Probable Number-polymerase Chain Reaction Research Articles

The Impact of Water Intrusion on Pathogenic Vibrio Species to Inland Brackish Waters of China.

The estuary is the ecological niche of pathogenic Vibrio spp. as it provides abundant organic and inorganic nutrients from seawater and rivers. However, little is known about the ecology of these Vibrio species in the inland brackish water area. In this study, their co-occurrence and relationships to key environmental constraints (salinity and temperature) in the Hun-Tai River of China were examined using the most probable number polymerase chain reaction (MPN-PCR) approach. We hereby report 2-year continuous surveillance based on six water indices of the Hun-Tai River. The results showed that seawater intrusion maximally reached inland as far as 26.5 km for the Hun-Tai River. Pathogenic Vibrio spp. were detected in 21.9% of the water samples. In particular, V. cholerae, V. parahaemolyticus, and V. vulnificus were isolated in 10 (10.4%), 20 (20.8.5%), and 2 (2.08%) samples, respectively. All V. parahaemolyticus strains were tdh gene negative, 10% were positive for the trh gene. Multi-locus sequence typing (MLST) divided V. parahaemolyticus strains into 12 sequence types (STs) for the Hun-Tai River. Five STs were respectively present in various locations along the Hun-Tai River. The PCR assay for detecting six virulence genes and Vibrio seventh pandemic island I and II revealed three genotypes in 12 V. cholerae isolates. The results of our study showed that seawater intrusion and salinity have profound effects on the distribution of pathogenic Vibrio spp. in the inland river, suggesting a potential health risk associated with the waters of the Hun-Tai River used for irrigation and drinking.

Specific detection and quantification of Ralstonia pseudosolanacearum race 4 strains from Zingiberaceae plant cultivation soil by MPN-PCR

The most probable number-polymerase chain reaction (MPN-PCR) method was assessed for the specific detection and quantification of Ralstonia pseudosolanacearum race 4 strains in soil from Zingiberaceae fields in Japan. Based on the genome sequence analysis of two bacterial strains [MAFF 211479 (type I) and MAFF 211472 (type II)] isolated from ginger, race 4 type I- and type II-specific nested PCR primer sets were designed, respectively. The MPN-PCR using these primer sets efficiently detected and quantified the pathogen in naturally and artificially infested soils.

Detection and Quantification of Total and Pathogenic Vibrio parahaemolyticus in Anadara subcrenata in the Zhoushan Archipelago

This study aimed to investigate the prevalence of total and pathogenic Vibrio parahaemolyticus in Anadara subcrenata sampled from aquafarms and retail markets in the Zhoushan islands during June 2013 to March 2015, using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 265 (83.86%) samples with the density 0.3 to 2400 MPN/g. In total, 30.70% and 17.41% of the samples exceeded 100 MPN/g and 1,000 MPN/g, respectively. Both highest positive rate (98.99%) and highest prevalence (median = 210.0 MPN/g) were recorded in summer. Samples from aquafarms had a higher positive rate and median than those from retail markets. Pathogenic V. parahaemolyticus was detected both in aquafarms and retail markets in all seasons but not in winter. Among the 265 tlh-positive samples, 20 (7.55%) of the samples harbored tdh, and 5 (1.89%) of the samples harbored both tdh and trh. These results indicate that the Zhoushan archipelago is severely contaminated with V. parahaemolyticus in Anadara subcrenata; these results are applicable in risk assessment and to control the risk of food-borne disease caused by V. parahaemolyticus.

