Detection of the key enzyme of alginate biosynthesis in <Emphasis Type="Italic">Vibrio</Emphasis> sp. QY102

Alginate is an important component of biofilms of many pathogens, but its presence in Vibrio has not been reported. The GDP-mannose dehydrogenase gene (algD), which is the kinetic control point in alginate biosynthesis, was cloned for the first time from Vibrio species using degenerated PCR and inverse PCR. Sequence analysis showed that algD was also localized in an alginate biosynthesis cluster, as it is in Pseudomonas aeruginosa. In addition, the existence of mannuronic acid, a component of alginate, was supported by the NMR spectrum of Vibrio sp. QY102 exopolysaccharide.     

Detection of Salmonella enterica serovar Typhimurium by selective amplification of fliC, fljB, iroB, invA, rfbJ, STM2755, STM4497 genes by polymerase chain reaction in a monoplex and multiplex format

The aim of the work was to specifically differentiate S. typhimurium from other closely related Salmonella serovars by monoplex or multiplex PCR and to detect it from water and food samples. Genes targeted were invA, iroB, STM4497, STM2755, fliC, fljB and rfbJ and evaluated on 58 Salmonella standard serovars/strains including 9 S. typhimurium strains, 7 suspected Salmonella isolates and 8 other organisms as negative controls. Both invA and iroB showed a uniform amplification with all serovars of S. enterica group. STM2755 and STM4497 gene based PCR’s specifically exhibited amplification in all the nine confirmed S. typhimurium strains. The rfbJ PCR produced amplification with confirmed S. typhimurium strains, in addition showed reaction with S. abony. Both STM4497, STM2755 PCR’s and rfbJ could identify two of the seven biochemically suspected Salmonella isolates that were later confirmed to be S. typhimurium on the basis of sequence data. PCR for fliC genes had amplification exhibited by a large number of serovars of the S. enterica group, including S. typhimurium strains but not to S. brunei, S. newporti, S. abony and S. weltevreden. fljB was detected in all strains of S. enterica and E. coli with the exception of S. typhi. fljB and fliC were amplified in 6/7 and 5/7 presumptive Salmonella isolates. The same PCR’s were converted into two multiplex formats for simultaneous identification of the Salmonella genus, S. enterica group and S. typhimurium as a species. The first multiplex set comprised on invA, iroB, STM4497, STM2755 and the IAC. The second multiplex set comprised of invA, iroB, fljB, fliC, rfbJ along with IAC. The detection limit for S. typhimurium in the two multiplex PCR sets was in the range of 350–400 cfu/PCR reaction and that of DNA around 2 pg. The multiplex PCR (format 1) was first evaluated on spiked water, chicken and mutton samples and the detection limit for S. typhimurium was in the range of 100 cfu/100 ml, <60 and <50 cfu/gm, respectively. Further evaluation of multiplex PCR (format 1) was undertaken on 50 natural samples of chicken, eggs, litter, soil etc. and the comparison done with conventional culture isolation and identification procedure. The multiplex PCR could identify the presence of Salmonellla in three samples and the same three samples also yielded Salmonella by the conventional method. Therefore, the presently described multiplex PCR can serve as an alternative to the tedious time-consuming procedure of Salmonella culture and identification in food safety laboratories.     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

The impact of biofilm-forming potential and tafi production on transport of environmental <Emphasis Type="Italic">Salmonella</Emphasis> through unsaturated porous media

Understanding the factors influencing the transport of microbial pathogens, such as Salmonella and Escherichia coli, through porous media is critical to protecting drinking water supplies. The production of biofilms, along with individual biofilm-associated components, such as tafi, is believed to hinder transport of microorganisms through soil. This study investigated the relationship between biofilm formation and tafi production and the transport of environmental Salmonella through porous media. Thirty-two Salmonella isolates were initially assayed for their ability to form biofilms, from which a subset of these was selected to represent a range of high and low biofilm-formation potential and tafi formation capabilities. These were subsequently examined in unsaturated sand columns for transport characteristics. No obvious correlation was observed between Salmonella phenotypes and column retention. The results indicated that while transport of well-characterized laboratory E. coli strains can often be hindered by the presence of tafi and the potential to form biofilms, the presence of tafi did not retard the transport of the Salmonella strains.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.     

