A comparison of molecular typing methods for Moraxella catarrhalis

AIMS: Three molecular typing techniques were examined to determine which method was the most discriminatory in order to perform epidemiological typing of Moraxella catarrhalis. METHODS AND RESULTS: Twenty-five Mor. catarrhalis isolates obtained from nasopharyngeal aspirates collected from Aboriginal and non-Aboriginal children were subjected to random amplified polymorphic DNA (RAPD) analysis, automated ribotyping and pulsed field gel electrophoresis (PFGE). RAPD analysis determined two Mor. catarrhalis types, automated ribotyping with PstI determined four Mor. catarrhalis ribogroups and PFGE analysis with NotI determined 21 pulse field groups within the 25 isolates examined. CONCLUSIONS: Analysis of discrimination index and typeability demonstrated that PFGE is the most discriminatory method for typing Mor. catarrhalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that PFGE is the most appropriate molecular tool for the epidemiological study of Mor. catarrhalis.     

Detection of peptidoglycan and β-glucan with silkworm larvae plasma test

Abstract Discrimination of 70 Salmonella strains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S. typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint.     

Identification and genetic characterisation of orthopaedic Staphylococcus isolates collected in Italy by automated EcoRI ribotyping

The aim of this study was to evaluate the possibility to use automated EcoRI ribotyping to address, during the same analysis, both identification and genetic characterisation of 38 Staphylococcus aureus and 64 coagulase-negative staphylococci collected from surgical injuries. The ribotyping identification results confirmed those obtained using the API Staph system for 96% of the isolates. All strains were successfully genotyped and the ribotyping discriminatory power, calculated using the Simpson's index of discrimination, was very high for both groups of staphylococci tested. The same, as well as different biotypes, were identified among isolates with the identical ribotyping profile.     

Genetic diversity of multi-resistant Salmonella enterica serotype Typhimurium isolates from animals and humans

In this study, the genetic diversities of multi-resistant Salmonella typhimurium (ST) isolates were analyzed via the application of both pulsed field gel electrophoresis (PFGE) and Polymerase chain reaction (PCR) analysis methods, using 6 kinds of primers (REP, ERIC, SERE, BOX, P-1254 and OPB-17). And their discriminative abilities (DA) were also compared in order to determine the most effective and reliable analysis method. 118 S. typhimurium isolates, cultured from diverse animals and human patients in Korea beginning in 1993, were analyzed and subjected to a comparison of Simpson's index of diversity (SID), using both PFGE and PCR methods. PFGE by XbaI enzyme digestion allowed for discrimination into 9 pulsotypes, with high SID values (0.991) on the genomic DNA level. This shows that PFGE is a very discriminative genotypic tool, and also that multiple clones of S. typhimurium isolates had existed in domestic animals and humans in Korea since 1993. However, we could ultimately not to trace the definitive sources or animal reservoirs of specific S. typhimurium isolates examined in this study. Depending on the SID values, the combined method (7 kinds of method) was found to be the most discriminative method, followed by (in order) SERE-PCR, REP-PCR, ERIC-PCR, PFGE & OPB-17 (RAPD), P-1254 (RAPD), and BOX-PCR at the 80% clone cut-off value. This finding suggests that the REP-PCR method (which utilizes 4 primer types) may be an alternative tool to PFGE for the genotyping of S. typhimurium isolates, with comparable cost, time, and labor requirement. The establishment of a highly reliable and discriminatory method for epidemiologic analysis is considered necessary in order for researchers to trace the sources of specific pathogens and, consequently, to control and prevent the spread of epidemic S. typhimurium isolates to humans.     

Molecular fingerprinting of Salmonella serotype Glostrup

Thirteen isolates of Salmonella serotype Glostrup (antigenic formula, 6.8:z10:e,n,z15) from various sources and countries were analysed by ribotyping and IS200 fingerprinting. Both methods provided a high index of strain discrimination by allowing detection of three ribotypes and eight IS200 fingerprints which, though generally related, were readily distinguishable. The findings of this analysis confirm the usefulness of ribotyping and IS200 fingerprinting for studying the epidemiology of rarely isolated salmonellae of serogroup C.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Molecular markers with potential to replace phage typing for Salmonella enterica serovar typhimurium

Using Amplified Fragment Length Polymorphism (AFLP) analysis of isolates from 23 phage types, we isolated 11 molecular markers that are potentially useful for molecular typing of Salmonella enterica serovar typhimurium. We tested these and 11 previously studied markers for their ability to discriminate among isolates and for correlation of their distribution with phage types. The Simpson's index of discriminatory power for the molecular markers is 0.96. One hundred and twenty one isolates from 33 phage types tested were divided into 51 types which are further grouped into 24 patterns. Eight patterns can unambiguously identify 8 phage types and a further 12 correlated with phage type distribution, showing the usefulness of these markers for molecular phage typing.     