Characterization of the genes involved in nitrogen cycling in wastewater treatment plants using DNA microarray and most probable number-PCR

To improve nitrogen removal performance of wastewater treatment plants (WWTPs), it is essential to understand the behavior of nitrogen cycling communities, which comprise various microorganisms. This study characterized the quantity and diversity of nitrogen cycling genes in various processes of municipal WWTPs by employing two molecular-based methods:most probable number-polymerase chain reaction (MPN-PCR) and DNA microarray. MPN-PCR analysis revealed that gene quantities were not statistically different among processes, suggesting that conventional activated sludge processes (CAS) are similar to nitrogen removal processes in their ability to retain an adequate population of nitrogen cycling microorganisms. Furthermore, most processes in the WWTPs that were researched shared a pattern:the nirS and the bacterial amoA genes were more abundant than the nirK and archaeal amoA genes, respectively. DNA microarray analysis revealed that several kinds of nitrification and denitrification genes were detected in both CAS and anaerobic-oxic processes (AO), whereas limited genes were detected in nitrogen removal processes. Results of this study suggest that CAS maintains a diverse community of nitrogen cycling microorganisms; moreover, the microbial communities in nitrogen removal processes may be specific. Open image in new window

Detection and Quantification of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Coastal Waters of Guinea-Bissau (West Africa).

V.cholerae, V.parahaemolyticus, and V.vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V.cholerae (ISR 16S-23S rRNA) ranged 0-1.2×10(4) MPN/L, whereas V.parahaemolyticus (toxR) varied from 47.9 to 1.2×10(5) MPN/L. Although the presence of genotypes associated with virulence was found in environmental V.cholerae isolates, ctxA+ V.cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V.parahaemolyticus were detected at concentrations up to 1.2×10(3) MPN/L. V.vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2×10(5) MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.

Temporal and Spatial Variation in the Abundance of Total and Pathogenic Vibrio parahaemolyticus in Shellfish in China.

We investigated the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish sampled from four provinces in China during May 2013 and March 2014 using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 67.7% of 496 samples. A total of 38.1% and 10.1% of samples exceeded 1,000 MPN g-1 and 10,000 MPN g-1, respectively. V. parahaemolyticus densities followed a seasonal and geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels that were 100-fold higher than those of the Liaoning and Shandong regions. Moreover, the levels of V. parahaemolyticus were at least 10-fold higher in the summer and autumn than in the cooler seasons. Pathogenic V. parahaemolyticus levels were generally lower than total V. parahaemolyticus levels by several log units and tended to be high in samples contaminated with high total V. parahaemolyticus levels. The aqua farms had a lower prevalence but higher abundance of total V. parahaemolyticus compared to retail markets. The catering markets showed the lowest levels of total V. parahaemolyticus, but 20.0% of samples exceeded 1,000 MPN g-1. The levels of both total and pathogenic V. parahaemolyticus in oysters were higher than in clams. The log-transformed abundance of V. parahaemolyticus was significantly correlated with both water temperature and air temperature but not water salinity. These results provide baseline contamination data of V. parahaemolyticus in shellfish in China, which can be applied to local risk assessments to prioritize risk control to key sectors and evaluate the effectiveness of future control measures.

MPN 및 H-NS 유전자를 표적으로 하는 PCR assay를 병용한 장염비브리오(Vibrio parahaemolyticus)의 정량

【We applied a combination of most probable number-polymerase chain reaction (MPN-PCR) methods using a PCR procedure targeting the H-NS (VP1133) gene to detect Vibrio parahaemolyticus presence and density in seawater as well as within short-necked clam Ruditapes philippinarum tissues collected from Gomso Bay, Korea. In 30 seawater samples, V. parahaemolyticus levels ranged from less than 1.8 to $1.1{\times}10^3MPN/100mL$ , and samples from August showed higher than those from other months. Furthermore, the levels of V. parahaemolyticus in six short-necked clam samples ranged from $7.8{\times}10^2$ to $2.1{\times}10^3MPN/100g$ , approximately 2.5 times higher than in seawater samples from the corresponding month. Our results provide data on V. parahaemolyticus contamination in seawater and short-necked clam tissues, and help to improve quantitative methods of assessing V. parahaemolytcius levels.】

Sensitive quantitative detection of Ralstonia solanacearum in soil by the most probable number-polymerase chain reaction (MPN-PCR) method

