Molecular epidemiological tools for Salmonella Dublin typing

Abstract A total of 32 strains of Salmonella Dublin recovered from cattle were differentiated by electrophoretic typing of their esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), arbitrarily primed PCR (AP-PCR) using five primers, PCR based on repetitive extragenic palindromic sequences (REP-PCR) and PCR based on enterobacterial repetitive intergenic consensus sequences (ERIC-PCR). ERIC-PCR and REP-PCR each gave one type, zymotyping gave three, AP-PCR gave five and ribotyping gave seven types. Combination of ribotyping and AP-PCR produced a total of 11 types, whereas 14 different types were obtained by all five methods. Thus a combination of several methods enhanced the discrimination of cattle-adapted strains among the genotypically homogeneous serovar Salmonella Dublin.     

Actinobacillus actinomycetemcomitans genetic heterogeneity: amplification of JP2-like ltx promoter pattern correlated with specific arbitrarily primed polymerase chain reaction (AP-PCR) genotypes from human but not marmoset Brazilian isolates

Specific clonal types of Actinobacillus actinomycetemcomitans, a major human periodontal pathogen, may be responsible for clinical manifestations and the production of leukotoxin virulence factors. Leukotoxicity is associated with genetic polymorphism at the promoter region of the leukotoxin (lItx) gene. Here, we describe the use of arbitrarily primed polymerase chain reaction (AP-PCR) and ltx promoter PCR to molecularly characterise 35 A. actinomycetemcomitans Brazilian isolates: 21 of human origin and 14 from captive marmosets (Callitrix spp., primates commonly used as animal models for periodontal research). The discriminative capacity of each of 12 arbitrary primers was found to be variable, yielding between 3 and 24 PCR amplitypes. Combination of the results for all primers led to characterisation of 14 genotypes that grouped into four major clusters based on genetic similarity. Clusters 2, 3, and 4 were discriminative to host origin. A correlation with periodontal disease was suggested for strains belonging to clusters 3 and 4. The JP2-like PCR amplification pattern, associated with highly leukotoxic strains, was exclusive to human isolates and present in 29% of human isolates where it occurred in close relationship with AP genotypes L and J (cluster 3).     

Genetic Relatedness among Environmental, Clinical, and Diseased-Eel Vibrio vulnificus Isolates from Different Geographic Regions by Ribotyping and Randomly Amplified Polymorphic DNA PCR

Genetic relationships among 132 strains of Vibrio vulnificus (clinical, environmental, and diseased-eel isolates from different geographic origins, as well as seawater and shellfish isolates from the western Mediterranean coast, including reference strains) were analyzed by random amplified polymorphic DNA (RAPD) PCR. Results were validated by ribotyping. For ribotyping, DNAs were digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene. Random amplification of DNA was performed with M13 and T3 universal primers. The comparison between ribotyping and RAPD PCR revealed an overall agreement regarding the high level of homogeneity of diseased-eel isolates in contrast to the genetic heterogeneity of Mediterranean isolates. The latter suggests the existence of autochthonous clones present in Mediterranean coastal waters. Both techniques have revealed a genetic proximity among Spanish fish farm isolates and a close relationship between four Spanish eel farm isolates and some Mediterranean isolates. Whereas the differentiation within diseased-eel isolates was only possible by ribotyping, RAPD PCR was able to differentiate phenotypically atypical isolates of V. vulnificus. On the basis of our results, RAPD PCR is proposed as a better technique than ribotyping for rapid typing in the routine analysis of new V. vulnificus isolates.     

Relationships between arginine degradation, pH and survival in Lactobacillus sakei

Ribotyping and RAPD profiling of a collection of 18 Streptococcus parauberis strains isolated from diseased turbot in Galicia (NW Spain) was performed in order to analyze the possible genetic variability within this bacterial fish pathogen. In addition, the value of this technique for intraspecific classification and epidemiological studies was evaluated. Ribopatterns of DNA digested with three endonucleases and hybridized with a cDNA probe complementary to highly conserved sequences in the 16S and 23S rRNA genes showed a great homogeneity among the turbot isolates. Compared with ribotyping, RAPD appeared to be a reliable and fast technique for discriminating between isolates of S. parauberis on the basis of their farm of isolation and, therefore, represents a powerful tool for epidemiological studies of this fish pathogen.     

Arbitrary-primed PCR for genomic fingerprinting and identification of differentially regulated genes in Indian isolates of Leishmania donovani

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within the Indian isolates and to identify differentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could be differentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, differences within the Indian isolates could also be identified. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern blot analysis. Three such fragments were found to represent transcribed sequences that were differentially expressed in the two stages of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in genomic fingerprinting and in identifying differentially transcribed sequences that could potentially contribute to parasite virulence.     

Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides.

