Diversity and health risk potentials of the Enterococcus population in tropical coastal water impacted by Hurricane Lane.

Hurricane-caused stormwater runoffs transport diverse terrestrial pollutants, adversely impact microbiological water quality, and introduce fecal and other pathogens to coastal water environments. This study investigated the genotypic diversity, phylogenetic composition, antibiotic resistance patterns, and virulence gene repertoire of the Enterococcus population in the Hilo Bay coastal water after the immediate impact of Hurricane Lane. DNA fingerprinting of Enterococcus isolates exhibited large genotypic diversity, while 16S rRNA gene sequencing identified four major species, including E. faecalis (34.7%), E. faecium (22.4%), E. hirae (22.4%), and E. durans (18.4%). Four common enterococcal virulence genes (cylA, esp, asa1, and gelE) were detected in the Enterococcus population, with significant portions of E. durans (33.3%), E. faecalis (41.2%), E. faecium (36.4%), and E. hirae (27.3%) isolates possessing two or more virulence genes. Considerable antibiotic resistance to rifampin, erythromycin, tetracycline, and nitrofurantoin was detected in the Enterococcus population, with one E. durans isolate showing vancomycin resistance. The results indicate considerable health implications associated with Enterococcus spp. in the hurricane-impacted tropical coastal water, illustrating the needs for more comprehensive understanding of the microbiological risks associated with storm-impacted coastal water.

High genetic diversity of Vibrio parahaemolyticus isolated from tidal water and mud of southern coast of South Korea.

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) DNA fingerprinting technique was adapted to examine the genotypic richness and source differentiation of Vibrio parahaemolyticus (n = 1749) isolated from tidal water and mud of southern coast of South Korea. The number of unique genotypes observed from June (163, 51.9%), September (307, 63.9%), December (205, 73.8%) and February (136, 74.7%), indicating a high degree of genetic diversity. Contrary, lower genetic diversity was detected in April (99, 46.8%), including predominant genotypes comprised >30 V. parahaemolyticus isolates. Jackknife analysis indicated that 65.1% tidal water isolates and 87.1% mud isolates were correctly assigned to their source groups. Sixty-nine isolates of pathogenic V. parahaemolyticus were clustered into two groups, separated by sampling month, source of isolation and serogroups. Serotypes O1, O4, O5, O10/O12 and O11 were the dominant serovariants, while serotypes O3/O13 were highly detected in April where there were no pathogenic V. parahaemolyticus isolates. Most of the V. parahaemolyticus isolates were resistant to ampicillin, ceftazidime and sulfamethoxazole. Interestingly, four V. parahaemolyticus isolates resistant to carbepenem did not contain the known carbapenemase-encoding gene, but possess an extended-spectrum ß-lactamase blaTEM.

Emergence of Klebsiella variicola positive for NDM-9, a variant of New Delhi metallo-ß-lactamase, in an urban river in South Korea.

Objectives: To examine the presence of pathogenic bacteria carrying New Delhi metallo-ß-lactamase in the environment and to characterize the genome structures of these strains. Methods: Phenotypic screening of antimicrobial susceptibility and WGS were conducted on three Klebsiella variicola strains possessing NDM-9 isolated from an urban river. Results: Three carbapenem-resistant K. variicola isolated from Gwangju tributary were found to possess bla NDM-9 genes. Antimicrobial susceptibility testing indicated resistance of these strains to aminoglycosides, carbapenems, cephems, folate pathway inhibitors, fosfomycin and penicillins, but susceptibility to fluoroquinolones, phenicols, tetracyclines and miscellaneous agents. WGS revealed that the 108 kb IncFII(Y)-like plasmids carry bla NDM-9 sandwiched between IS 15 for the GJ1 strain, IS 26 for the GJ2 strain, IS 15D1 for the GJ3 strain and IS Vsa3 , and further bracketed by IS 26 and Tn AS3 along with the mercury resistance operon upstream and the class 1 integron composed of gene cassettes of aadA2 , dfrA12 and sul1 downstream. An aph(3')-Ia gene conferring resistance to aminoglycosides is located after the integrons. Chromosomally encoded bla LEN-13 , fosA , aqxA and oqxB genes, as well as plasmid-mediated bla TEM-1B and bla CTX-M-65 encoding ESBL, ant(3')-Ia and mph (A) genes, were also identified. Conclusions: The findings of the present study provide us with the information that NDM-9 has been spreading into the environment. Dissemination of NDM-9 in the environment has raised a health risk alarm as this variant of NDM carries MDR genes with highly transferable mobile genetic elements, increasing the possibility of resistance gene transfer among microorganisms in the environment.