We developed a sensitive quantitative assay for detecting Ralstonia solanacearum in soil by most probable number (MPN) analysis based on bio-PCR results. For development of the detection method, we optimized an elution buffer containing 5g/L skim milk for extracting bacteria from soil and reducing contamination of polymerase inhibitors in soil extracts. Because R. solanacearum can grow in water without any added nutrients, we used a cultivation buffer in the culture step of the bio-PCR that contained only the buffer and antibiotics to suppress the growth of other soil microorganisms. To quantify the bacterial population in soil, the elution buffer was added to 10g soil on a dry weight basis so that the combined weight of buffer, soil, and soil-water was 50g; 5mL of soil extract was assumed to originate from 1g of soil. The soil extract was divided into triplicate aliquots each of 5mL and 500, 50, and 5μL. Each aliquot was diluted with the cultivation buffer and incubated at 35 °C for about 24h. After incubation, 5μL of culture was directly used for nested PCR. The number of aliquots showing positive results was collectively checked against the MPN table. The method could quantify bacterial populations in soil down to 3cfu/10g dried soil and was successfully applied to several types of soil. We applied the method for the quantitative detection of R. solanacearum in horticultural soils, which could quantitatively detect small populations (9.3cfu/g), but the semiselective media were not able to detect the bacteria.

Depth-resolved abundance and diversity of arsenite-oxidizing bacteria in the groundwater of Beimen, a blackfoot disease endemic area of southwestern Taiwan

The role of arsenite oxidizers in natural attenuation of arsenic pollution necessitates studies on their abundance and diversity in arsenic-contaminated aquifers. In this study, most probable number-polymerase chain reaction (MPN-PCR) and denaturing gradient gel electrophoresis (DGGE) was applied to monitor depth-wise abundance and diversity of aerobic arsenite oxidizers in arsenic-enriched groundwater of Beimen, southwestern Taiwan. The results revealed that the abundance of arsenite oxidizers ranged from 0.04 to 0.22, and the lowest ratio was observed in the most arsenic-enriched and comparatively more reduced groundwater (depth 200 m) of Beimen 1. The highest ratio was observed in the less arsenic-enriched and less reduced groundwater (depth 60 m) of Beimen 2B. DGGE profiles showed a shift in diversity of arsenite oxidizers, consisting of members of the Betaproteobacteria (61%), Alphaproteobacteria (28%) and Gammaproteobacteria (11%), depending on mainly arsenic concentration and redox level in groundwater. Groundwater with the lowest arsenic and highest dissolved oxygen at Beimen 2B harbored 78% of the arsenite oxidizers communities, while groundwater with the highest arsenic and lowest dissolved oxygen at Beimen 1 and Beimen–Jinhu harbored 17 and 22% of arsenite oxidizers communities, respectively. Pseudomonas sp. was found only in groundwater containing high arsenic at Beimen 1 and Beimen–Jinhu, while arsenite oxidizers belonging to Alpha- and Betaproteobacteria were dominated in groundwater containing low arsenic.

Changes in Bacterial Community Structure and Abundance in Agricultural Soils under Varying Levels of Arsenic Contamination

Arsenic contamination from groundwater used to irrigate crops is a major issue across several agriculturally important areas of Asia. Assessing bacterial community composition in highly contaminated sites could lead to the identification of novel bioremediation strategies. In this study, the bacterial community structure and abundance are assessed in agricultural soils with varying levels of arsenic contamination at Ambagarh Chauki block, Chhattisgarh, India, based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S rRNA gene and the most probable number-polymerase chain reaction (MPN-PCR). The results revealed that the bacterial communities of arsenic-contaminated soils are dominated by β-proteobacteria (36%), γ-proteobacteria (21%), δ-proteobacteria (11%), α-proteobacteria (11%), and Bacteroidetes (11%). The bacterial composition of high arsenic-contaminated soils differed significantly from that of low arsenic-contaminated soils. The Proteobacteria appeared to be more resistant to arsenic contamination, while the Bacteroidetes and Nitrospirae were more sensitive to it. The bacterial abundance determined by MPN-PCR decreased significantly as As-toxicity increased. In addition to As, other trace metals, like Pb, U, Cu, Ni, Sn, Zn and Zr, significantly ( p < 0.01) explain the changes in bacterial structural diversity in agricultural soils with different level of arsenic contamination, as determined by canonical correspondence analysis (CCA).

