Sang-Jong Kim

Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea.

We investigated the viral contamination of tap water at 11 urban sites in Korea between 1997 and 1998 over a period of 11 months. A total of 23 tap water samples were examined for infectious enteroviruses and adenoviruses by a cell culture technique followed by polymerase chain reaction (PCR) amplification. To identify the recovered viruses, sequence analysis of PCR products was performed. Infectious enteroviruses and adenoviruses were detected in 11 (47.8%) and 9 (39.1%) of the samples, respectively. Both enteroviruses and adenoviruses were detected in five samples (21.7%). The level of viral contamination was quite high, ranging from 2 x 10(-3) to 2.9 x 10(-2) Most Probable Number of Infectious Unit L(-1), far above the recommended virus level in drinking water set by the US EPA. Poliovirus type I derived from vaccine was frequently detected and the remainder comprised coxsackievirus B type or echovirus type 6, which were causative agents of aseptic meningitis in Korea in 1997 and 1998, respectively. Several types of adenovirus were detected in tap water samples and some water samples were found to contain adenoviruses which were closely related to enteric adenovirus types 40 and 41.

Increase in Bacterial Community Diversity in Subsurface Aquifers Receiving Livestock Wastewater Input

ABSTRACT Despite intensive studies of microbial-community diversity, the questions of which kinds of microbial populations are associated with changes in community diversity have not yet been fully solved by molecular approaches. In this study, to investigate the impact of livestock wastewater on changes in the bacterial communities in groundwater, bacterial communities in subsurface aquifers were analyzed by characterizing their 16S rDNA sequences. The similarity coefficients of restriction fragment length polymorphism (RFLP) patterns of the cloned 16S ribosomal DNAs showed that the bacterial communities in livestock wastewater samples were more closely related to those in contaminated aquifer samples. In addition, calculations of community diversity clearly showed that bacterial communities in the livestock wastewater and the contaminated aquifer were much more diverse than those in the uncontaminated aquifer. Thus, the increase in bacterial-community diversity in the contaminated aquifer was assumed to be due to the infiltration of livestock wastewater, containing high concentrations of diverse microbial flora, into the aquifer. Phylogenetic analysis of the sequences from a subset of the RFLP patterns showed that the Cytophaga-Flexibacter-Bacteroidesand low-G+C gram-positive groups originating from livestock wastewater were responsible for the change in the bacterial community in groundwater. This was evidenced by the occurrence of rumen-related sequences not only in the livestock wastewater samples but also in the contaminated-groundwater samples. Rumen-related sequences, therefore, can be used as indicator sequences for fecal contamination of groundwater, particularly from livestock.

Enteric Viruses in Raw Vegetables and Groundwater Used for Irrigation in South Korea

ABSTRACT Raw vegetables irrigated with groundwater that may contain enteric viruses can be associated with food-borne viral disease outbreaks. In this study, we performed reverse transcription-PCR (RT-PCR) and cell culture-PCR to monitor the occurrence of enteric viruses in groundwater samples and in raw vegetables that were cultivated using that groundwater in South Korea. Samples were collected 10 times from three farms located in Gyeonggi Province, South Korea. RT-PCR and cell culture-PCR were performed to detect adenoviruses (AdVs), enteroviruses (EVs), noroviruses (NoVs), and rotaviruses, followed by sequence analyses of the detected strains. Of the 29 groundwater samples and the 30 vegetable samples, five (17%) and three (10%) were positive for enteric viruses, respectively. AdVs were the most frequently detected viruses in four groundwater and three vegetable samples. EVs and NoVs were detected in only one groundwater sample and one spinach sample, respectively. The occurrence of enteric viruses in groundwater and vegetable samples was not correlated with the water temperature and the levels of indicator bacteria, respectively. Phylogenetic analysis indicated that most of the detected AdVs were temporally distributed, irrespective of sample type. Our results indicate that raw vegetables may be contaminated with a broad range of enteric viruses, which may originate from virus-infected farmers and virus-contaminated irrigation water, and these vegetables may act as a potential vector of food-borne viral transmission.

Use of Cell Culture-PCR Assay Based on Combination of A549 and BGMK Cell Lines and Molecular Identification as a Tool To Monitor Infectious Adenoviruses and Enteroviruses in River Water

ABSTRACT Viral contamination in environmental samples can be underestimated because a single cell line might reproduce only some enteric viruses and some enteric viruses do not exhibit apparent cytopathic effects in cell culture. To overcome this problem, we evaluated a cell culture-PCR assay based on a combination of A549 and Buffalo green monkey kidney (BGMK) cell lines as a tool to monitor infectious adenoviruses and enteroviruses in river water. Water samples were collected 10 times at each of four rivers located in Gyeonggi Province, South Korea, and then cultured on group 1 cells (BGMK cells alone) and group 2 cells (BGMK and A549 cells). Reverse transcription and multiplex PCR were performed, followed by phylogenetic analysis of the amplicons. Thirty (75.0%) of the 40 samples were positive for viruses based on cell culture, and the frequency of positive samples grown on group 2 cells (65.0%) was higher than the frequency of positive samples grown on group 1 cells (50.0%). The number of samples positive for adenoviruses was higher with A549 cells (13 samples) than with BGMK cells (one sample); the numbers of samples positive for enteroviruses were similar with both types of cells. By using phylogenetic analysis, adenoviral amplicons were grouped into subgenera A, C, D, and F, and enteroviral amplicons were grouped into coxsackieviruses B3 and B4 and echoviruses 6, 7, and 30, indicating that A549 and BGMK cells were suitable for recovering a wide range of adenoviral and enteroviral types. The cell culture-PCR assay with a combination of A549 and BGMK cells and molecular identification could be a useful tool for monitoring infectious adenoviruses and enteroviruses in aquatic environments.

