Repetitive Element Sequence-based PCR Research Articles

Prevalence and Typing of Listeria monocytogenes in Ready-to-Eat Food Products on the Belgian Market

Listeria monocytogenes is a major concern to producers of ready-to-eat foods because of the high mortality rate associated with listeriosis and the widespread nature of the organism. To investigate the prevalence of this pathogen in different ready-to-eat food products on the Belgian market, a variety of 252 ready-to-eat food products, mainly fish and meat products, were analyzed. Overall, L. monocytogenes was detected in 23.4% of the samples. The highest prevalence of L. monocytogenes was found in prepared minced meat (42.1%) and smoked halibut (33.3%). Contamination levels were in most cases low (<10 CFU/g); however, levels higher than 100 CFU/g were detected in some samples of smoked salmon, smoked halibut, and prepared minced meat. A high prevalence of Listeria innocua (15.8%) and Listeria welshimeri (36.8%) was detected in prepared minced meat. L. monocytogenes strains isolated from different contaminated products were subjected to repetitive element sequence-based PCR (REP-PCR) typing to determine possible associations with product type, producer, or market. REP-PCR patterns were analyzed using BioNumerics software, and seven different groups with at least 90% similarity were identified. The cluster analysis indicates that cross-contamination occurred at the producer and retail level. Serotype identification of the strains by PCR revealed that most belonged to the 1/2a(3a) serotype group.

Bacillus farraginis sp. nov., Bacillus fortis sp. nov. and Bacillus fordii sp. nov., isolated at dairy farms.

Forty-eight bacterial strains were isolated at dairy farms from raw milk, the milking apparatus, green fodder or feed concentrate after a heat treatment of 30 min at 100 degrees C. In this way, spore-forming bacteria with a very high intrinsic heat resistance were selected for. The aerobic spore-forming isolates were subjected to a polyphasic taxonomical study, including repetitive element sequence-based PCR typing, whole-cell protein profiling, 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base composition, fatty acid analysis, and morphological and biochemical characteristics. A comparison of the REP- and (GTG)5-PCR and whole-cell protein SDS-PAGE profiles resulted in three clusters of similar strains. Analysis of the 16S rDNA sequences and DNA-DNA relatedness data showed that these clusters represented three novel species. The highest 16S rDNA similarity to a recognized species found for the three groups was around 94% with Bacillus lentus and Bacillus sporothermodurans. Further phenotypic characterization supported the proposal of three novel species in the genus Bacillus, Bacillus farraginis, Bacillus fortis and Bacillus fordii. The respective type strains are R-6540T (=LMG 22081T=DSM 16013T), R-6514T (=LMG 22079T=DSM 16012T) and R-7190T (=LMG 22080T=DSM 16014T); their G+C DNA base contents are 43.7, 44.3 and 41.9 mol%, respectively. Although in variable amounts, a predominance of the branched fatty acids iso-C(15 : 0) and anteiso-C(15 : 0) was observed in all three novel species.

Increasing resistance to antimicrobial agents of Gram-negative organisms isolated at a London teaching hospital, 1995-2000.

To investigate the changes in resistance frequencies of common Gram-negative bacteria in a London teaching hospital. Antimicrobial susceptibilities were analysed for the 6 years 1995-2000. Gentamicin-resistant isolates from 1995 and 2000 were typed by a repetitive element sequence-based PCR (Rep-PCR) method. Resistance rates for all agents and all organisms were higher in isolates from inpatients than in those from outpatients or general practice. For most agents and most species there was a trend for a highly significant linear increase in resistance over the study period, and there was significant cross-resistance between different agents. Increases in resistance were especially marked in Klebsiella, Enterobacter and Acinetobacter spp., organisms that tend to cause outbreaks of hospital cross-infection. For example, the increases in gentamicin resistance in isolates from inpatients was from 2.9% to 23.5% for Klebsiella spp., from 0.3% to 20.8% for Enterobacter spp. and from 10.1% to 42.2% for Acinetobacter spp. There was much less increase in acquired resistance in Escherichia coli and Pseudomonas aeruginosa, organisms that tend to cause endogenous infections, with gentamicin resistance in isolates from inpatients increasing from 0.4% to 3.2% for E. coli and decreasing from 4.6% to 3.6% for P. aeruginosa. Rep-PCR typing showed considerable diversity amongst gentamicin-resistant isolates of E. coli and P. aeruginosa, but dominance by a limited number of presumably epidemic types of gentamicin-resistant isolates of the other species. Multiple antibiotic resistance has increased dramatically in some hospital isolates, and appears to be associated with hospital cross-infection.