Quantification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae in French Mediterranean coastal lagoons

Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae are human pathogens. Little is known about these Vibrio spp. in the coastal lagoons of France. The purpose of this study was to investigate their incidence in water, shellfish and sediment of three French Mediterranean coastal lagoons using the most probable number-polymerase chain reaction (MPN-PCR). In summer, the total number of V. parahaemolyticus in water, sediment, mussels and clams collected from the three lagoons varied from 1 to >1.1 × 103 MPN/l, 0.09 to 1.1 × 103 MPN/ml, 9 to 210 MPN/g and 1.5 to 2.1 MPN/g, respectively. In winter, all samples except mussels contained V. parahaemolyticus, but at very low concentrations. Pathogenic (tdh- or trh2-positive) V. parahaemolyticus were present in water, sediment and shellfish samples collected from these lagoons. The number of V. vulnificus in water, sediment and shellfish samples ranged from 1 to 1.1 × 103 MPN/l, 0.07 to 110 MPN/ml and 0.04 to 15 MPN/g, respectively, during summer. V. vulnificus was not detected during winter. V. cholerae was rarely detected in water and sediment during summer. In summary, results of this study highlight the finding that the three human pathogenic Vibrio spp. are present in the lagoons and constitute a potential public health hazard.

Quantification of Escherichia coli O157:H7 in milk by most probable number –polymerase chain reaction (MPN-PCR) method

Escherichia coli O157:H7 has been recognized as one of the most important causes of potentially life-threatening human diseases such as haemorrhagic colitis, haemolytic ureamic syndrome and thrombotic thrombocytopenic purpura. To combine the principles of most-probable-number (MPN) statistics and the conventional polymerase chain reaction (PCR) technique to enumerate E. coli O157:H7 in milk, from 101 to 105 ml-1 of bacterial cells were inoculated in sterilized milk. Different background microorganisms including Gram positive and Gram negative bacteria were also inoculated. Modified MPN dilutions from inoculated milk sample with three replicates per dilution were prepared and enumeration was performed by DNA extraction from tubes showing turbidity and performing multiplex-PCR (m-PCR) using primers specific for O157 and H7 antigens gene. This MPN-PCR proved to be a rapid and reliable method for enumerating E. coliO157:H7 in milk at the lowest level (101 cfu.ml-1), even in the presence of different Gram positive and Gram negative background microorganisms. It may facilitate the enumeration of E. coli O157:H7 for routine analyses in milk without excessive work and could be considered as an alternative to MPN- culture techniques. Key words: E. coli O157:H7, milk, most probable number-polymerase chain reaction (MPN-PCR), most probable number (MPN) - culture, multiplex- polymerase chain reaction (m-PCR).

Listeria monocytogenes in raw salad vegetables sold at retail level in Malaysia

A range of commercially available vegetables ( n = 306) that are consumed in the minimally processed state in Malaysia was examined for the presence of Listeria spp. and Listeria monocytogenes to provide information on the occurrence of such organisms in these vegetables. Analysis was carried out using the most probable number–polymerase chain reaction (MPN–PCR) method. It was found that Listeria spp. and L. monocytogenes could be detected in 33.3% and 22.5% of the vegetables respectively. L. monocytogenes was more frequently detected in Vigna unguiculata (Japanese parsley) at 31.3% and Oenanther stolonifera (yardlong bean) at 27.2%.