We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma. The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively. Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P. syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms. The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively. All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393. Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P. syringae pv. syringae. A total of 100 randomly chosen P. syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique. An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups. The plasmid profiles were specific to isolates belonging to particular AP-PCR groups. Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied.     

Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis

Several molecular methods are currently available for identification and discrimination of bacterial strains within the same species, which vary in efficiency and required labour. Here we applied a novel method for fingerprinting genomes, called arbitrarily primed PCR (AP-PCR), to the delineation of strains within the species Porphyromonas gingivalis. Using a single primer on a set of nine strains, nine simple distinct banding patterns, indicative of genetic polymorphism, were observed. Common amplicons and amplicons shared by only some strains were also observed, the latter suggesting that AP-PCR can be used to generate polymorphic markers. Genomic fingerprinting obtained by AP-PCR was independent of the quality of DNA. Assays performed directly using whole cells as a source of DNA template indicated that AP-PCR from colony is a quick, simple and accurate procedure.     

Identification of enterococci by ribotyping with horseradish-peroxidase-labelled 16S rDNA probes.

Enterococci are frequently associated with hospital-acquired infection. Identification of enterococci using conventional biochemical tests are often tedious to perform in a routine diagnostic laboratory and may give equivocal results. This study evaluates the usefulness of ribotyping by DNA hybridisation to identify 68 members of the bacterial genus Enterococcus characterised by a conventional test scheme. DNA probes (830 bp in size) were derived from the 16S rRNA gene of E. coli or E. faecalis by PCR, labelled with horseradish peroxidase and used in Southern blot hybridisations of enterococcal DNA digested with EcoRI. Unique ribotypes were obtained for 11 different species using 12 Enterococcus type strains. Ribotyping identified 44 E. faecalis isolates, 19 E. faecium isolates, two E. durans isolates and one E. avium isolate in concordance with results of the biochemistry tests. Two isolates that had ribotype patterns identical to the E. faecium type strain were unable to be definitively identified by biochemical tests. The results show that ribotyping is able to differentiate between E. faecium and E. faecalis and may be useful for identifying other enterococci in the hospital setting. In addition, ribotyping using DNA probes and enhanced chemiluminescence is a safe and more reproducible alternative to radiolabelling RNA in a clinical microbiology laboratory.     

Molecular identification of vaginal lactobacilli isolated from Bulgarian women

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in␣comparison with a set of 21 reference strains based␣on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of␣them to be classified as belonging to one of␣the␣following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.     

Investigations of genetic variation between olive (Olea europaea L.) cultivars using arbitrarily primed polymerase chain reaction (AP-PCR)

Characterization and selection of olive clones for the production of olive oil is essential in Turkey because of its profitable exploitation. AP-PCR (Arbitrarily-Primed PCR) is a technique that can distinguish the genetic relationship among plant species and other organisms. In this study, AP-PCR approach was used in order to determine the genetic relationship of different six olive clones. The purity of DNA is one of the most important factors affecting the product of the AP-PCR method. In this respect, modified genomic DNA isolation procedure from Oleae europaea clones was developed so that this procedure can be used to obtain plant genomic DNA from diverse aromatic plants, which produce essential oils and secondary metabolites. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each olive clone were produced. AP-PCR-generated unique DNA fingerprint profiles can be used in the identification, distribution and diversity of various olive cultivars.     

Can composition and structural features of oligonucleotides contribute to their wide-scale applicability as random PCR primers in mapping bacterial genome diversity?

Among current genotypic methodologies, random amplification of polymorphic DNA (RAPD or AP-PCR) represents a widely employed assay for the evaluation of bacterial genomic diversity. A common bottleneck of this technique, however, is represented by the screening of useful informative primers to discriminate among isolates of a particular bacterial species. In an attempt to simplify this process, we evaluated here the utility of degenerate oligonucleotides to act as informative AP-PCR primers. For this purpose, a number of features (G+C contents, degeneracy rate, modifications at the 5′ end) of related degenerate primers was tested for their effects in the generation of informative arrays from a set of bacterial genomes. Our results indicate that a combination of a wide base composition and a common palindromic structure at the 5′ end of the sequences that compose the degenerate primers tested here beneficially resulted for the generation of informative arrays aimed to evaluate the bacterial genome heterogeneity.     

Genetic variability in Metarhizium flavoviride revealed by telomeric fingerprinting

Previous studies using arbitrarily primed PCR (AP-PCR/RAPD) analysis have shown only little genetic variation among isolates of the entomopathogenic fungus Metarhizium flavoviride. In the current study, however, telomeric fingerprinting unambiguously differentiated several Brazilian strains of M. flavoviride as well as strains from Africa and Australia. Using this technique, similarity estimates of telomeric DNA among distinct strains were less than 50%, showing this locus to be highly mutable in this species.     