Comparison of repetitive element sequence-based PCR with multilocus enzyme electrophoresis and pulsed field gel electrophoresis for typing Listeria monocytogenes food isolates

Repetitive element sequence-based PCR (rep-PCR) was compared with multilocus enzyme electrophoresis (MEE) and pulsed field gel electrophoresis (PFGE) for typing Listeria monocytogenes food isolates. All 45 L. monocytogenes food isolates were typable by rep-PCR using two primer sets (REP 1R-I REP 2-I and ERIC 1R ERIC 2), with good reproducibility though with less discriminatory power than MEE or PFGE. MEE and rep-PCR types were more stable than PFGE types over a given time period. All three typing methods usefully differentiated between closely related L. monocytogenes strains. Results obtained using rep-PCR concurred broadly with those obtained using MEE, while the former method proved to be more reproducibly and conveniently performed. The greater discriminatory power of PFGE enabled subtyping of rep-PCR and MEE types. When examining L. monocytogenes isolates collected over an extended period, the slightly less discriminatory but more stable electrophoretic types (ETs) distinguished by MEE are useful for confirmation and interpretation of PFGE data. However MEE is a cumbersome technique requiring a high degree of technical expertise in order to obtain reproducible results. In the present study rep-PCR conveniently and reliably produced typing data broadly congruent with that obtained using MEE which should prove useful in aiding interpretation of PFGE data for L. monocytogenes isolates obtained over extended time periods.

Aplicaciones de las técnicas de PCR a la epidemiología molecular de las enfermedades infecciosas

The development of new PCR-based typing methods in the last years have supposed an important advance in the study of infectious diseases. Arbitrarily primed PCR (AP-PCR) and repetitive element sequence-based PCR (rep-PCR) are the most widely used PCR-based fingerprinting methods for bacteria and fungi. Major advantages of these methods are flexibility, technical simplicity and high discriminatory power. The AP-PCR presents problems of low inter-run and inter-laboratory reproducibility which make necessary the optimization of the protocol and reagents. PCR-RFLP is based in the enzymatic digestion of polymorphic genes amplified by PCR. This method is easy to perform and discriminatory, although less than AP-PCR or rep-PCR. Amplified fragment length polymorphism (AFLP) is a highly reproducible and discriminatory typing method based on the amplification by PCR of restriction fragments obtained from chromosomic DNA. This method is more discriminative and reproducible than AP-PCR, rep-PCR and PCR-RFLP, but it is more time-consuming and expensive, and requires specialised personnel. Most of these PCR-based typing methods are less time-consuming, rapid and easy to perform and of interpretation than pulsed-field gel electrophoresis (PFGE; gold standard method for typing most bacterium and fungi), but they usually are less discriminative and reproducible than PFGE, depending on the species studied and the method of PCR used. In summary, there are several PCR-based methods which are useful as a primary approach to the study of the clonal relationship among microbial isolates. The selection of the method to be used depend on technical (rapid, low time-consuming, easy to perform and to interpret, reproducible and discriminatory) and economical (low cost) factors.

The rabbit as a new reservoir host of enterohemorrhagic Escherichia coli.

We investigated the prevalence of enterohemorrhagic Escherichia coli (EHEC) in rabbits acquired from two commercial vendors and a local petting zoo. Fecal samples from 34 Dutch Belted (DB) and 15 New Zealand White (NZW) rabbits were cultured; and isolates were biotyped, serotyped, tested by polymerase chain reaction (PCR), and genotyped by repetitive-element sequence–based PCR (Rep-PCR). Seven (25%) of 28 DB rabbits acquired from one commercial source were positive for EHEC, including O153:H- and O153:H7. One (9%) of 11 NZW rabbits from the same source was positive for eae-, stx1+ O153 strains. In contrast, six DB rabbits from another commercial source and four rabbits from a petting zoo were negative for EHEC. Rep-PCR demonstrated that the O153 EHEC and O145 enteropathogenic E. coli were two distinct clones. Our study indicates that rabbits are a new reservoir host of EHEC that may pose a zoonotic risk for humans.