Season-Specific Occurrence of Potentially Pathogenic Vibrio spp. on the Southern Coast of South Korea.

Vibrio species are widely distributed in warm estuarine and coastal environments, and they can infect humans through the consumption of raw and mishandled contaminated seafood. In this study, we aimed to isolate and observe the distribution of enteropathogenic Vibrio spp. from environments of the southern coast of South Korea over a season cycle. A total of 10,983 isolates of Vibrio spp. were obtained from tidal water and mud samples over a 1-year period from five sampling sites along the southwest coast of South Korea. We found that Vibrio alginolyticus (n = 6,262) and Vibrio parahaemolyticus (n = 1,757) were ubiquitous in both tidal water and mud year round, whereas Vibrio cholerae (n = 24) and Vibrio vulnificus (n = 130) were seasonally specific to summer. While all V. cholerae isolates were nontoxigenic (non-O1 and non-O139), more than 88% of V. vulnificus isolates possessed the virulence factor elastolytic protease (encoded by vvp). Interestingly, V. parahaemolyticus, which was omnipresent in all seasons, contained the virulence factors thermostable direct hemolysin (encoded by tdh) and thermostable direct hemolysin-related hemolysin (encoded by trh) in larger amounts in June (29 trh-positive strains) and September (14 tdh-, 36 trh-, and 12 tdh- and trh-positive strains) than in December (4 trh-positive strains) and February (3 tdh-positive strains), and virulence factors were absent from isolates detected in April. To understand why virulence factors were detected only in the warm season and were absent in the cold season although the locations are static, long-term monitoring and particularly seasonal study are necessary. IMPORTANCE: The presence of enteropathogenic Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus), which cause acute diarrheal infection, septicemia, and wound infections upon ingestion through food and water, is usually associated with temperature. The World Health Organization (WHO) has estimated that there are 1.4 to 4.3 million cases and 28,000 to 142,000 deaths per year worldwide caused by cholera disease. In South Korea alone, consumption is as much as 52.4 kg of fish and shellfish per year per capita. Our findings suggested that seasonally specific acceleration of these possible pathogenic Vibrio spp. may threaten seafood safety and increase the risk of illness in South Korea, where local people consume raw fish during warmer months.

Dynamic changes in the population structure of Escherichia coli in the Yeongsan River basin of South Korea.

Although Escherichia coli has been used as an indicator to examine fecal contamination of aquatic environment, it also has been reported to become naturalized to secondary habitats, including soil, water and beach sand. A total of 2880 E. coli isolates obtained from surface water and sediment samples from the Yeongsan River in 2013 were genotyped by using the horizontal fluorophore-enhanced rep-PCR DNA fingerprinting technique. Although different E. coli genotypic groups were observed between surface water and sediments in the dry season, they were mingled and undifferentiated from each other in the rainy season. This indicates that there are frequent sediment resuspension events in the river basin. Moreover, the genotypic composition of the E. coli population in the Yeongsan River basin changes over months and years, implying that genotypic structure of E. coli populations dynamically fluctuates in the river environment. Consequently, our data suggests that the use of E. coli libraries for fecal source tracking needs to be reassessed to account for the changing structure of riverine E. coli populations.

Seasonal and genotypic changes in escherichia coli phylogenetic groups in the Yeongsan River basin of South Korea.