An eight-hour PCR-based technique for detection of <Emphasis Type="Italic">Salmonella</Emphasis> serovars in seafood

A rapid and sensitive 8-h PCR assay has been developed for detection of Salmonella serovars in seafood. A total of 110 fresh and raw seafood samples were analysed for the presence of Salmonella using different enrichment periods prior to PCR assay. Seafood samples included in this study were fish, shrimps, mussels, crabs, edible oysters, and clams, collected from local fish markets in Cochin (India). The assay was performed with a Salmonella-specific 284 bp invA gene amplicon. Specificity and sensitivity of the assay were ascertained with seafoods spiked with viable Salmonella cells to a level of 10⁶ to 2 CFU per 25 g. Detection efficiency of the assay increased with increasing enrichment period for seafood, and 33.6% of seafood samples were found positive for Salmonella by 8-h PCR assay. Detection limit for the 8-h PCR assay showed visible 284 bp amplicon from seafood homogenates spiked with 2 CFU per 25 g. Seafood samples spiked with different Salmonella serovars, namely Salmonella typhi, Salmonella typhimurium, Salmonella enteritidis, Salmonella mbandka, Salmonella bareilly, and Salmonella weltevreden, were detected, confirming this technique would be ideal for detection of the Salmonella serovars prevalent in seafood. This study also covered inhibition by the seafood matrix and the detection limit for dead Salmonella cells during the PCR assay. There was no visible inhibition of this Salmonella PCR assay by seafood matrices. The detection limit for dead Salmonella cells by 8-h PCR assay was 2 × 10³ CFU per 25 g seafood. The data indicated that dead cells of Salmonella in naturally contaminated seafood samples do not interfere with the assay resulting in false positives.     

鹌鹑源鼠伤寒沙门氏菌的分离鉴定与耐药性分析

【背景】在鹌鹑养殖过程中,抗菌药物和消毒剂的不规范使用加剧了耐药菌株在动物、场所和食品之间的相互传播,因此,掌握致病菌株在养殖动物中的耐药状况至关重要。【目的】检测北京周边地区鹌鹑蛋源致病菌株的耐药特征和耐药基因的流行情况。【方法】在天津市武清区部分鹌鹑养殖场采集鹌鹑泄殖腔粪便、鹌鹑蛋表、养殖环境和鹌鹑饮水的样品,通过细菌分离培养、菌落形态观察、染色镜检、生化鉴定、血清分型、沙门氏菌inv A基因序列测定等方法对分离菌株进行鉴定。同时进行小鼠攻毒试验,测定小鼠半数致死量(median lethal dose, LD50)。再通过药敏试验和PCR方法对分离菌的耐药表型、耐药基因及毒力基因进行检测。【结果】分离菌株菌落颜色、镜检形态和生化试验结果符合沙门氏菌特性,沙门氏菌inv A基因序列测定与鼠伤寒沙门氏菌参考株相似度为99.44%,鉴定为鼠伤寒沙门氏菌,血清型为1,4,[5],[12]:i:l,2。该菌株对小鼠有致病作用,小鼠LD50为2.10×107 CFU/mL;药敏试验结果显示该菌株对氨苄西林、阿莫西林/克拉维酸、头孢噻呋、链霉素、磺胺甲啞唑、磺胺异啞唑、诺氟沙星、环丙沙星表现耐...     

