Comparison of rpoA and pheS Gene Sequencing to 16S rRNA Gene Sequencing in Identification and Phylogenetic Analysis of LAB from Probiotic Food Products and Supplements

An investigation of the relevance of sequenced protein-coding genes in comparison with 16S rRNA gene sequences to correctly delineate and discriminate between closely related Lactobacillus isolates is presented. These reportedly probiotic Lactobacillus strains were obtained from probiotic supplements and food products. Probiotic Lactobacillus species have been found to have several potential health benefits and technological properties which may be strain-dependent. It was deemed necessary to investigate molecular techniques for the identification and discrimination between closely related isolates. The 16S rRNA, pheS, and rpoA genes were amplified in a polymerase chain reaction using the oligonucleotide pairs 27F/1492R, pheS-21-F/pheS-23-R, and rpoA-21-F/rpoA-23-R. The PCR amplicons were separated using gel agarose electrophoresis and observed by the Bio-Rad Gel Doc^TM XR+ Imaging system transilluminator. Sequencing of the PCR amplicons was done in an ABI PRISM^TM 3100 genetic analyzer. MEGA 5.05 software was employed in doing the phylogenetic analysis. Identification by 16S rRNA gene sequencing was confirmed by pheS and rpoA gene sequencing. Subsequent phylogenetic analysis and concatenation showed that interspecies and intraspecies degrees of diversity and similarity were better enabled by pheS and rpoA primers. Overall, findings revealed cases of inaccurate identification of Lactobacillus strains in probiotic supplements on the market, highlighting the relevance of the multigenic approach in more accurately identifying Lactobacillus strains. Supplemental materials are available for this article; see the publisher's online edition of Food Biotechnology to view the supplemental file.     

Cloning,Expression, and Genus-Specificity Analysis of 28-kDa OmpK from Vibrio alginolyticus

ABSTRACT: Some Vibrio species are universal marine pathogens and Vibrio infections are often encountered due to consumption of raw or uncooked seafood. The outer membrane proteins, playing a key role in interaction between bacteria and hosts, are potential candidates for development of vaccine and markers of the genus Vibrio. In this study, the ompK (outer membrane protein K) genes of Vibrio alginolyticus, V. vulnificus, V. parahaemolyticus, V. fluvialis, and V. mimicu were cloned with 798 to 822 nucleotides encoding 266 to 274 amino acids. The ompK gene from V. alginolyticus was expressed in Escherichia coli using pET-22b expression vector. The recombinant fusion OmpK protein with 6×His tag was purified with nickel chelate affinity chromatography. The polyclonal antibody (titer, 1:102400) against V. alginolyticus OmpK was developed in guinea pigs and it positively reacted with each of 5 Vibrio species but negative to other 18 Gram-negative bacterial strains. The result suggests that Vibrio OmpK protein could be a genus-specific antigen, which can be used for developing vaccines and rapid detection of multiple Vibrio species.     

Primers for Amplifying an Alanine Racemase Gene Fragment to Detect E. coli Strains in Foods

Specific oligonucleotide primers for detecting Escherichia coli in various foods were designed based upon the conserved sequences of the E. coli air gene from positions 322 to 345 and from 664 to 687. Bacteria and food samples were treated at 100°C for 10 min in 1% Tween 20 containing 5% NaCl and 1 mM EDTA, then used as templates for polymerase chain reaction (PCR). The oligonucleotide primers were specific to E. coli, except for Shigella species, when tested with 67 strains of E. coli, including such serotypes as O157:H7 and O111, and 32 strains of non-E. coli species. The oligonucleotide primers could prove useful for detecting E. coli in beef, chicken, pork, tomato, soybean, potato, cow's milk, and egg.     

