MassARRAY: a high-throughput solution for rapid detection of foodborne pathogens in real-world settings.

Introduction: Bacterial foodborne pathogens pose a substantial global public health concern, prompting government agencies and public health organizations to establish food safety guidelines and regulations aimed at mitigating the risk of foodborne illness. The advent of DNA-based amplification coupled with mass spectrometry, known as MassARRAY analysis, has proven to be a highly precise, sensitive, high-throughput, and cost-effective method for bacterial detection. This study aimed to develop, validate, and evaluate a MassARRAY-based assay for the detection and identification of significant enteropathogenic bacteria. Methods: The MassARRAY-based assay was developed for the detection of 10 crucial bacterial foodborne pathogens, including Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Salmonella spp., Shigella spp., and Staphylococcus aureus. The assay was optimized using the reference gDNA (n = 19), followed by validation using gDNA (n = 85) of reference and laboratory isolates. Additionally, the evaluation of the assay's reaction using a mixture of gDNA from all nine targeted species was performed. The limit of detection of the developed MassARRAY-based assay was determined using bacterial cells. Moreover, the validation method for field samples was evaluated by comparing it with standard microbiological testing methods routinely analyzed. Results: The developed MassARRAY-based assay demonstrated 100% concordance with known bacterial pure cultures. The assay's reaction using a mixture of gDNA from all nine targeted species revealed the MassARRAY's capability to detect all targeted species in a single assay with the lowest concentration of 1 ng/µL of gDNA. The limits of detection of the assay range from 357 ± 101 to 282,000 ± 79,196 cells. Moreover, the validation of the assay in field samples revealed a 100% correlation between the data obtained from the standard microbiological method and the MassARRAY-based assay. Discussion: These findings suggested that the developed MassARRAY-based assay exhibited the excellence in high-throughput detection of foodborne bacterial pathogens with high accuracy, reliability, and potential applicability within real-world field samples.

Nanopore Sequencing Discloses Compositional Quality of Commercial Probiotic Feed Supplements.

The market for the application of probiotics as a livestock health improvement supplement has increased in recent years. However, most of the available products are quality-controlled using low-resolution techniques and un-curated databases, resulting in misidentification and incorrect product labels. In this work, we deployed two workflows and compared results obtained by full-length 16S rRNA genes (16S) and metagenomic (Meta) data to investigate their reliability for the microbial composition of both liquid and solid forms of animal probiotic products using Oxford Nanopore long-read-only (without short-read). Our result revealed that 16S amplicon data permits to detect the bacterial microbiota even with the low abundance in the samples. Moreover, the 16S approach has the potential to provide species-level resolution for prokaryotes but not for assessing yeast communities. Whereas, Meta data has more power to recover of high-quality metagenome-assembled genomes that enables detailed exploration of both bacterial and yeast populations, as well as antimicrobial resistance genes, and functional genes in the population. Our findings clearly demonstrate that implementing these workflows with long-read-only monitoring could be applied to assessing the quality and safety of probiotic products for animals and evaluating the quality of probiotic products on the market. This would benefit the sustained growth of the livestock probiotic industry.

Whole-genome sequence analysis for evaluating the safety and probiotic potential of Lactiplantibacillus pentosus 9D3, a gamma-aminobutyric acid (GABA)-producing strain isolated from Thai pickled weed.