Evaluation of different species-specific PCR protocols for the detection of Vibrio tapetis

In this study the specificity and sensitivity of three primer pairs, Jvt1–Jvt2, VtF–VtR and VtKF–VtKR, for the detection of Vibrio tapetis were evaluated in parallel using 23 V. tapetis strains isolated from different mollusc and fish species and with different geographical origin, as well as 29 representatives of related Vibrio species. The three primer pairs amplified all the V. tapetis strains, regardless of their host or geographical origin. However, with primer sets VtF–VtR and VtKF–VtKR amplification products of the expected size were obtained from chromosomal DNA of some of the non-V. tapetis bacteria tested. The sensitivity of the three PCR detection methods was also different. The detection limit obtained with primer pairs Jvt1–Jvt2 and VtF–VtR was between 1 and 10 pg DNA/PCR tube (2–20 bacterial cells per reaction). The primer set VtKF–VtKR showed a reduction of sensitivity in at least one order of magnitude. The results were highly reproducible with all primer sets when using the same thermal cycler, although some differences were observed in the results obtained in different PCR machines. Based on the findings reported here, we propose the Jvt1–Jvt2 PCR protocol as the most adequate for an accurate detection of V. tapetis in diagnostic pathology as well as in epidemiological studies of this clam pathogen.     

The applicability of hns and fimA primers for detecting Salmonella in bioaerosols associated with animal and municipal wastes

The ability to rapidly screen environmental samples for specific pathogens such as Salmonella spp., is of particular importance in molecular epidemiology. Although gene amplification reactions allow the rapid detection of microorganisms, the use of appropriate oligonucleotide primers targeted against specific microbial genes is critical for accurate detection specificity and sensitivity. Primers such as fimA and hns have been previously shown to be specific for pure cultures of Salmonella. However, the analysis of environmental samples requires post-amplification hybridizations to detect amplicons, since the presence of inhibitory environmental components reduces amplification efficiency of the target organism. These sensitive post-amplification approaches also enable the detection of spurious amplification from non-target sequences. Bioaerosols associated with animal facilities and municipal wastes contain a diverse array of pathogens including Clostridium spp. In our studies, hybridization sensor data revealed spurious amplification of clostridial species with Salmonella hns primers. Specificity checks using type cultures of Clostridium spp. revealed non-specific amplification by hns primers. These results suggest that fimA primers may be better suited to screen Salmonella-specific sequences in environmental samples, especially those obtained from animal and municipal waste facilities.     

PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation

The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonellacells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.     

Multivariate drug resistance and microbial risk assessment in tropical coastal communities

Multivariate water quality parameters and statistical analysis were used to evaluate the factors controlling coastal drinking water quality and associated health risks among fisherfolks. Multidrug-resistant strains noticed in 400 isolates show 62% Salmonella; 53% Shigella sp.; 48% E. coli; and 36% Vibrio sp. in groundwater sample. In component analysis seawater intrusion, redox reaction, anthropogenic pollution, and weather factors were responsible for more than 93.3% in postmonsoon and 89.4% in summer season, respectively, for Cumulative %. In epidemiology study, 66% and 76% of municipally supplied drinking water were used in Pondicherry and Rameshwaram, respectively, compared to the amount of groundwater (34% and 20%) used in the study area. Similarly, Pondicherry and Rameshwaram areas recorded open defecation instances of 94% and 82%, respectively where less than 5% of the population used hygienic sanitation as part of the Clean India Mission in rural areas.     

Validation of ERIC PCR as a tool in epidemiologic research of Salmonella in slaughter pigs

The purpose of this study was to test a protocol for a standardized ERIC PCR for its capability of genotyping Salmonella, isolated from pigs and their environment, in an epidemiologic approach. To test repeatability, four different Salmonella isolates were subjected to PCR three times. Furthermore, it was tested if the profiles on gel differed when a higher annealing temperature was used. Four Salmonella isolates were subjected to four different annealing temperatures (36, 40, 48 and 55°C). Moreover it was tested if the differentiation of Salmonella isolates, based on the genotypes, differed when a higher annealing temperature was used. Eight Salmonella isolates were tested at normal (36°C) and high (55°C) annealing temperatures. The results showed that this standardized ERIC PCR protocol was an efficient tool for typing many Salmonella isolates within a short period of time. The profiles were repeatable within one PCR reaction, but some profiles differed when they were compared between reactions. A higher annealing temperature resulted in profiles that contained more or fewer bands. The differentiation between isolates, when comparing profiles, remained the same. It was concluded that the standardized ERIC PCR protocol is useful for genotyping Salmonella. Received 26 January 1998/ Accepted in revised form 3 August 1998     