Development of novel species‐specific primers for species identification of the Lactobacillus casei group based on RAPD fingerprints

BACKGROUND: It is difficult to clearly distinguish and identify specific species of the Lactobacillus casei group using phenotypic and genotypic (16S ribosomal DNA sequence analysis) techniques alone. Some species of this group are probiotic and are widely used in the food and feed industries. The objective of this study was to develop species‐specific primers based on randomly amplified polymorphic DNA (RAPD) fingerprinting for species identification within the closely related L. casei group of bacteria. RESULTS: Three random primers termed OPT‐14, OPA‐11 and OPT‐16 were developed for analysis. The primer pairs each produced a species‐specific band found only in the tested Lactobacillus rhamnosus, Lactobacillus paracasei subsp. tolerans and Lactobacillus zeae isolates respectively. These specific fragments were then sequenced for further analysis. The species‐specific primers were designed according to cloned sequencing, which was employed for polymerase chain reaction (PCR) with the template DNA of Lactobacillus strains. Single 102, 179 and 451 bp species‐specific bands were found only in L. rhamnosus, L. paracasei subsp. tolerans and L. zeae respectively. CONCLUSION: Using PCR, the novel species‐specific primers have been shown to rapidly, accurately and effectively identify species of L. rhamnosus, L. paracasei subsp. tolerans and L. zeae from within the L. casei group of probiotic bacteria. Copyright © 2009 Society of Chemical Industry     

A PCR-Based Assay for the Detection and Differentiation of Potential Fumonisin-Producing Fusarium verticillioides Isolated from Indian Maize Kernels

One hundred and three Fusarium isolates from maize samples collected from different districts of Karnataka state, India, were analyzed with genus-specific, species-specific, and potential fumonisin specific oligonucleotide primers. One set of genus-specific primers ITS F and ITS R based on a highly conserved ITS region of the genus Fusarium were used to differentiate Fusarium species from closely related genera. All the Fusarium species tested scored positive with the ITS pair of primers. Detection and identification of Fusarium verticillioides species was done by using a newly designed reverse primer VERT-R (5′- CGA CTC ACG GCC AGG AAA CC ?3′) based on an intergenic spacer sequence (IGS) combined with an already designed forward primer VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) published previously. Out of 103 Fusarium species tested, 83 isolates of F. verticillioides scored positive for VERTF-1/ VERT-R species-specific pair of primers. Further to discriminate potential fumonisin-producing and nonproducing strains of F. verticillioides, the VERTF-1/VERTF-2 set of primers [VERTF-1 (5′-GCG GGA ATT CAA AAG TGG CC -3′) and VERTF-2 (5′-GAG GGC GCG AAA CGG ATC GG -3′)] were used. 64 isolates of F. verticillioides scored positive for VERTF-1/ VERTF-2 pair of primers. In total, three primers, one forward primer VERTF-1 and two reverse primers VERT-R and VERTF-2, were used for the confirmation of F. verticillioides up to the species level and the second pair of primers were used to confirm the potential for fumonisin production. The developed PCR assay should provide a powerful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in a population.     

Species identification of Wickerhamomyces anomalus and related taxa using β‐tubulin (β‐tub) DNA barcode marker

Wickerhamomyces anomalus is used in food and feed processing, although the species has been reported as an opportunistic human pathogen, predominantly in neonates. Neither phenotypic nor the most frequently applied genotypic marker (D1/D2 LSU ribosomal DNA) provide sufficient resolution for accurate identification of this yeast. In this study, the β‐tubulin gene was used for species identification by direct DNA sequencing and as marker in a species‐specific PCR assay. The results showed that all examined W. anomalus strains were clearly distinguished from the closely related species by comparative sequence analysis of the β‐tubulin gene. In addition, the species‐specific primers were also developed based on the β‐tubulin gene, which was employed for polymerase chain reaction with the template DNA of Wickerhamomyces strains. A single 218 bp species‐specific band was found only in W. anomalus. Our data indicate that the phylogenetic relationships between these strains are easily resolved by sequencing of the β‐tubulin gene and combined with species‐specific PCR assay. Copyright © 2012 John Wiley & Sons, Ltd.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene

BACKGROUND: To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species‐specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. RESULTS: The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species‐specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231‐bp species‐specific band was found only in L. delbrueckii. A SNaPshot mini‐sequencing assay using recA as a target gene was also developed. The specificity of the mini‐sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. CONCLUSION: The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species‐specific polymerase chain reaction combined with SNaPshot mini‐sequencing. Copyright © 2012 Society of Chemical Industry     

Identification of spoilage yeasts in a food-production chain by microsatellite polymerase chain reaction fingerprinting