Rapid Quantitative Detection of Vibrio parahaemolyticus in Seafood by MPN-PCR

This study aimed to adopt MPN-PCR (most probable number-polymerase chain reaction) for rapid detection of the quantity of Vibrio parahaemolyticus in seafood. V. parahaemolyticus in seafood could be quantitated by MPN statistics according to PCR products. The sensitivity of MPN-PCR was 100 times higher than that of direct PCR. Of 225 seafood samples from Qingdao, 165 were positive for the presence of V. parahaemolyticus, with an MPN value of >719 per gram, and about 41.5% of samples were positive for tdh gene-possessing cells. Eighty muscle tissues from the 225 seafood samples were investigated by direct PCR and MPN-PCR, but no V. parahaemolyticus was detected. The MPN-PCR test could be completed in less than 16 h from the time of sample preparation. It was rapid, sensitive, and reliable for comprehensive detection and quick quantitative determination of V. parahaemolyticus in seafood and it revealed the potential risk of illness associated with their consumption.

Quantification of anammox populations enriched in an immobilized microbial consortium with low levels of ammonium nitrogen and at low temperature

Anaerobic ammonium oxidizing (anammox) bacteria present in microbial communities in two laboratory-scale upflow anoxic reactors supplied with small amounts of ammonium (<3 mg/l) at low temperature were detected and quantified. The reactors, operated at 20 degrees C, were seeded with an immobilized microbial consortium (IMC) and anaerobic granules (AG) from an upflow anaerobic sludge blanket (UASB) treating brewery wastewater. Our results showed that complete ammonium and nitrite removal with greater than 92% total nitrogen removal efficiency was achieved in the reactor inoculated with both the IMC and AG, while that of the reactor inoculated with only the IMC was lower than 40%; enrichment was successful after the addition of AG. Quantitative fluorescence in situ hybridization (FISH) analysis confirmed that anammox bacteria were present only in the reactor inoculated with an IMC and AG. The copy number of the 16S-rRNA gene of the anammox bacteria calculated by most probable number-polymerase chain reaction (MPN-PCR) from the total DNA extracted from both reactors (2.5 x 10(4) copies/mug of DNA) was two orders lower than that of the domain bacteria (2.5 x 10(6) copies/mug of DNA). The results revealed that immobilized multiple seed sludges were optimal for anammox enrichment at low temperature and ammonium concentrations.

Levels of Vibrio parahaemolyticus and Thermostable Direct Hemolysin Gene-positive Organisms in Retail Seafood Determined by the Most Probable Number-polymerase Chain Reaction (MPN-PCR) Method

The incidence and levels of Vibrio parahaemolyticus and thermostable direct hemolysin gene (tdh)-positive organisms in retail seafood were determined. The most probable number-polymerase chain reaction (MPN-PCR) method using a PCR procedure targeting the species-specific thermolabile hemolysin gene (tlh) and tdh was used to determine the levels of V. parahaemolyticus and tdh-positive organisms, respectively. In seafood for raw consumption, V. parahaemolyticus was found in four (13.3%) of 30 fish samples, 11 (55.0%) of 20 crustacean samples, and 29 (96.7%) of 30 mollusc samples. Levels of V. parahaemolyticus were below 10(4) MPN/100 g in all fish and crustacean samples tested. However, they were above 10(4) MPN/100 g in 11 (36.7%) of the 30 mollusc samples. In all seafood for raw consumption, the level of tdh-positive organisms was below the limit of detection (< 30 MPN/100 g). In seafood for cooking, V. parahaemolyticus was found in 15 (75.0%) of 20 fish samples, nine (45.0%) of 20 crustacean sample, and 20 (100%) of 20 mollusc samples. Levels of V. parahaemolyticus were above 10(4) MPN/100 g in only three (15.0%) and one (5.0%) of the 20 fish and 20 crustacean samples, respectively. However, they were above 10(4) MPN/100 g in 18 (90.0%) of the 20 mollusc samples. In seven (35.0%) of the 20 mollusc samples, tdh-positive organisms were found and their levels ranged from 3.6x10 to 1.1 x 103 MPN/100 g. From four of seven tdhpositive samples, tdh-positive V. parahaemolyticus was isolated.