With 3,480 E. coli strains isolated from the Yeongsan River basin, South Korea, correlations between phylogenetic groups and horizontal fluorophore enhanced rep-PCR (HFERP) genotypes were examined, and environmental factors affecting E. coli phylogenetic groups in the river water were determined. Interestingly, multidimentional scaling (MDS) analyses based on HFERP DNA fingerprint data indicated that E. coli in phylogenetic groups A and B1 were uniquely clustered. Results of self-organized maps (SOMs) analyses also indicated that E. coli phylogenetic groups were seasonally affected by water temperature, with greater occurrences of phylogenetic groups A and B1 in low and high temperature seasons, respectively. The presence of E. coli in phylogenetic groups A and B1 were inversely related. Furthermore, redundancy analysis (RDA) revealed that phylogenetic group B1 correlated positively with temperature, strain diversity, and biochemical oxygen demand (BOD) but negatively with phylogenetic group A. Results of this study indicated that while E. coli strains could be clustered based on their genotypes and environment conditions, their phylogenetic groups did not change in relation to the same conditions. The distributional differences of phylogenetic groups among E. coli populations in different environments may be caused by different genomic adaptability and plasticity of E. coli strains belonging to each phylogenetic group. Although several previous studies have reported different E. coli ecological structures depending on their origins, this study is a first description of the specific environmental factors affecting E. coli phylogenetic groups in river water.

Pathogenic Escherichia coli strains producing extended-spectrum ß-lactamases in the Yeongsan River basin of South Korea.

A total of 3564 E. coli isolates obtained from Yeongsan River basin of South Korea were investigated for their production of extended-spectrum ß-lactamases (ESBLs) and potential pathogenicity to better understand the linkage between antibiotic-resistant pathogens in the environment and their public health risks. Interestingly, 60% (53 of 89) of the screen-positive ESBL producers were determined to be potentially one or both of the diarrheagenic and extraintestinal pathogenic (ExPEC) pathotypes, suggesting that trade-off between resistance and virulence of E. coli may not apply to this study. In addition, 67% (60 of 89) of the screen-positive ESBL producers possessed more than one ß-lactamase gene, and most (59 of 63) of the ESBL producers had the CTX-M-14 enzyme, which is the most dominant ESBL and seems to be related to urban anthropogenic activities. About 68% (36 of 53) of the potential pathogenic strains were resistant to more than 2 non-ß-lactam antibiotics. Results from this study indicate that the Yeongsan River basin has been contaminated with antibiotic-resistant and potential pathogenic E. coli strains. While few studies have examined pathogenecity of ESBL-producing bacteria, this study reports the possible public health risk which could be caused by the fecal indicator bacterium itself containing both ESBL genes and virulence factors. This will likely impact the dissemination of potential pathogenic E. coli producing ESBLs in the environment and suggests the need for further investigations of antibiotic-resistant pathogens to prevent public health impacts in the Yeongsan River basin.

Integrated online system for a pyrosequencing-based microbial source tracking method that targets Bacteroidetes 16S rDNA.

Genotypic microbial source tracking (MST) methods are now routinely used to determine sources of fecal contamination impacting waterways. We previously reported the development of a pyrosequencing-based MST method that assigns contamination sources based on shared operational taxonomic units (OTUs) between fecal and environmental bacterial communities. Despite decreasing sequencing costs, pyrosequencing-based MST approaches are not used in routine water quality monitoring studies due in large part to difficulties in handling massive data sets and difficulties in determining sources of fecal contamination. In the studies presented here we describe the development of an online MST tool, PyroMiST ( http://env1.gist.ac.kr/∼aeml/MST.html) that uses total bacterial or Bacteroidetes 16S rDNA pyrosequencing reads to determine fecal contamination of waterways. The program cd-hit was used for OTU assignment and a Perl script was used to calculate the number of shared OTUs. The analyses require only a small number of pyrosequencing reads from environmental samples. Our results indicate that PyroMiST provides a user-friendly web interface for pyrosequence data that significantly reduces analysis time required to determine potential sources of fecal contamination in the environment.