A survey of yeast strains present in the production chain of mayonnaise and salad dressings was carried out over a period of 14 months. Attempts were made to identify the isolated yeasts with the API system, but identification of all species involved was not possible. In the investigation the performance of the microsatellite polymerase chain reaction (PCR) fingerprinting analysis with the microsatellite oligonucleotide primers (GAC)₅and (GTG)₅appeared to be superior. Several yeast species were encountered in the production lines but only the speciesZygosaccharomyces bailiiandZygosaccharomyces bisporuswere present in the final products. Only microsatellite PCR fingerprinting analysis allowed discrimination between species of theZygosaccharomycesgenus. In addition, the PCR-based technique allowed the discrimination of different types within theZ. bailiispecies.Z. bailiistrains isolated from spoiled products displayed PCR-fingerprinting types that appeared to be identical to some of those generated by strains isolated from one specific production line. This suggested that microsatellite PCR fingerprinting is useful in tracing back the origin of spoilage outbreaks, and that it can be applied in microbiological quality assurance monitoring systems in industrial environments.     

The Use of Molecular Methods for Detecting and Discriminating Salmonella Associated with Foods — A Review

The genus Salmonella is composed of two species, Salmonella enterica and Salmonella bongori. Only S. enterica subsp. enterica is considered of human clinical significance and consists of 1478 serotypes. A large number of virulence genes and virulence-enhancing genes have been described for Salmonella. There are more than 30 Salmonella specific genes that have been used for the polymerase chain reaction to detect and characterize Salmonella. The sensitivity of detection of Salmonella from complex matrices such as food and feces by PCR is invariably enhanced using nonselective or selective enrichment, particularly if followed by immuno-magnetic separation in addition to coupling the PCR with ELISA formats. R-plasmids are considered to be the main factors responsible for the horizontal transfer of antibiotic resistance genes in Salmonella. A sizeable number of primer pairs are available for determining by the PCR the presence of many antibiotic resistance genes in Salmonella isolates that are not necessarily specific for Salmonella. The collective PCR detection of members of the genus Salmonella in foods and environmental samples has been achieved by amplification of invA gene sequences that are highly conserved among all Salmonella serotypes in addition to the amplification of his gene sequences also present throughout the genus Salmonella. Amplification of 16S rDNA sequences have also been found useful for genus specific detection of Salmonella. d-Tartrate (dT⁺) fermenting strains have been found to result in less severe gastrointestinal infections than d-tartrate-nonfermenting (dT^-) strains. Primers have therefore been developed for distinguishing between (dT⁺) and (dT^-) strains. Among the molecular techniques available for strain discrimination of Salmonella isolates, pulsed field gel electrophoresis, random amplified polymporphic DNA analysis, ribotyping, multilocus sequence typing, subtracted finger printing, and enterobacterial repetitive intergeneric consensus typing have been found useful. Multiplex PCR has been found effective for simultaneously detecting Salmonella and other pathogens in foods, particularly with real-time PCR.     

Development of a genus-specific PCR combined with ARDRA for the identification of Leuconostoc species in kimchi

With Leuconostoc species being increasingly isolated from many fermented kimchi, a systematic identification of this genus is required, for which an amplified ribosomal DNA restriction analysis (ARDRA) has been developed. This method applies both polymerase chain reaction and restriction pattern analysis using a restriction enzyme. First, using specific primers that were designed from lactic acid bacteria (LAB) sequences, 217 bp specific to LAB was produced. Then, restriction analysis using the MspI was applied to identify these bacteria at the species level. This study demonstrates novel ARDRA technique for the identification of Leuconostoc species was successful in identifying according to the genomic species.     

PCR‐Based Differentiation and Homology of Brewing and Type Strains of the Genus Saccharomyces

Restriction fragment length polymorphism (RFLP) patterns of PCR‐amplified ribosomal RNA gene fragments (rDNA) and randomly amplified polymorphic DNA (RAPD) were applied for the analysis of 15 brewing and 6 related yeast strains of the genus Saccharomyces. One five‐base (ScrFI) and two four‐base cutting (HaeIII, MspI) restriction enzymes were used. The primers 21 and M13 core sequence were selected for RAPD analysis. PCR‐RFLP rDNA analysis with HaeIII, ScrFI and MspI differentiated the strains tested into four, five and four types of patterns, respectively and the analyses of the profiles showed 100% homology, between the yeast strains. One strain was an exception. Homological groups were observed for strains used in breweries globally, from a local production strain and from the isolates identified as S. cerevisiae. Using RAPD analysis, and according to discrete differences in the profiles, it was possible to divide twenty one strains into 15 and 20 groups with primer 21 and M13 respectively. RFLP‐PCR rDNA analysis was used to show similarities in closely related brewing strains, while RAPD analysis was used for differentiation of strains.     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Development of Molecular Typing Methods for Bacillus spp. and Paenibacillus spp. Isolated from Fluid Milk Products