DNA fingerprinting of Lactobacillus crispatus strain CTV-05 by repetitive element sequence-based PCR analysis in a pilot study of vaginal colonization.

Lactobacillus crispatus is one of the predominant hydrogen peroxide (H(2)O(2))-producing species found in the vagina and is under development as a probiotic for the treatment of bacterial vaginosis. In this study, we assessed whether DNA fingerprinting by repetitive element sequence-based PCR (rep-PCR) can be used to distinguish the capsule strain of L. crispatus (CTV-05) from other endogenous strains as well as other species of vaginal lactobacilli. Vaginal and rectal lactobacilli were identified to the species level by using whole-chromosome probe DNA hybridization. The DNAs from L. crispatus, L. jensenii, L. gasseri, and an as-yet-unnamed H(2)O(2)-negative Lactobacillus species designated 1086V were subjected to rep-PCR. The results of gel electrophoresis and ethidium bromide staining of the DNA fingerprints obtained were compared. L. crispatus CTV-05 had a unique DNA fingerprint compared to all other lactobacilli. DNA fingerprints for 27 production lots of L. crispatus sampled from 1994 through 2001 were identical to that of the original strain isolated in 1993, suggesting strain stability. In a pilot study of nine women, this DNA fingerprinting method distinguished CTV-05 from other endogenous vaginal lactobacilli prior to and after vaginal capsule use. rep-PCR DNA fingerprinting is useful for strain typing and for evaluating longitudinal loss or acquisition of vaginal lactobacilli used as probiotics.

Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system

A new method for isolation of methane oxidising bacteria (methanotrophs) is presented. Soil samples from a wetland area and a landfill were plated on polycarbonate membranes, which were incubated in a methane–air atmosphere using a non-sterile soil suspension as the medium. The membrane acted as a permeable growth support. The membrane method resulted in selective growth conditions, which allowed isolation of methane oxidising bacteria. The method resulted in isolation of both type I and type II methanotrophs from natural wetland and landfill soils. The isolates obtained from the landfill were dominated by type II methanotrophs and included several isolates carrying the gene for soluble methane monooxygenase (sMMO). Repetitive element sequence-based PCR fingerprinting documented genotypic diversity at the strain level. The presented method is a promising tool for easy and rapid isolation of different indigenous methanotrophs from an environment of interest.

Prevalence of Listeria monocytogenes during Production and Postharvest Processing of Cabbage

From November 1999 to May 2000, analyses of 425 cabbage, 205 water, and 225 environmental sponge samples from four cabbage farms with packing sheds and from two packing sheds in the Rio Grande Valley and Uvalde, Tex., were conducted to determine whether Listeria monocytogenes was present. Samples were tested by the Food and Drug Administration method for the isolation of Listeria spp., and confirmed isolates were DNA fingerprinted by repetitive-element sequence-based polymerase chain reaction (rep-PCR). L. monocytogenes was isolated from 3% (26 of 855) of the samples. Twenty of these isolates were obtained from cabbage (7 isolates from farms and 13 from packing sheds). Three isolates were from water samples (two from farms and one from a packing shed), and three were from environmental sponge samples of packing shed surfaces. Rep-PCR–generated fingerprints of 21 of the isolates revealed 18 distinctive banding patterns. Four isolates from environmental sponge samples of conveyor belts and from cabbage samples shared identical banding patterns, suggesting common sources of contamination. These identical environmental isolates suggest that contact with packing shed surfaces may be a source of contamination of cabbage. However, the cabbage samples could have arrived contaminated, since they were not washed.

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.

