefuroxime axetil (65.97%) resistances had the highest frequencies among the ExPEC isolates. In terms of phylogenetic classification of ExPEC, D (38.14%, 37/97) was the most prevalent group after A (29.89%, 29/97), B2 (20.61%, 20/97), and B1 (11.34%, 11/97). The sequence types of the 20 ExPEC isolates belonging to the B2 phylogenetic group were analyzed by Multi Locus Sequence Typing. Ten isolates out of 20 (50.0%) were identified as ST131. The other STs were ST95 (n = 1), ST14 (n = 1), ST10 (n = 1), ST69 (n = 1), ST1722 (n = 2), ST141 (n = 1), ST88 (n = 1), ST80 (n = 1), and ST998 (n = 1). Of the ST131 strains, six (60%, 6/10) represented serogroup O25. The most common virulence factor genes were serum resistance factor gene, traT (55.7%) aerobactin siderophore receptor and yersiniabactin encoding genes iutA (45.3%) and fyuA (50.5%), respectively. In addition, PAI (41.2%), iroN (23.7%), hlyA (15.4%), kpsII (13.4%), ompT (13.4%), papG (12.4%), iss (9.3%), cnf1 (7.2%), ibeA (2.06%), and sfaS (2.06%) genes were present in the ExPEC isolates. CONCLUSION: The 16S rRNA-based phylogenetic relationship tree analysis showed that a large cluster was present among 97 ExPEC isolates along with related reference strains. There were 21 main clusters with 32 closely related subclusters. Based on our findings, different clonal lineages of ExPEC can display different antibiotic susceptibilities and virulence properties. We also concluded that virulence factors were not distributed depending on phylogenetic groups (A, B1, B2, and D). The ExPEC isolates belonging to the same phylogenetic group and sequence type could display different resistance and virulence characteristics.

LuxCDE-luxAB-based promoter reporter system to monitor the Yersinia enterocolitica O:3 gene expression in vivo.

It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice.

Investigation of enteropathogenic Escherichia coli and Shiga toxin-producingEscherichia coli associated with hemolytic uremic syndrome in Izmir Province, Turkey.

BACKGROUND/AIM: The purpose of this study was to investigate Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) strains originating from diarrheagenic patients. MATERIALS AND METHODS: A total of 102 patients with diarrhea between October 2012 and January 2013 were enrolled in this study. Multiplex and standard polymerase chain reactions were performed to detect and distinguish STEC and EPEC strains. O serotyping of EPEC was carried out by monovalent antisera. The O and H serotyping of STEC strains was performed at the Refik Saydam Institute, Ankara. RESULTS: A total of 5 (3.42%) strains were identified as STEC, and 3 strains (2.05%) were atypical EPEC. One of the STEC serotypes was O157:H7 carrying VT1, Stx1A, and escv genes. The other STEC strain was identified as O174:H21, which is associated with hemolytic uremic syndrome and consists of VT2 and Stx2A genes. One of the EPEC and three of the STEC serotypes were nontypeable. The serotypes of the atypical EPEC strains were identified as O114 and O26. CONCLUSION: To the best of our knowledge, this is the first report of O174:H21 from the Izmir region that was shown to be a Shiga toxin-producing non-O157 serotype of STEC.

Isolation of pathogenic Yersinia enterocolitica strains from different sources in Izmir region, Turkey.

Yersinia enterocolitica is a foodborne pathogen that is very rarely encountered in Turkey. In this work, several human, porcine, and environmental samples collected from Izmir region in Turkey were examined for the presence of Y. enterocolitica using different cultivation and enrichment methods. A total of nine pathogenic Y. enterocolitica strains were isolated; five strains from pig stool and manure samples and four strains from waste water samples. On the other hand, no Y. enterocolitica was isolated from human diarrheal stool samples (n = 102) and from 12 gulf, canal, municipal pool, and well water samples. Biochemical and serological characterization of the nine Y. enterocolitica strains revealed that they belonged to three different bioserotypes: 4/O:3, 2/O:9, and 2/O:5,27. All the strains were deemed pathogenic based on virulence factor-specific PCR analysis. Detection of pathogenic Y. enterocolitica strains from the pig and waste water samples from the Izmir region indicates that Y. enterocolitica is a potential risk for public health.