Bacillus spp. and related sporeformers are important food spoilage organisms. While use of molecular subtyping methods has provided important information on the ecology and transmission of foodborne pathogens, the lack of rapid, reliable, and affordable subtyping methods for Bacillus spp. has limited our ability to understand and control their transmission throughout the food chain. We used a previously described collection of Bacillus spp. and Paenibacillus spp. isolated from dairy products to develop a DNA sequencing‐based subtyping approach for these spoilage microorganisms. After optimization of polymerase chain reaction (PCR) parameters, primers targeting the rpoB housekeeping gene allowed for successful amplification in all isolates. rpoB sequencing allowed differentiation of 29 subtypes (that is, sequence types) among the 57 isolates characterized. Phylogenetic analyses of rpoB sequences revealed distinct monophyletic lineages that correlated with bacterial genera (Bacillus and Paenibacillus) as well as with species or species‐like assemblages within each genus. rpoB sequencing provided improved subtype discrimination over 16S rDNA sequencing; therefore, rpoB sequencing allows for both sensitive subtype discrimination as well as for species and genus identification. Analysis of subtypes isolated over time in dairy products revealed the presence of both persistent and transient bacterial subtypes, indicating that application of these methods can improve our understanding of the ecology of these spoilage organisms and can help in identification of bacterial niches that may contribute to the persistence of these spoilage organisms in food systems.     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

Nucleotide Sequence Identities of horA Homologues and Adjacent DNA Regions Identified in Three Species of Beer‐Spoilage Lactic Acid Bacteria

The horA homologues and adjacent DNA regions identified in beer‐spoilage Lactobacillus lindneri DSM 20690^Tand L. paracollinoides DSM15502^Twere examined and compared with the corresponding DNA region of beer‐spoilage L. brevis ABBC45, a strain in which the hop‐resistance gene horA was originally identified. The PCR analysis suggests ORFB1‐B5 regions surrounding horA are conserved in all of the strains. The nucleotide sequence comparison of the conserved DNA regions revealed extremely high levels of identities among the three beer‐spoilage strains that are not typical for distinct species. The PCR methods using primers specific to the adjacent ORFs were found to be able to differentiate beer‐spoilage Lactobacillus strains from non‐spoilers, indicating these ORFs are also useful genetic markers for determining the beer‐spoilage ability of lactobacilli. The presence or absence of the adjacent ORFs in 92 bacterial strains was completely identical with that of horA homologues, indicating the ORFB1‐B5 regions are generally conserved in beer‐spoilage Lactobacillus strains. Taken together, these results suggest the ORFB1‐B5 regions have been acquired by beer‐spoilage lactobacilli through horizontal gene transfer and provide a theoretical basis for applying a trans‐species genetic marker such as horA to deal with unencountered species of beer‐spoilage lactobacilli.     

Comparison and evaluation of molecular methods used for identification and discrimination of lactic acid bacteria

BACKGROUND: Lactobacillus and Bifidobacterium strains are present in a great variety of habitats, including fermented products, probiotic concoctions and the human colon. Some species are so closely related that it is difficult to distinguish them by microbiological techniques. Nevertheless, discrimination of isolates is an important issue in respect of application, and molecular methods such as restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD) or species‐specific polymerase chain reaction (PCR) might help in resolving this problem. In this study, PCR, RFLP and sequencing were applied to identify lactobacilli and bifidobacteria originating from various sources and the DSMZ strain collection. RESULTS: The microbiological composition of foods was analysed by molecular methods. Using species‐specific PCR primers, three restriction enzymes (AluI, HhaI and RsaI) and sequencing, three Bifidobacterium and six Lactobacillus reference strains could be distinguished and four additional lactobacilli of food origin were identified. CONCLUSION: A combination of three molecular methods resulted in successful discrimination of nine reference strains and four isolates of food origin. Since these methods are not always accurate owing to their high genetic homogeneity, it is advisable to use more than one method for the identification of L. casei and closely related species. Copyright © 2012 Society of Chemical Industry     

