Antibiotic resistance in shellfish and major inland pollution sources in the drainage basin of Kamak Bay, Republic of Korea.

Shellfish-growing areas in marine environments are affected by pollutants that mainly originate from land, including streams, domestic wastewater, and the effluents of wastewater treatment plants (WWTPs), which may function as reservoirs of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs). The objective of this study was to identify the occurrence and distribution of antibiotic resistance at five oyster sampling sites and 11 major inland pollution sources in the drainage basin of Kamak Bay, Republic of Korea. Culture-based methods were used to estimate the diversity and abundance of antibiotic-resistant Escherichia coli strains isolated from oysters and major inland pollution sources. The percentages of ARB and multiple antibiotic resistance index values were significantly high in discharge water from small fishing villages without WWTPs. However, the percentages of antibiotic-resistant E. coli isolates from oysters were low, as there was no impact from major inland pollutants. Fourteen ARGs were also quantified from oysters and major inland pollution sources. Although most ARGs except for quinolones were widely distributed in domestic wastewater discharge and effluent from WWTPs, macrolide resistance genes (ermB and msrA) were detected mainly from oysters in Kamak Bay. This study will aid in tracking the sources of antibiotic contamination in shellfish to determine the correlation between shellfish and inland pollution sources.

Level of contamination in the feces of several species at major inland pollution sources in the drainage basin of Yeoja Bay, Republic of Korea.

We assessed the levels of fecal contamination and the originating species of 12 major inland pollutants in the drainage basin of Yeoja Bay. The presence of the human-specific (HF183), ruminant-specific (BacR and Rum-2-Bac), pig-specific (Pig-Bac-2 and Pig-2-Bac), avian-specific (GFD), and gull-specific (Gull2) markers in water samples (n = 34) from 12 inland pollution sources around Yeoja Bay was analyzed. HF183 was detected in 97% of the water samples, and all major inland pollution sources were contaminated with human feces. BacR and Rum-2-Bac were detected in 94% and 11%, respectively, of the water samples. Pig-2-Bac was not detected in the inland pollution sources, but site L5 might be contaminated with swine feces. Gull2 was not detected, whereas GFD was detected in 26% of the water samples. This study highlights the utility of a MST toolbox approach for characterizing the water quality of inland pollution sources and identifying the feces producing species.

Winogradskyella flava sp. nov., isolated from the brown alga, Sargassum fulvellum.

An aerobic, gliding and yellow-pigmented bacterium, designated strain SFD31T, was isolated from brown alga collected from the South Sea, Republic of Korea. Cells were Gram-stain-negative, and catalase- and oxidase-positive. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain SFD31T forms an independent lineage within the genus Winogradskyella. Strain SFD31T was related distantly to Winogradskyella echinorum KMM 6211T (97.9 %, 16S rRNA gene sequence similarity), Winogradskyellalitoriviva KMM 6491T (97.4 %), Winogradskyellapulchriflava EM106T (97.2 %) and Winogradskyellaeckloniae EC29T (96.9 %). The major fatty acids of strain SFD31T were iso-C15 : 0, iso-C15 : 1 G, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and unknown 13.565. The only isoprenoid quinone of the isolate was menaquinone 6. The major polar lipids were phosphatidylethanolamine, four unidentified aminolipids and two unidentified lipids. The DNA G+C content of strain SFD31T was 36.0 mol%. Phenotypic characteristics distinguished strain SFD31T from the related species of the genus Winogradskyella. On the basis of the evidence presented in this study, a novel species, Winogradskyella flava sp. nov., is proposed for strain SFD31T (=KCTC 52348T=JCM 31798T).

An ELISA-on-a-chip biosensor system coupled with immunomagnetic separation for the detection of Vibrio parahaemolyticus within a single working day.