Real-time PCR method to detect Enterococcus faecalis in water

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat

The current surveillance protocol for Karnal bunt of wheat in most countries, including the USA, European Union (EU), and Australia, involves the tentative identification of the spores based on morphology followed by a molecular analysis. Germination of spores is required for confirmation which incurs a delay of about two weeks, which is highly unsatisfactory in a quarantine situation. A two-step PCR protocol using FRET probes for the direct detection and identification of Tilletia indica from a very few number of spores (≤10) is presented. The protocol involves amplification of the ITS1 DNA segment in the highly repeated rDNA unit from any Tilletia species, followed by FRET analysis to detect and unequivocably distinguish T. indica and the closely related T. walkeri. This rapid, highly sensitive, fluorescent molecular tool is species-specific, and could supersede the conventional microscopic diagnosis used in a quarantine surveillance protocol for Karnal bunt which is often confounded by overlapping morphological characters of closely related species.     

Identification of environmental plasmid-bearing Vibrio species isolated from polluted and pristine marine reserves of Hong Kong, and resistance to antibiotics and mercury

Fifty environmental isolates of Vibrio species were isolated from water samples of Mai Po Nature Reserve and the Cape d’Aguilar Marine Reserve in Hong Kong and screened for the presence of plasmid. Mai Po is a wastewater-impacted area while the Cape d’Aguilar Marine Reserve is pristine natural marine water. Plasmid was found in Vibrio isolates from both sites at similar frequencies and each site showed distinctive plasmid profiles. These plasmid-bearing Vibrio isolates were identified as different species of the Vibrio genus by both biochemical test and subsequently full-length 16S rRNA sequences. Antibiotic resistance test showed that all these plasmid-bearing Vibrio isolates showed multiple resistance to 21 antibiotics tested. In addition, selective isolates also showed tolerance to 10 M Hg²⁺ in culture medium and they generally harbored large plasmid(s) (>‰30 kb). Our results show that the high frequency of plasmid in Vibrio species of both polluted and pristine environments may be ecologically important to the survival of these bacteria in the environment. The specific functioning of the cryptic plasmids remains the focus of current investigations.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

Detection of Viable Toxigenic Vibrio cholerae from Environmental Water Sources by Direct Cell Duplex PCR Assay

Summary Environmental monitoring is important to enable effective resource management and public health protection as well as rapid and accurate identification of Vibrio cholerae in drinking-water sources. Traditional methods employed in identification are laborious, time-consuming and practically not viable for screening of a large number of samples. In this study, a direct cell duplex PCR assay for the detection of viable toxigenic V. cholerae in environmental water samples was developed. In the PCR assay, two gene sequences were amplified together, one of outer membrane protein (ompW), which is species-specific and another of cholera toxin (ctxAB). The detection limit of duplex PCR was 5 × 10⁴ V. cholerae/reaction. Different environmental water samples were artificially spiked with V. cholerae O1 cells and filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly in the duplex PCR assay. The PCR procedure coupled with enrichment could detect as few as 1.2 c.f.u./ml in ground water, 1.2 × 10² c.f.u. ml⁻¹ in sewer water and 1.2 × 10³c.f.u. ml⁻¹ in tap water. The assay was successfully applied directly for screening of environmental potable water samples collected from a cholera-affected area. The proposed method is simple and can be used for environmental monitoring of toxigenic as well as non-toxigenic V. cholerae.     

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.