Serotyping and RAPD profiles of Salmonella enterica isolates from Mauritius

AIMS: The genus Salmonella is a common agent of gastroenteritis in Mauritius, generating more cases of the disease during summer than during winter. The aims of this study were to assess the genetic diversity of isolates of Salmonella enterica by RAPD fingerprinting, and to establish the relationship between human and chicken isolates. METHODS: Twenty-six isolates were obtained from hospital laboratories and commercial poultry producers locally. RESULTS: The RAPD profiles, biochemical and serological analyses showed that two of the chicken isolates were mistakenly identified as Salmonella. The genetic diversity of the remaining 24 isolates (five chicken and 19 human), confirmed as Salmonella, was analysed using four arbitrary primers, OPA-10, OPR-03, OPI-06 and OPJ-09, chosen from an initial set of 10 decamers. Seventy RAPD markers were generated in four individual DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Cluster analysis (UPGMA) performed using the NTSYS-pc V 1.8 computer software, confirmed that some strains of Salmonella isolated from chicken were genetically similar to those isolated from humans. Furthermore, a 1 kbp band amplified using primer OPA-10 was specific for the Salmonella genus as it was not amplified in any of the control bacteria.     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

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.     

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.     

Restriction enzyme analysis of randomly amplified polymorphic DNA amplicons of Salmonella enterica ser. Enteritidis PT4 and Typhimurium DT104

Salmonella enterica serotype Enteritidis PT4 and Typhimurium DT104 isolates were characterized using a random amplification of polymorphic DNA (RAPD) protocol found previously to be highly discriminatory for isolates of Salmonella . Profiles generated with a single primer 1254, and independently 1283, successfully characterized an outbreak strain of Enteritidis PT4 but could not differentiate epidemiologically unrelated strains of Enteritidis PT4 from the outbreak strains. Primer 1254 differentiated one strain, and 1283 two strains of Typhimurium DT104 previously undifferentiated on the basis of biochemical and physical properties. Subsequent analysis using a combination of RAPD and restriction enzyme analysis could not provide additional differentiation of Enteritidis PT4 and Typhimurium DT104 isolates but did, however, exhibit the potential to be a useful combination of molecular techniques.     

Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England,Wales and Ireland

AIMS: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. METHODS AND RESULTS: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI-SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. CONCLUSIONS: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates.     

Comparison of five molecular subtyping methods for differentiation of Salmonella Kentucky isolates in Tunisia

Salmonellosis is one of the most common causes of food-borne infection worldwide. In the last decade, Salmonella enterica serovar Kentucky has shown an increase in different parts of the world with the concurrent emergence of multidrug-resistant isolates. These drug-resistant types spread from Africa and the Middle East to Europe and Asia. Although S. Kentucky serovar is of potential human relevance, there is currently no standardized fingerprinting method for it, in Tunisia. In the present study, a collection of 57 Salmonella Kentucky isolates were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC) fingerprinting, and Random Amplification of Polymorphic DNA. Plasmid profiling showed a discriminatory index (D) of 0.290, and only 9 out of 57 (16 %) isolates carried plasmids, which represents a limitation of this technique. Fingerprinting of genomic DNA by PFGE and ribotyping produced 4 and 5 patterns, respectively. Distinct PFGE patterns (SX1, SX2, SX3, and SX4) were generated for only 28 strains out of 57 (49.1 %) with a D value of 0.647. RAPD fingerprinting with primers RAPD1 and RAPD2 produced 4 and 20 patterns, respectively. ERIC fingerprinting revealed 14 different patterns with a high discriminatory index (D) of 0.903. When the methods were combined, the best combination of two methods was ERIC-2 with RAPD2. These results indicates that a single method cannot be relied upon for discriminating between S. Kentucky strains, and a combination of typing methods such ERIC2 and RAPD2 allows further discrimination.     

Genetic heterogeneity and functional properties of intestinal bifidobacteria

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

Analysis of Salmonella Agona and Salmonella Weltevreden in Malaysia by PCR fingerprinting and antibiotic resistance profiling

Forty-eight strains of Salmonella enterica subsp. enterica serovar Agona and 33 strains of Salmonella enterica subsp. enterica serovar Weltevreden were characterized by random amplified polymorphic DNA (RAPD) fingerprinting using 3 different arbitrary primer, Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) and antimicrobial susceptibility testing. By using RAPD, 81 strains (44 strains of S. Agona and 33 strains of S. Weltevreden) can be clustered into 14 groups and 6 single isolates whereas ERIC-PCR produced 7 clusters and 3 single isolates. Thirteen antimicrobial agents were used and all the isolates were resistant to erythromycin and showed Multiple Antimicrobial Resistance indexes, ranging from 0.08 to 0.62. Poultry still remain as the common reservoir for multi-drug-resistant Salmonella. On the other hand, vegetables contaminated with S. Weltevreden showed a gain in antimicrobial resistance. Besides that, consistent antibiograms were observed from S. Weltevreden isolated at Kajang wet market on 2000/08/02.     

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.     

Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Random amplification of polymorphic DNA (RAPD) of Salmonella: strain differentiation and characterization of amplified sequences

Random amplification of polymorphic DNA (RAPD) was evaluated for its ability to differentiate Salmonella strains from various sources. Under defined conditions RAPD using a 10-mer primer (1254) produced a series of amplification products able to reproducibly distinguish strains representing 20 different serotypes of Salmonella. Primer 1254 also proved capable of discrimination between some but not all isolates of Salm. ser. Enteritidis and Salm. ser. Typhimurium, phage typing proving to be most discriminatory for the latter serotype. Cloning of fragments into a vector allowed sequencing and database searching for identification of fragments and an indication of criteria for primer template interaction in RAPD. Southern blotting using a digoxigenin-labelled probe allowed identification of related bands between RAPD profiles. These observations demonstrate the potential of rapid molecular typing by RAPD for the genomic typing of Salmonella strains.