Lactiplantibacillus pentosus 9D3, a prominent gamma-aminobutyric acid (GABA)-producing bacteria isolated from Thai pickled weed was characterized for its safety and probiotic properties via whole-genome analysis and in vitro testing. The whole-genome sequence of L. pentosus 9D3 was determined using a hybrid-sequencing approach, combining PacBio and Illumina technologies. A 3.81-Mbp genome of L. pentosus 9D3 consisting of one 3.65-Mbp chromosome and six plasmids (1.9-71.9 Kbp) was identified with an estimated GC content of 46.09% and 3,456 predicted genes. The strain was confirmed to be Lactiplantibacillus pentosus according to the high average nucleotide identity value of >95% and digital DNA-DNA hybridization scores of >70% to the L. pentosus type strain. Comparative genome analysis with other L. pentosus strains showed that the GABA-producing capability was specific to the strain 9D3. Genes related to GABA biosynthesis and transport were identified on a plasmid, pLPE-70K, indicating the acquired nature of this property. The safety of L. pentosus 9D3 was demonstrated through the lack of genes related to the production of toxins, biogenic amines, and antimicrobial drugs. Although the strain exhibited resistance to ampicillin and chloramphenicol, none of the antimicrobial resistance (AMR) genes were associated with mobile elements, i.e., plasmids and prophages. Therefore, the strain is considered to have low risk of transferring the AMR genes to other, potentially pathogenic bacteria. In addition, L. pentosus 9D3 showed good survivability in the gastrointestinal tract environment and was able to adhere to the intestinal cell in vitro. Therefore, L. pentosus 9D3 is concluded to be safe, with the potential to be used as a probiotic, exerting its health benefit through GABA production in the food system. The GABA-producing capability of the strain in vivo is the subject of further investigation.

Characterisation of Salmonella enterica clones carrying mcr-1 plasmids in meat products and patients in Northern Thailand using long read sequencing.

Salmonella spp. is an important foodborne pathogen associated with consumption of contaminated food, especially food of livestock origin. Antimicrobial resistance (AMR) in Salmonella has been reported globally and increasing AMR in food production is a major public health issue worldwide. The objective of this study was to describe the genetic relatedness among Salmonella enterica isolates, which displayed identical DNA fingerprint profiles. Ten S. enterica isolates were selected from meat and human cases with an identical rep-PCR profile of serovars Rissen (n = 4), Weltevreden (n = 4), and Stanley (n = 2). We used long-read whole genome sequencing (WGS) on the MinION sequencing platform to type isolates and investigate in silico the presence of specific AMR genes. Antimicrobial susceptibility testing was tested by disk diffusion and gradient diffusion method to corroborate the AMR phenotype. Multidrug resistance and resistance to more than one antimicrobial agent were observed in eight and nine isolates, respectively. Resistance to colistin with an accompanying mcr-1 gene was observed among the Salmonella isolates. The analysis of core genome and whole genome MLST revealed that the Salmonella from meat and human salmonellosis were genetically related. Hence, it could be concluded that meat is one of the important sources for Salmonella infection in human.

KITSUNE: A Tool for Identifying Empirically Optimal K-mer Length for Alignment-Free Phylogenomic Analysis.

Genomic DNA is the best "unique identifier" for organisms. Alignment-free phylogenomic analysis, simple, fast, and efficient method to compare genome sequences, relies on looking at the distribution of small DNA sequence of a particular length, referred to as k-mer. The k-mer approach has been explored as a basis for sequence analysis applications, including assembly, phylogenetic tree inference, and classification. Although this approach is not novel, selecting the appropriate k-mer length to obtain the optimal resolution is rather arbitrary. However, it is a very important parameter for achieving the appropriate resolution for genome/sequence distances to infer biologically meaningful phylogenetic relationships. Thus, there is a need for a systematic approach to identify the appropriate k-mer from whole-genome sequences. We present K-mer-length Iterative Selection for UNbiased Ecophylogenomics (KITSUNE), a tool for assessing the empirically optimal k-mer length of any given set of genomes of interest for phylogenomic analysis via a three-step approach based on (1) cumulative relative entropy (CRE), (2) average number of common features (ACF), and (3) observed common features (OCF). Using KITSUNE, we demonstrated the feasibility and reliability of these measurements to obtain empirically optimal k-mer lengths of 11, 17, and ∼34 from large genome datasets of viruses, bacteria, and fungi, respectively. Moreover, we demonstrated a feature of KITSUNE for accurate species identification for the two de novo assembled bacterial genomes derived from error-prone long-reads sequences, and for a published yeast genome. In addition, KITSUNE was used to identify the shortest species-specific k-mer accurately identifying viruses. KITSUNE is freely available at https://github.com/natapol/kitsune.

Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis.

The safety of microbial cultures utilized for consumption is vital for public health and should be thoroughly assessed. Although general aspects on the safety assessment of microbial cultures have been suggested, no methodological detail nor procedural guideline have been published. Herein, we propose a detailed protocol on microbial strain safety assessment via whole-genome sequence analysis. A starter culture employed in traditional fermented pork production, nham, namely Lactobacillus plantarum BCC9546, was used as an example. The strain's whole-genome was sequenced through several next-generation sequencing techniques. Incomplete plasmid information from the PacBio sequencing platform and shorter chromosome size from the hybrid Oxford Nanopore-Illumina platform were noted. The methods for 1) unambiguous species identification using 16S rRNA gene and average nucleotide identity, 2) determination of virulence factors and undesirable genes, 3) determination of antimicrobial resistance properties and their possibility of transfer, and 4) determination of antimicrobial drug production capability of the strain were provided in detail. Applicability of the search tools and limitations of databases were discussed. Finally, a procedural guideline for the safety assessment of microbial strains via whole-genome analysis was proposed.

PSO-LocBact: A Consensus Method for Optimizing Multiple Classifier Results for Predicting the Subcellular Localization of Bacterial Proteins.

Several computational approaches for predicting subcellular localization have been developed and proposed. These approaches provide diverse performance because of their different combinations of protein features, training datasets, training strategies, and computational machine learning algorithms. In some cases, these tools may yield inconsistent and conflicting prediction results. It is important to consider such conflicting or contradictory predictions from multiple prediction programs during protein annotation, especially in the case of a multiclass classification problem such as subcellular localization. Hence, to address this issue, this work proposes the use of the particle swarm optimization (PSO) algorithm to combine the prediction outputs from multiple different subcellular localization predictors with the aim of integrating diverse prediction models to enhance the final predictions. Herein, we present PSO-LocBact, a consensus classifier based on PSO that can be used to combine the strengths of several preexisting protein localization predictors specially designed for bacteria. Our experimental results indicate that the proposed method can resolve inconsistency problems in subcellular localization prediction for both Gram-negative and Gram-positive bacterial proteins. The average accuracy achieved on each test dataset is over 98%, higher than that achieved with any individual predictor.

Core genome sequence analysis to characterize Salmonella enterica serovar Rissen ST469 from a swine production chain.

Salmonella enterica subsp. enterica serotype Rissen is the predominant serotype found in Thai pork production and can be transmitted to humans through contamination of the food chain. This study was conducted to investigate the genetic relationships between serovar Rissen isolates from all levels of the pork production chain and evaluate the ability of the in silico antimicrobial resistance (AMR) genotypes to predict the phenotype of serovar Rissen. A total of 38 serovar Rissen isolates were tested against eight antibiotic agents by a disk diffusion method and the whole genomes of all isolates were sequenced to detect AMR genetic elements using the ResFinder database.A total of 86.84% of the isolates were resistant to tetracycline, followed by ampicillin (78.96%) and sulfonamide-trimethoprim (71.05%). Resistance to more than one antimicrobial agent was observed in 78.95% of the isolates, with the most common pattern showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide-trimethoprim, and tetracycline. The results of genotypic AMR indicated that 89.47% of the isolates carried tet(A), 84.22% carried blaTEM-1B, 78.95% carried sul3, and 78.95% carried dfrA12. The genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.45% and specificity of 75.48%. Analysis by core genome multilocus sequence typing (cgMLST) demonstrated that the Salmonella isolates from various sources and different locations shared many of the same core genome loci. This implies that serovar Rissen has infected every stage of the pork production process and that contamination can occur in every part of the production chain.