Ribotypes and AP-PCR fingerprints of thermophilic campylobacters from marine recreational waters

Thirty-two strains of thermophilic campylobacters isolated from marine recreational waters and seven reference strains were biotyped and analysed by chromosomal DNA Hae III ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3'). The majority of seawater isolates (90%) were Campylobacter coli , and three strains were Camp. jejuni. Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp. jejuni subsp. jejuni, Camp. coli , and a combination of Camp. coli and Camp. jejuni subsp. doylei. AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method. Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp. jejuni subsp. jejuni, Camp. coli (two clusters) and Camp. lari. The study shows that strains within each species are diverse genomically. Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.     

Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills

The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.     

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.     

Rapid differentiation of Staphylococcus aureus from staphylococcal species by arbitrarily primed-polymerase chain reaction

An arbitrarily primed-polymerase chain reaction (AP-PCR) method was optimized to differentiate Staphylococcus aureus from other staphylococcal species, using DNA from crude cell extract. From the different assays carried out, the best resolution of the band patterns was obtained when the reaction mixture contained 200 micromol l(-1) dNTPs, 200 ng primer, 1 U Taq DNA polymerase and 3 mmol l(-1) MgCl2 and the amplification conditions were: initial denaturation of 94 degrees C for 1 min, primer annealing of 30 degrees C for 1.5 min, DNA extension at 55 degrees C for 5 min and final extension at 55 degrees C for 5 min. The results of the characterization of the staphylococcal isolates by AP-PCR are in accordance with those of the biochemical identification by the API Staph System, time of analysis of the AP-PCR being only 6-7 h. Thus, this technique could be a useful method for microbial quality assurance.     

Ribotyping and TCP gene cluster analysis of environmental and clinical Vibrio cholerae strains isolated in Iran

The aim of this study was to investigate the presence of TCP gene clusters among clinical and environmental Vibrio cholerae isolates and to explore the genetic relatedness of isolates using ribotyping technique. A total of 50 V. cholerae strains (30 clinical and 20 environmental) were included in this study. Three clinical isolates were negative for TCP cluster genes while the cluster was absent in all of the environmental strains. Ribotyping of rRNA genes with BglI produced 18 different ribotype patterns, three of which belonged to clinical O1 serotype isolates. The remaining 15 ribotypes belonged to clinical non-O1, non-O139 serogroups (two patterns) and environmental non-O1, non-O139 serogroups (13 patterns). Clinical V. cholerae O1 strains from 2004 through 2006 and several environmental non-O1, non-O139 V. cholerae strains from 2006 showed 67.3 % similarity and fell within one single gene cluster. Ribotyping analysis made it possible to further comprehend the close originality of clinical isolates as very little changes have been occurred within rRNA genes of different genotypes of V. cholerae strains through years. In conclusion, ribotyping analysis of environmental V. cholerae isolates showed a substantial genomic diversity supporting the fact that genetic changes within bacterial genome occurs during years in the environment, while only little changes may arise within the genome of clinical isolates.     

Use of ribotyping and random amplified polymorphic DNA to differentiate isolates of Burkholderia andropogonis

AIMS: The aim of this study was to determine the genetic diversity among isolates of Burkholderia andropogonis from various host plant species and geographic locations. METHODS AND RESULTS: Both random amplified polymorphic DNA (RAPD) and ribotyping analyses were used to assess the diversity of B. andropogonis isolates and compare these results with pathogenicity assays carried out on a number of common hosts of the organism. CONCLUSIONS: Both RAPD and ribotyping analyses revealed a high level of genetic diversity between isolates of B. andropogonis. Both methods demonstrated a similar clustering of isolates. However, there was no strict correlation between the genetic diversity revealed and the original host, geographic location or pathogenicity of the isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the genetic diversity of isolates of B. andropogonis. The great degree of diversity revealed in this study contrasts with the lack of phenotypic diversity within this species.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Automated ribotyping and random amplified polymorphic DNA analysis for molecular typing of Salmonella enteritidis and Salmonella typhimurium strains isolated in Italy

AIMS: The ability of automated ribotyping and random amplified polymorphic DNA (RAPD) analysis to differentiate Salmonella enteritidis and Salmonella typhimurium isolates in relation to their origin was evaluated. METHODS AND RESULTS: The restriction enzymes EcoRI, PvuII and PstI, and the random primers OPB17 and P1254, were tested for ribotyping and RAPD analysis, respectively. Seventeen subtypes were identified among the isolates of the two pathogenic Salmonella serovars using the RiboPrinter, and 25 subtypes using RAPD. CONCLUSIONS: The greatest degree of genetic diversity was observed among Salm. typhimurium isolates using both automated ribotyping (Simpson's index of discrimination 0878) and RAPD (Simpson's index of discrimination 0886). SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results of this research, automated ribotyping and RAPD are two useful genotyping techniques for identifying unique and common subtypes associated with a specific source and location, and provide powerful tools for epidemiological investigations.