Characterisation of ropy slime-producing Lactobacillus sakei using repetitive element sequence-based PCR

Eighteen previously characterised Lactobacillus sakei strains exhibiting varying slime production capabilities in vacuum-packaged meat products were analysed using repetitive element sequence-based PCR (rep-PCR). The single primers BOXA1R and RW3A and the primer pair REP1R-Dt and REP2R-Dt were evaluated for their applicability in L. sakei genotyping. The five different patterns produced by RW3A were the least useful, with the discriminatory power equal to ribotyping. BOXA1R and REP–primer pair both produced six different banding patterns and the combination of these results yielded seven different rep-types. Rep-PCR was concluded to have approximately the same discriminatory power as randomly amplified polymorphic DNA (RAPD) analysis, but was inferior to pulsed-field gel electrophoresis (PFGE). However, if the results of rep-PCR and RAPD were combined, the discrimination was comparable to PFGE, with the exception that within Ribogroup I non-slime-producing strains were indistinguishable from weak slime producers. It was concluded that the combination of the two PCR-based typing techniques, rep-PCR and RAPD, would be a valuable tool in large scale contamination studies at meat processing plants, since results can be obtained rapidly with fewer isolates needing further analysis by PFGE.

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads™ and Ready-To-Go PCR Beads™ were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.

Molecular genotyping of Staphylococcus aureus strains: comparison of repetitive element sequence-based PCR with various typing methods and isolation of a novel epidemicity marker.

Repetitive sequence-based (Rep)-PCR genotyping as described here is based on the presence of homologues of Mycoplasma pneumoniae repeat-like elements in Staphylococcus. In this study we comparatively evaluated the usefulness of rep-PCR typing with two sets of well-defined collections of Staphylococcus aureus strains. Rep-PCR analysis of the first collection of S. aureus strains (n = 59) and one Staphylococcus intermedius strain showed 14 different rep-PCR patterns, with each pattern harboring 6 to 15 DNA fragments. The discriminatory power of rep-PCR typing compared well to those of arbitrarily primed PCR (average of 20 types) and pulsed-field gel electrophoresis (11 types). S. aureus strain collection I comprised four outbreak-related groups of isolates. The isolates in only one group were found to have identical rep-PCR profiles. However, in an analysis of isolates from three additional independent local outbreaks (n for outbreaks 1 and 2 = 5, n for outbreak 3 = 12), identical rep-PCR types were found among strains isolated during each outbreak. Therefore, we conclude that rep-PCR genotyping may be an easy and fast method for monitoring of the epidemiology of nosocomial Staphylococcus infections. Rep-PCR analysis of strain collection II, which consisted of epidemic and nonepidemic methicillin-resistant S. aureus (MRSA) strains, revealed that a cluster of similar rep-PCR profiles was found among MRSA isolates which were more frequently isolated and which were most often associated with outbreaks.

Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR.

Listeria monocytogenes strains possess short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. We used repetitive element sequence-based PCR (rep-PCR) to evaluate the potential of REP and ERIC elements for typing L. monocytogenes strains isolated from humans, animals, and foods. On the basis of rep-PCR fingerprints, L. monocytogenes strains were divided into four major clusters matching origin of isolation. rep-PCR fingerprints of human and animal isolates were different from those of food isolates. Computer evaluation of rep-PCR fingerprints allowed discrimination among the tested serotypes 1/2a, 1/2b, 1/2c, 3b, and 4b within each major cluster. The index of discrimination calculated for 52 epidemiologically unrelated isolates of L. monocytogenes was 0.98 for REP- and ERIC-PCR. Our results suggest that rep-PCR can provide an alternative method for L. monocytogenes typing.

Typing of Bacillus sporothermodurans and other Bacillus species isolated from milk by repetitive element sequence based PCR.

When analysing raw milk for the presence of Bacillus sporothermodurans, 11 Bacillus strains were isolated which could be differentiated from known Bacillus, Brevibacillus and Paenibacillus spp. by different primer specificity in PCR experiments, indicating that they probably belong to Bacillus species as yet undescribed. Using repetitive element sequence based PCR (REP-PCR), these 11 strains could be clearly distinguished from B. sporothermodurans as well as from each other. Eighty-five B. sporothermodurans strains were characterized by a typical REP-pattern. Using REP-PCR combined with separation on non-denaturing polyacrylamide gel electrophoresis and silver staining, individual B. sporothermodurans strains could be discriminated, which was not possible by methods previously published.