Dynamics of biogenic amines and bacterial communities in a Thai fermented pork product Nham.

A traditional Thai fermented pork, nham, is a product popularly consumed in Thailand. Fermentation of the protein-rich product by uncontrolled bacterial community can result in high amounts of hazardous biogenic amines (BA). This study aimed to unveil dynamics of microbial community and its relation to BA accumulation in nham. Three batches of nham were analyzed for pH, lactic acid bacteria population, concentrations of organic acids and BA. Bacterial communities were analyzed by pyrosequencing of 16S rRNA gene amplicons. In all batches, pH dropped to the quality standard of nham (≤4.6) within 3-5 days by production of lactic acid and acetic acid. Initial BA levels varied batch-by-batch and increased with fermentation time. In the highest quality batch, levels of histamine, tyramine, and total BA were within the recommended safety limits (200, 100 and 1000 mg/kg, respectively) throughout the 10-days study. However, in other batches, unsafe levels of tyramine and total BA were found after 5 days of fermentation. The results indicated that over-fermentation and inferior conditions of ingredients increased risk due to high levels of BA. Lactobacillus, Lactococcus, Pediococcus and Weissella were prevalent and comprised >90% of total bacteria during fermentation. Weissella was predominant in the batch with low BA while Lactobacillus and Pediococcus were predominant in the higher BA batches. A negative correlation between Weissella dominance and total BA was observed (r = -0.90, p = .003). A 10% increase in dominance of Weissella was associated with 75-170 mg/kg decrease in total BA. W. hellenica was the species prevalent only in low BA batch. Therefore, W. hellenica isolates were suggested as subjects for future study to develop efficient starter culture securing safety of nham.

Quantification and rep-PCR characterization of Salmonella spp. in retail meats and hospital patients in Northern Thailand.

Human salmonellosis is a major public health problem worldwide. Infections can pass to humans by contact with contaminated substances in the food chain. This study aimed to determine the prevalence and contamination levels of Salmonella isolated from pork, chicken and beef sold in different types of retail stores in Chiang Mai and Lamphun provinces and to investigate the genetic relatedness among Salmonella isolates in food chains in that area. A total of 360 meat samples from supermarkets, mini-grocery stores and fresh markets were obtained. Salmonella Rissen and S. Weltevreden were found in all meat sample types and in human cases. The overall prevalence of Salmonella in the chicken, pork and beef samples was 34.17%, 32.50% and 3.33%, respectively. Quantitatively, Salmonella contamination was highest in pork (1.24 log10 MPN/g), followed by chicken (1.08 log10 MPN/g), and beef (0.75 log10 MPN/g). The highest frequency of Salmonella contamination was found at the fresh markets (85.71%), whereas the highest quantity of contamination level was from mini-grocery stores (1.27 log10 MPN/g). The rep-PCR analysis results revealed that some of the Salmonella from meat samples and human cases were identical clones.

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand.

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.

Class 1 integrons characterization and multilocus sequence typing of Salmonella spp. from swine production chains in Chiang Mai and Lamphun provinces, Thailand.

Pigs and pork products are well known as an important source of Salmonella, one of the major zoonotic foodborne pathogens. The emergence and spread of antimicrobial resistance is becoming a major public health concern worldwide. Integrons are genetic elements known to have a role in the acquisition and expression of genes conferring antibiotic resistance. This study focuses on the prevalence of class 1 integrons-carrying Salmonella, the genetic diversity of strains of those organisms obtained from swine production chains in Chiang Mai and Lamphun provinces, Thailand, using multilocus sequence typing (MLST) and comparison of genetic diversity of sequence types of Salmonella from this study with pulsotypes identified in previous study. In 175 Salmonella strains, the overall prevalence of class 1 integrons-carrying-Salmonella was 14%. The gene cassettes array pattern "dfrA12-orfF-aadA2" was the most frequently observed. Most of the antimicrobial resistance identified was not associated with related gene cassettes harbored by Salmonella. Six sequence types were generated from 30 randomly selected strains detected by MLST. Salmonella at the human-animal-environment interface was confirmed. Linkages both in the farm to slaughterhouse contamination route and the horizontal transmission of resistance genes were demonstrated. To reduce this problem, the use of antimicrobials in livestock should be controlled by veterinarians. Education and training of food handlers as well as promotion of safe methods of food consumption are important avenues for helping prevent foodborne illness.

Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.

Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction.

Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

Distribution, quantitative load and characterization of Salmonella associated with swine farms in upper-northern Thailand.

This study was conducted to analyze the prevalence and quantitative loads of Salmonella spp. on pig farms in Chiang Mai, Lamphun, Thailand to assess loading levels before slaughtering. The serotype diversity, antimicrobial-resistance pattern and pulse-field type of Salmonella spp. were also characterized to assess the dynamic propagation of the pathogen. The Salmonella-positive prevalence was 246/805 (30.56%), and the quantitative loads varied from 1.48 ˜ 4.04 Log10MPN/g, with a mean ± standard deviation of 2.11 ± 0.57. AMP/S/TE (ampicillin/streptomycin/tetracycline) was the highest frequency antimicrobial resistance pattern found in this study. In addition, Salmonella Rissen was the primary serotype in this region. PFGE results indicated the occurrence of infection by cross contamination among pig farms. Our study showed that pork is easily contaminated with this pathogen. Farm control programs must be based on strict biosecurity and hygienic measures, which could further reduce the contamination pressure at slaughterhouses or retail shops.

Comparison of multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) fingerprinting for differentiation of Campylobacter jejuni isolated from broiler in Chiang Mai, Thailand.

We compared rapid fingerprinting using repetitive sequence-based PCR (rep-PCR) for subtyping Campylobacter jejuni isolates to the widely used multilocus sequence typing (MLST). Representative C. jejuni isolates (n = 16) from broilers were analyzed using MLST and rep-PCR. Both techniques demonstrated an equal discriminatory power of 0.8917, and 9 subgroups were identified. Clonal identification of all 16 isolates was identical for both techniques. The rep-PCR as described in this study may be used as a rapid and cost-effective alternative for subtyping of C. jejuni isolates, or as an effective screening tool in large epidemiological studies.

Bacillus siamensis sp. nov., isolated from salted crab (poo-khem) in Thailand.

A Gram-positive, endospore-forming, rod-shaped bacterium, strain PD-A10(T), was isolated from salted crab (poo-khem) in Thailand and subjected to a taxonomic study. Phenotypic and chemotaxonomic characteristics, including phylogenetic analyses, showed that the novel strain was a member of the genus Bacillus. The novel strain grew in medium with 0-14 % (w/v) NaCl, at 4-55°C and at pH4.5-9. The predominant quinone was a menaquinone with seven isoprene units (MK-7). The major fatty acids were anteiso-C15:0 and anteiso-C17:0. Polar lipid analysis revealed the presence of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, lysylphosphatidylglycerol, glycolipid and unknown lipids. The DNA G+C content was 41.4 mol%. The 16S rRNA gene sequence similarities between strain PD-A10(T) and Bacillus amyloliquefaciens NBRC 15535(T), Bacillus subtilis DSM 10(T), Bacillus vallismortis DSM 11031(T) and Bacillus mojavensis IFO 15718(T) were 99.5, 99.4, 99.4 and 99.2 %, respectively. Strain PD-A10(T) showed a low degree similarity of rep-PCR fingerprints and low DNA-DNA relatedness with the above-mentioned species. On the basis of the data gathered in this study, strain PD-A10(T) should be classified as representing a novel species of the genus Bacillus, for which the name Bacillus siamensis sp. nov. is proposed. The type strain is PD-A10(T) (=BCC 22614(T)=KCTC 13613(T)).