In this study, we constructed a rapid detection system for a foodborne pathogen, Vibrio parahaemolyticus, by using enzyme-linked immunosorbent assay (ELISA)-on-a-chip (EOC) biosensor technology to minimize the risk of infection by the microorganism. The EOC results showed a detection capability of approximately 6.2x10(5) cells per ml, which was significantly higher than that of the conventional rapid test kit. However, this high level of sensitivity required cultivation of the pathogen prior to analysis, which typically exceeded a day. To shorten the test period, we combined the EOC technology with immunomagnetic separation (IMS), which could enhance the sensitivity of the biosensor. IMS was carried out with magnetic particles coated with a monoclonal antibody specific to the microbe. To test the performance of the IMS-EOC method, fish intestine samples were prepared by artificially inoculating less than 1 or 5 CFU/10 g, allowing for enrichment over predetermined times, and analyzing the sample by using the EOC sensor after concentrating the culture 86-fold via IMS. Using this approach, the bacterium was detected after (at most) 9 h, which approximately corresponds to standard working hours. Thus, the IMS-EOC method allowed for the rapid detection of V. parahaemolyticus, which is responsible for foodborne diseases, and this method could be used for early isolation of contaminated foods before distribution.

Fecal source tracking based on fecal coliform concentration and bacterial community structure in the Bong stream, Korea.

Fecal source tracking of the Bong stream, a representative inland pollutant around the drainage basin of Gangjin Bay (an area where shellfish are grown for export), was performed three times in four confluence areas with 13 sampling sites by analyzing fecal coliform concentrations and two types of bacterial community structures. Identification of the origin of major fecal pollution in the area that inflowed simultaneously via several branch streams was difficult using fecal source tracking based on fecal coliform concentration. Bacterial community analyses using high-throughput sequencing showed that the dominant groups in the entire bacterial community at the class level were Beta-, Gamma-, and Alpha-proteobacteria; Flavobacteriia; and Bacteroidia, and the most abundant groups in the Bacteroidales-specific community at the genus level were Prevotella and Bacteroides. Hierarchical clustering and Bray-Curtis dissimilarity analysis for fecal source tracking indicated that the Bacteroidales-specific community was superior in water environments compared with analysis of the entire bacterial community. Conversely, when the degree of fecal pollution in the sample was low, fecal source tracking based on the entire bacterial community was more reliable. These results suggest that fecal source tracking based on bacterial communities is a useful tool for identifying the origin of fecal pollution in a large stream and implementing systematic guidelines for the establishment of an effective management plan to reduce fecal pollution sources.

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda.

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.

Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda.

The full length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity) and Salmonella typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83→Arg). However, an alteration in the QRDR of ParC (Ser84→Ile) following an amino acid substitution in GyrA (Asp87→Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464→Leu) and GyrA (Asp87→Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.

Evaluation of the sensitivity and specificity of primer pairs and the efficiency of RNA extraction procedures to improve noroviral detection from oysters by nested reverse transcription-polymerase chain reaction.

Noroviruses (NoV) are the key cause of acute epidemic gastroenteritis, and oysters harvested from NoV-polluted sea areas are considered as the significant vectors of viral transmission. To improve NoV detection from oyster using nested reverse transcription-polymerase chain reaction (RT-PCR), we evaluated the sensitivity and specificity of previously published primer pairs and the efficiency of different RNA extraction procedures. Among the primer pairs used for RT-PCR, the sensitivity of GIF1/GIR1-GIF2/GIR1 and GIIF1/GIIR1-GIIF2/GIIR1 was higher than that of other primer pairs used in nested RT-PCR for the detection of NoV genogroup I (NoV GI) and NoV GII from both NoV-positive stool suspension and NoV-seeded oyster concentrates, respectively; the resulting products showed neither unspecific bands in the positive samples nor false-positive bands in the negative controls. The extraction of NoV RNA from oyster samples using a QIAamp® Viral RNA Mini kit with a QIAshredder™ Homogenizer pretreatment afforded more efficient recovery (mean recovery for NoV GI and GII, 6.4%) and the procedure was less time consuming (<30 min) than most other RNA extraction procedures. The results of RNA extraction procedure and primer pairs evaluated by nested RT-PCR assay in this study can be useful for monitoring NoV contamination in oysters, which is an indicator of possible public health risks.