Evaluating the use of fatty acid profiles to identify deep-sea Vibrio isolates

Capillary gas chromatography with flame ionisation detection (GC-FID) was used to determine the cellular fatty acid (CFA) profiles of a number of Vibrio strains obtained from ATCC and grown on various media. This initial determination for optimal growth conditions, including type of medium and incubation temperature, for these various ATCC Vibrio species, was important for use in the subsequent evaluation of deep-sea Vibrio strains. The deep-sea Vibrios were obtained from Harbor Branch Oceanographic Institution (Fort Pierce, FL), and GC-FID analysis was used to determine whole cell fatty acid methyl esters (FAMEs) from the cells. The Vibrio strains were cultured for 24 h on a specific medium which included brain heart infusion (BHI) agar, trypticase soy agar (TSA), trypticase soy broth agar (TSBA), and Luria–Bertani (LB) agar. The temperature of incubation was 28 °C for cells grown on TSA, TSBA, and LB and 35 °C for BHI. A data set for each Vibrio species was prepared, using fatty acid profiles for a specific medium. Major fatty acids of the Vibrio strains evaluated in this study were straight-chain C_12:0, C_14:0, C_16:0, and unsaturated summed C_16:1 ω7c/C_16:1 ω6c, C_18:1 ω7c, and summed C_14:0 3-OH/iso-C_16:1. Most of the deep-sea Vibrio isolates were identified as Vibrio parahaemolyticus and Vibrio harveyi. Analysis of FAMEs from Vibro strains grown on a specific medium by this rapid GC-FID method can provide a sensitive procedure for the identification of these organisms, and the differentiation between Vibrio species.     

Multiplex PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in rice and fingermillet collected from southern India

BACKGROUND: The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In the present study, a multiplex PCR was standardised for the group‐specific detection of fumonisin‐producing and trichothecene‐producing strains of Fusarium species. Primers for genus‐level recognition of Fusarium spp. were designed from the internal transcribed spacer regions 1 and 2 of rDNA. Primers for group‐specific detection were designed from the tri5 and tri6 genes involved in trichothecene biosynthesis and the fum1 and fum13 genes involved in fumonisin biosynthesis. RESULTS: Among the various genera and their strains tested, all the 85 confirmed Fusarium strains were positive for rDNA gene and the rest stayed negative. From among the Fusarium strains, 15 had amplification for trichothecene‐ and 20 for fumonisin‐encoding genes. All PCR positive trichothecene chemotypes of Fusarium species tested were positive for chemical analysis but in the case of fumonisins, of the 20 PCR positive cultures, only 13 showed positive for chemical analysis by HPTLC. CONCLUSION: The assay described here provided a rapid and reliable detection of trichothecene‐ and fumonisin‐producing Fusarium directly from natural food grains and the results were always comparable with a conventional HPTLC detection method. It can, therefore, be used by the food industry to monitor quality and safety. Copyright © 2011 Society of Chemical Industry     

PCR-RFLP analysis of the rpoB gene to distinguish the five species of Cronobacter

Members of the genus Cronobacter are opportunistic pathogens associated with life-threatening infections in immuno-compromised individuals. Polyphasic analysis has facilitated the classification of the novel genus Cronobacter containing five species. However, since this recent reclassification there are not many identification methods optimised for differentiation between the five Cronobacter species. This differentiation between the species is of importance as there are indications that the species may be diverse regarding their virulence. The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol to differentiate between the five Cronobacter species. The rpoB gene of 49 Enterobacteriaceae strains, including 33 Cronobacter strains was amplified using conventional PCR, followed by digestion of these PCR products with restriction endonucleases MboI, HinP1I and Csp6I. The PCR-RFLP analysis with single digestions of each of the restriction endonucleases did not distinguish between all five Cronobacter species. This study describes the successful differentiation of the five Cronobacter species based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. This PCR-RFLP assay is an accurate identification method that ensures rapid differentiation between the five species of Cronobacter.