Incidence of Staphylococcus aureus and associated risk factors in Nham, a Thai fermented pork product.

Staphylococcus aureus is one of the most prevalent bacterial pathogens causing food-borne disease worldwide. Staphylococcal food poisoning is caused by ingestion of staphylococcal enterotoxins (SEs) pre-formed in the implicated food. In this study, the incidences of S. aureus and classical SEs (SEA-SEE) contamination in 'Nham', a traditional Thai fermented pork product, were determined. Among 155 Nham samples tested, as high as 39.35% of the samples were positive for S. aureus (2-3500 MPN/g), but none were positive for the SEs. The risk factors for S. aureus contamination were highly correlated with the manufacturer and the pH of the product. A predictive model determined the probability of the presence of S. aureus to be < or = 0.24 at the pH < or = 4.6. During the fermentation process, the number of S. aureus slightly increased in the first day and decreased afterward. S. aureus counts continued to decrease when Nham was stored refrigerated. The negative result for enterotoxins and low counts of S. aureus in Nham surveyed in this study, and reduction of the pathogen counts during fermentation and storage suggested that there is very low risk of staphylococcal food poisoning from consuming properly fermented Nham.

Diversity and seasonality of bioluminescent Vibrio cholerae populations in Chesapeake Bay.

Association of luminescence with phenotypic and genotypic traits and with environmental parameters was determined for 278 strains of Vibrio cholerae isolated from the Chesapeake Bay during 1998 to 2000. Three clusters of luminescent strains (A, B, and C) and two nonluminescent clusters (X and Y) were identified among 180 clonal types. V. cholerae O1 strains isolated during pandemics and endemic cholera in the Ganges Delta were related to cluster Y. Heat-stable enterotoxin (encoded by stn) and the membrane protein associated with bile resistance (encoded by ompU) were found to be linked to luminescence in strains of cluster A. Succession from nonluminescent to luminescent populations of V. cholerae occurred during spring to midsummer. Occurrence of cluster A strains in water with neutral pH was contrasted with that of cluster Y strains in water with a pH of >8. Cluster A was found to be associated with a specific calanoid population cooccurring with cyclopoids. Cluster B was related to cluster Y, with its maximal prevalence at pH 8. Occurrence of cluster B strains was more frequent with warmer water temperatures and negatively correlated with maturity of the copepod community. It is concluded that each cluster of luminescent V. cholerae strains occupies a distinct ecological niche. Since the dynamics of these niche-specific subpopulations are associated with zooplankton community composition, the ecology of luminescent V. cholerae is concluded to be related to its interaction with copepods and related crustacean species.

Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR.

A high-throughput method which is applicable for rapid screening, identification, and delineation of isolates of Vibrio cholerae, sensitive to genome variation, and capable of providing phylogenetic inferences enhances environmental monitoring of this bacterium. We have developed and optimized a method for genomic fingerprinting of V. cholerae based on long-range PCR. The method uses a primer set directed to enterobacterial repetitive intergenic consensus sequences, a high-fidelity DNA polymerase, and analysis via conventional agarose gel electrophoresis. Long ( approximately 10 kb), highly reproducible amplicons were generated from V. cholerae isolates, including those from different geographical locations and historical strains isolated during the period 1931-2000. The amplicons yielded reduced variability in their densitometric band patterns to /=90% similarity, discriminating O serotypes and biotypes (classical versus El Tor) as well as pathogenic and nonpathogenic strains. Compared to genome similarity measured by DNA-DNA hybridization, the results showed good correlation (r = 0.7; P < 0.001), with five times less measurement error and without bias. The method permits both phylogenetic inference and clonal differentiation of individual V. cholerae strains, enables robust, high-throughput analysis, and does not require specialized equipment to perform. With access to a curated public database furnished with appropriate analytical software applications, the method should prove useful in large-scale multilaboratory surveys, especially those designed to detect specific pathogens in the natural environment.