Diversity of DNA sequences among Vibrio cholerae O1 and non‐O1 isolates detected by whole‐cell repetitive element sequence‐based polymerase chain reaction

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.

Genomic fingerprints of Salmonella species generated with repetitive element sequence-based PCR

We describe the use of repetitive element sequence-based PCR (rep-PCR) on the two repetitive sequences, REP and ERIC elements, to distinguish members of closely related Salmonella species. Within the species, ERIC–PCR showed a higher discriminative potential than REP–PCR, but by using a combination of the two PCR methods it was possible to distinguish all the isolates examined. The rep-PCR fingerprints of Salmonella organisms were distinctly different from some Gram-positive bacteria, for example Staphylococcus, Bacillus megaterium, and even the closely related Escherichia coli and Serratia marcescens. Identical fingerprints were observed with whole-cell preparations. Rapid specimen preparation has enhanced the value of rep-PCR in timely analysis of epidemiological relationships.

Repetitive element sequence-based PCR for species and strain discrimination in the genus Listeria

Repetitive element sequence-based PCR (rep-PCR) was used to generate DNA fingerprints for Listeria spp. Two primer sets (REP 1R-I REP 2-I and ERIC 1R ERIC 2) used in respectively REP- and ERIC-PCR revealed that bacteria of the genus Listeria possess short repetitive extragenic palindromic elements and enterobacterial repetitive intergenic consensus sequences. Specific band profiles obtained by ERIC-PCR enabled the identification of Listeria species. With both REP- and ERIC-PCR the L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3b and 4b could be clearly distinguished from each other. Within the serotype 1/2a, REP-PCR showed a higher discriminative potential than ERIC-PCR and a comparable discriminative potential as RAPD combining 3-4 primers.

Genetic diversity of Neisseria gonorrhoeae IB-2 and IB-6 isolates revealed by whole-cell repetitive element sequence-based PCR.

Phenotypic characterization of 19 Neisseria gonorrhoeae serovar IB-2 and 8 serovar IB-6 isolates by the combined use of auxotypes, serological characterization, and penicillin susceptibility testing indicated intraserovar genetic diversity. In the present study, we applied whole-cell repetitive element sequence-based PCR (rep-PCR) analysis which allows a rapid assessment of the clonal relationships of IB-2 and IB-6 isolates. DNA templates were prepared by boiling cells harvested directly from plate cultures, eliminating the need for time-consuming phenol extraction. Six different rep-PCR profiles were established among the 19 IB-2 isolates. Rep-PCR typing results had a good correlation with pulsed-field gel electrophoresis patterns. It is slightly less discriminatory than BglII-generated macrorestriction pattern analysis by pulsed-field gel electrophoresis. It is capable of discriminating epidemiologically related from epidemiologically unrelated IB-2 isolates. It should serve as a rapid and useful subtyping tool for epidemiologic investigations in which there is a predominance of major serovar groups.

Molecular genotyping of methicillin-resistant Staphylococcus aureus via fluorophore-enhanced repetitive-sequence PCR.

Methicillin resistance in Staphylococcus aureus is a frequent cause of nosocomial and community-acquired infections. Accurate, rapid epidemiologic typing is crucial to the identification of the source and spread of infectious disease and could provide detailed information on the generation of methicillin-resistant S. aureus (MRSA) strains. The high degree of genetic relatedness of MRSA strains has precluded the use of more conventional methods of genetic fingerprinting. A rapid DNA fingerprinting method that exploits PCR amplification from a DNA repeat sequence in MRSA is described. The random chromosomal distribution of this repeat sequence provides an ideal target for detecting DNA fragment patterns specific to individual MRSA strains. Two PCR fingerprinting methods which use an oligonucleotide primer based on a repetitive sequence found in Mycoplasma pneumoniae are presented. The repetitive element sequence-based PCR (rep-PCR) and fluorophore-enhanced rep-PCR (FERP) can identify epidemic strains among background MRSA. The combination of oligonucleotide primers labeled with different fluorescent dyes allowed simultaneous FERP fingerprinting and mecA gene detection. Eight different fingerprint patterns were observed in MRSA strains collected from different sources. These techniques provide a rapid discriminatory means of molecular epidemiologic typing of MRSA involved in nosocomial infections.