Development of Faecalibacterium 16S rRNA gene marker for identification of human faeces

Aims:  The focus of this study was to identify a bacterial 16S rRNA gene sequence, unique to microbiota in the human gut, for use in development of a dependable PCR assay to detect human faecal pollution in water.
Methods and Results:  Suppression subtractive hybridization (SSH) and bioinformatics were used to identify a genetic marker, within the 16S rRNA gene of Faecalibacterium , for the detection of human faeces. DNA sequencing analysis demonstrated that a majority (16) of 74 clones of the SSH library contained insertion sequences identified as Faecalibacterium 16S rRNA genes . Human faeces-specific sequences were derived and six PCR primer sets designed and tested against faecal DNA samples from human and nonhuman sources. One PCR primer set, HFB-F3 and HFB-R5, was exclusively associated with human faeces. These primers generated a human faeces-specific amplicon of 399 bp from 60·2% of human faecal samples and 100% of sewage samples.
Conclusions:  The subject Faecalibacterium marker is specific for sewage.
Significance and Impact of the Study:  This study represents the initial report of a Faecalibacterium marker for human faeces, which may prove useful for microbial source tracking.     

A real-time polymerase chain reaction assay for quantitative detection of the human-specific enterococci surface protein marker in sewage and environmental waters

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Use of a Bacteroides thetaiotaomicron-specific α-1-6, mannanase quantitative PCR to detect human faecal pollution in water

Aims: The aims of this work were to develop a quantitative test, based on Bacteroides thetaiotaomicron, for human faecal pollution in water and to evaluate test performance. Methods and Results: qPCR primers, based on the complete genomic sequence of B. thetaiotaomicron VPI 5482, were designed and tested. The single-copy putative mannanase homologue, α-1-6 mannanase, was selected as the particular target and sequences within this gene chosen as the qPCR primers by Blast search for specificity to B. thetaiotaomicron. The average concentration of B. thetaiotaomicron in human faeces was 1·39 × 10⁸ cells per gram faeces and the detection limit was 9·3 B. thetaiotaomicron copies per qPCR procedure. Comparison of B. thetaiotaomicron content in sewage vs pooled nonhuman faecal samples indicated that the current assay is specific for sewage. Conclusion: The subject assay is potentially useful for quantification of sewage pollution in water. Significance and Impact of the Study: Bacteroides-associated markers, proposed for faecal source tracking, have exclusively been based on gene sequences related to generally classified and uncultured bacteria. However, genes associated with host-microbe interaction have been suggested as more specific markers. The present assay targets such a gene of B. thetaiotaomicron which is considered to be a symbiont in the human gut.     

A quantitative real-time PCR assay for the highly sensitive and specific detection of human faecal influence in spring water from a large alpine catchment area

AIMS: The aim of the study was the development of a sensitive human-specific quantitative real-time PCR assay for microbial faecal source tracking (MST) in alpine spring water. The assay detects human-specific faecal DNA markers (BacH) from 16S rRNA gene sequences from the phylum Bacteroidetes using TaqMan minor groove binder probes. METHODS AND RESULTS: The qualitative and quantitative detection limits of the PCR assay were 6 and 30 marker copies, respectively. Specificity was proved by testing 41 human faeces and waste water samples and excluding cross-amplification from 302 animal faecal samples from Eastern Austria. Marker concentrations in human faecal material were in the range from 6.6 x 10(9) to 9.1 x 10(10) marker equivalents per gram. The method was sensitive enough to detect a few 100 pg of faeces in faecal suspensions. The assay was applied on water samples from an alpine karstic spring catchment area and the results reflected the expected levels of human faecal influence. CONCLUSIONS: The method exhibited sufficient sensitivity to allow quantitative source tracking of human faecal impact in the investigated karstic spring water. Significance AND IMPACT OF THE STUDY: The developed method constitutes the first quantitative human-specific MST tool sensitive enough for investigations in ground and spring water.     

New genotypes and factors associated with Cryptosporidium detection in mussels (Mytilus spp.) along the California coast

A 3 year study was conducted to evaluate mussels as bioindicators of faecal contamination in coastal ecosystems of California. Haemolymph samples from 4680 mussels (Mytilus spp.) were tested for Cryptosporidium genotypes using PCR amplification and DNA sequence analysis. Our hypotheses were that mussels collected from sites near livestock runoff or human sewage outflow would be more likely to contain the faecal pathogen Cryptosporidium than mussels collected distant to these sites, and that the prevalence would be greatest during the wet season when runoff into the nearshore marine environment was highest. To test these hypotheses, 156 batches of sentinel mussels were collected quarterly at nearshore marine sites considered at higher risk for exposure to livestock runoff, higher risk for exposure to human sewage, or lower risk for exposure to both faecal sources. Cryptosporidium genotypes detected in Haemolymph samples from individual mussels included Cryptosporidium parvum, Cryptosporidium felis, Cryptosporidium andersoni, and two novel Cryptosporidium spp. Factors significantly associated with detection of Cryptosporidium spp. in mussel batches were exposure to freshwater outflow and mussel collection within a week following a precipitation event. Detection of Cryptosporidium spp. was not associated with higher or lower risk status for exposure to livestock faeces or human sewage sources. This study showed that mussels can be used to monitor water quality in California and suggests that humans and animals ingesting faecal-contaminated water and shellfish may be exposed to both host-specific and anthropozoonotic Cryptosporidium genotypes of public health significance.     