Monitoring of alkane-degrading bacteria in a sea-water microcosm during crude oil degradation by polymerase chain reaction based on alkane-catabolic genes.

Behaviour of microbial populations responsible for degrading n-alkanes, a major component of crude oil, was monitored during crude oil degradation in a sea-water microcosm by both traditional colony culturing and molecular techniques. A DNA extraction method applicable to crude oil-amended sea-water samples was developed to obtain DNA applicable to most probable number (MPN) polymerase chain reaction (PCR). The population of alkane-degrading bacteria responsible for degradation of n-alkanes in a crude oil-amended microcosm altered, so that shorter alkanes were degraded first by alkane-degrading bacteria possessing alkane hydroxylase genes from group I (Kohno et al., 2002, Microb Environ 17: 114-121) and longer ones afterwards by those possessing alkane hydroxylase genes from group II. Thus, the degradation mechanism of the n-alkanes can be clarified during crude oil degradation. Application of the method of detecting different types of alkane-catabolic genes, as shown in the present study, enabled bacterial groups preferring alkanes of either shorter or longer chain lengths to be enumerated selectively.

DNA-based and culture-based characterization of a hydrocarbon-degrading consortium enriched from Arctic soil.

A hydrocarbon-degrading consortium was enriched from fuel-contaminated soil from the northeastern tip of Ellesmere Island (82 degrees 30'N, 62 degrees 19'W). The enrichment culture was grown on Jet A-1 fuel at 7 degrees C. Bacterial 16S RNA gene (rDNA) fragments were amplified by polymerase chain reaction (PCR) from members of the above consortium and cloned into a plasmid vector. Partial sequences (approximately 500 bp) were determined for 29 randomly selected rDNA clones. The majority of sequences were most similar to the corresponding rDNA sequences of Rhodococcus erythropolis (15 sequences), Sphingomonas spp. (six sequences), and Pseudomonas synxantha (four sequences). Amplified ribosomal DNA restriction analysis confirmed that a larger set of 50 clones had frequencies of the three phylotypes similar to those above. Phylotype-specific PCR assays were developed and validated for the above three phylotypes. The consortium was plated and grown on Jet A-1 fuel vapors, and randomly selected isolated colonies were screened with the above PCR assays. Of 17 colonies, six matched the Rhodococcus phylotype, and three matched the Pseudomonas phylotype. A representative strain of each phylotype was physiologically characterized. Both isolates grew on alkanes at low temperature and had general characteristics consistent with their respective phylotypes. During growth of the consortium, the three phylotype populations were monitored by a most probable number PCR assay. All three phylotypes were detected, but their relative abundance was not consistent with that of the phylotypes in the clone library. The relative abundance of all three phylotypes changed substantially during long-term incubation of the consortium. The DNA-based approach used identified phylotypes consistently present in the consortium, but it failed to predict the relative abundance of their populations.

Quantitative and specific detection of a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, by a most probable number-polymerase chain reaction method.

We developed a rapid and specific enumeration method for a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, based on a most probable number-polymerase chain reaction method for monitoring the bacterium at bioremediation sites. The primers designed for the mmoC gene of the soluble methane monooxygenase gene cluster were specific to strain M. Recovery of the cells with a membrane filter enabled us to detect strain M in trichloroethylene-contaminated groundwater. We used the enumeration method to monitor the number of strain M cells in effluent from soil columns supplied with trichloroethylene-contaminated groundwater. The number of strain M cells in the effluent depended on the amount of the strain M inoculated and the number of cells measured by the most probable number-polymerase chain reaction method was correlated with that measured by a culture method. The detection limit for strain M in effluent detected by MPN-PCR method was 4 to 8 x 10(2) cells/ml.