Heavy contamination of a subsurface aquifer and a stream by livestock wastewater in a stock farming area, Wonju, Korea

A survey of groundwater and stream water quality was undertaken in a stock farming area where livestock wastewater infiltrates into sandy unsaturated zones and saturated bedrock aquifers containing fractures. To determine the degree of contamination and track the effect of livestock wastewater on groundwater and stream water quality, the population of indicator bacteria (total coliforms, fecal coliforms, fecal streptococci, Staphylococcus spp., and sulfite-reducing clostridia) together with relevant physicochemical parameters were monitored along the wastewater flow-pathways over a 19-month period. The stream water was severely contaminated with livestock wastewater. Nearly all physicochemical and bacteriological parameters in the stream water were much greater than those in the groundwater. Nitrate-N concentrations ranged from 10.0 to 20.0 mg l(-1) in boreholes located downstream (site C) from the livestock waste disposal site, while those in the background borehole (W2) were below 1.0 mg l(-1). Densities of indicator bacteria in boreholes at site C were two or three orders of magnitude higher than those in W2 borehole. In boreholes located downstream from the livestock waste disposal site, the concentration of ammonium-N, nitrate-N, and pollution indicator bacteria increased as groundwater level rose due to infiltration of rainwater. In W2 borehole, however, physicochemical parameters and the number of pollution indicator bacteria had no correlation with the groundwater level. Collectively, these results suggest that the deep aquifers were heavily contaminated with infiltrated livestock wastewater, which consequently must be adequately treated to minimize groundwater pollution.

The genetic diversity of human noroviruses detected in river water in Korea.

We studied the genetic diversity of human noroviruses in river waters by RT-nested PCR and phylogenetic analysis. During 2002-2003, water samples were collected from four rivers in Gyeonggi Province, South Korea. Among the 58 samples, 32 (55.2%) and 26 (44.8%) showed positive results with noroviruses belonging to genogroups I (GI) and II (GII), respectively. The phylogenetic analysis grouped 8 and 7 genotypes in GI and GII, respectively. The major types were GI/1, GI/13, and GII/15, and GI/1 and GI/3 were temporarily distributed. Most GI- and GII-grouped strains were closely related to the reference strains from neighboring countries, China and Japan, and GII/4-related strains had similar sequences to strains recognized as worldwide epidemic outbreaks. The strains circulating between countries are of particular concern to the outbreaks of noroviral diseases in Korea and must be periodically monitored in the natural environments.

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples.

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.

Concentration Method for the Detection of Enteric Viruses from Large Volumes of Foods

Enteric viruses are the major cause of outbreaks of foodborne viral disease worldwide, and vegetables and fruits are considered significant vectors of virus transmission. In this study, we compared viral elution concentration methods in strawberry and lettuce and tested the secondary concentration step for concentrating viruses from large volumes of lettuce samples. Among the tested procedures, the combination of a 0.05 M glycine plus 100 mM Tris elution buffer (pH 9.5) and a polyethylene glycol precipitation concentration was most efficient for the detection of norovirus genogroup II from strawberries (50% of samples) and lettuce (2.9% of samples). The secondary concentration step using ultrafiltration devices could be applied to large lettuce samples without any decrease in detection limit and efficiency, and other cultivable enteric viruses including enteroviruses, adenoviruses, and rotaviruses were recovered from lettuce at efficiencies of 11.4, 9.05, and 11.3%, respectively. This method could be useful for detecting enteric viruses in fresh foods.

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.

Physiological, biochemical, and genetic characterization of an alicyclic amine-degrading Mycobacterium sp. strain THO100 isolated from a morpholine-containing culture of activated sewage sludge

Mycobacterium sp. strain THO100 was isolated from a morpholine-containing culture of activated sewage sludge. This strain was able to utilize pyrrolidine, morpholine, piperidine, piperazine, and 1,2,3,6-tetrahydropyridine as the sole sources of carbon, nitrogen, and energy. The degradation pathway of pyrrolidine as the best substrate for cellular growth was proposed based on the assays of substrate-induced cytochrome P450 and constitutive enzyme activities toward 4-aminobutyric acid (GABA) and succinic semialdehyde (SSA). Its 16S ribosomal RNA gene sequence (16S rDNA) was identical to that of Mycobacterium tokaiense ATCC 27282T. The morABC genes responsible for alicyclic amine degradation were nearly identical among different species of Mycobacteria. Remarkably, repetitive sequences at the intergenic spacer (IGS) region between morC and orf1’ were detected by comparison of the nearly identical mor gene cluster regions. Considering the strain activity for alicyclic amine degradation, the deleted 65-bp DNA segment did not significantly alter the open reading frames, and the expression and functions of the P450mor system remained unaltered. In addition, we found a spontaneous deletion of P450mor from another strain HE5 containing the archetypal mor gene cluster, which indicated a possible occurrence of DNA recombination to rearrange the DNA.