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.     

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.     

Real-time quantitative PCR for enteric adenovirus serotype 40 in environmental waters

Adenoviruses 40 and 41 have been recognized as important etiological agents of gastroenteritis in children. A real-time PCR method (TaqMan assay) was developed for rapid quantification of adenovirus 40 (Ad40) by amplifying an 88 bp sequence from the hexon gene. To establish a quantification standard curve, a 1090 bp hexon region of Ad40 was amplified and cloned into the pGEM-T Vector. A direct correlation was observed between the fluorescence threshold cycle number (Ct) and the starting quantity of Ad40 hexon gene. The quantification was linear over 6-log units and the amplification efficiency averaged greater than 95%. Seeding studies using various environmental matrices (including sterile water, creek water, brackish estuarine water, ocean water, and secondary sewage effluent) suggest that this method is applicable to environmental samples. However, real-time PCR was sensitive to inhibitors present in the environmental samples. Lower efficiency of PCR amplification was found in secondary sewage effluent and creek waters. Application of the method to fecal contaminated waters successfully quantified the presence of Ad40. The sensitivity of the real-time PCR is comparable to the traditional nested PCR assay for environmental samples. In addition, the real-time PCR assay offers the advantage of speed and insensitivity to contamination during PCR set up. The real-time PCR assay developed in this study is suitable for quantitative determination of Ad40 in environmental samples and represents a considerable advancement in pathogen quantification in aquatic environments.     

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.     

Detection and identification by real time PCR/RFLP analyses of Cryptosporidium species from human faeces

AIMS: To detect a wide range of Cryptosporidium species from human faeces by analysis of the Cryptosporidium oocyst wall protein gene by PCR. METHODS AND RESULTS: The nested-assay comprised an initial amplification using a conventional thermocycler followed by real time PCR using a LightCycler with SYBR Green I for the characterization of the amplicons. The technique uses four sets of primers composed of five to six oligonucleotides with one to six base differences corresponding to the inter-species sequence differences of the gene fragment. Restriction fragment length polymorphism analysis identified Cryptosporidium hominis and C. parvum. The assay was evaluated using DNA extracted from purified material and faecal specimens containing a range of potential pathogens (including Cryptosporidium). The assay was specific, sensitive, reproducible and rapid. CONCLUSIONS: This unique technique enables the rapid detection of a range of polymorphic COWP gene sequences directly from faeces using real time PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a novel approach to identification of Cryptosporidium species and the identification of C. hominis and C. parvum. The technique may be especially useful for the analysis of environmental samples which are likely to contain heterogeneous mixtures of Cryptosporidium species.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli, faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli/faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the microtiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Detection of Helicobacter pylori DNA in human faeces and water with different levels of faecal pollution in the north-east of Spain

AIMS: To assess the role of water in the faecal transmission of Helicobacter pylori by detecting the DNA of this pathogen in human faecal samples and environmental water samples with a range of faecal pollution from the north-east of Spain. METHODS AND RESULTS: Semi-nested PCR was used to detect H. pylori in stools and water, both matrices with a complex biota. DNA was detected using highly specific primers of an ureA gene fragment. In addition, antigens were used to detect the bacteria in stools. Helicobacter pylori was detected in 33% of 36 human faecal samples and in 66% of wastewater samples, and 11% of river samples, but in none of the spring waters samples. Faecal pollution of the aquatic environment was tested analysing the presence of microbial indicators. CONCLUSIONS: We report the presence of H. pylori DNA in stools and in aquatic environments with different levels of faecal pollution, from the north-east of Spain. In this study a higher number of positive results were obtained in the more faecally polluted waters. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that water may be a vector of H. pylori in its faecal-oral route.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli , faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli /faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the mictrotiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR

Aims:  To develop a strain-specific rapid assay for identification and quantification of Lactobacillus rhamnosus GG in human faecal samples.
Methods and Results:  A unique random amplified polymorphic DNA (RAPD) band of the L. rhamnosus GG strain was isolated and sequenced. Strain-specific polymerase chain reaction (PCR) primers and probes were designed based on the sequence. Quantification was performed by the real-time PCR using a fluorescent resonance energy transfer (FRET) system. The specificity of the assay was tested with DNA isolated from a set of known strains and human faecal samples. The analytical sensitivity of the method for L. rhamnosus GG was about 10 CFU per assay, which corresponds to 10⁵ CFU g⁻¹ of wet faeces.
Conclusions:  Quantitative real-time PCR is a suitable method for strain-specific identification of L. rhamnosus GG in human faecal samples.
Significance and Impact of the Study:  Lactobacillus rhamnosus GG is one of the most studied probiotic strains in clinical trials but still lacks a DNA-based identification method. This study describes a real-time PCR method for strain-specific identification and quantification of L. rhamnosus GG in human faecal samples.     

Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification.

A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.     

A high‐throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)‐based approach to identify sources of faecal contamination in water bodies

As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides‐Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human‐associated Bacteroides spp. and three clusters of cow‐, pig‐ and dog‐specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high‐throughput method that can identify sources of faecal contamination.     

Detection of low numbers of Salmonella in environmental water, sewage and food samples by a nested polymerase chain reaction assay

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of <10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.     

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.