Typing of group A streptococci by random amplified polymorphic DNA analysis.

Random amplified polymorphic DNA (RAPD) analysis was evaluated in comparison with restriction endonuclease analysis (REA) of genomic DNA and serotyping in the typing of 160 epidemiologically unrelated group A streptococci (GAS). Amplification of genomic DNA of GAS was performed with a single primer with an arbitrarily selected nucleotide sequence of 12 nucleotides. In total, 31 RAPD patterns and 15 REA patterns were observed among the isolates studied. The results of RAPD analysis were in accordance with the results of REA for 86% of the isolates, as both methods identified 15 different strains among 138 isolates. However, RAPD analysis differentiated 16 additional strains among 22 isolates. RAPD analysis was somewhat better than REA for differentiation of isolates of the same and different serotypes. However, not all of the serotypes were differentiated by RAPD analysis either. In conclusion, RAPD analysis provides a practical alternative for genomic typing of GAS. It can be recommended for the typing of GAS, especially if used in parallel with serotyping.     

Typing of Listeria strains by random amplification of polymorphic DNA.

The polymerase chain reaction (PCR) was used to obtain randomly amplified polymorphic DNA (RAPD) profiles for typing of Listeria strains. In this procedure, whole cells were incubated in the reaction mixture. The discriminating ability of a randomly designed 10-mer primer, HLWL74, was assessed. A total of 60 collection strains of Listeria, encompassing all 7 Listeria species and all known serovars was submitted to PCR with the primer HLWL74. Upon agarose gel electrophoresis, 29 different banding profiles were reproducibly obtained. No common profiles were recorded for strains from different Listeria species. For various groups of strains sharing the same serotype (e.g. 4b, 1/2a, 1/2b), RAPD analysis could generate further subdivision. On the other hand, some strains from different serotypes produced identical RAPD profiles with the primer HLWL74. The RAPD typing method from whole cells is proposed as an attractive alternative for other Listeria typing systems, and the 10-mer HLWL74 as a primer to include in a forthcoming set of standard primers for RAPD typing of Listeria isolates.     

Rapid discrimination of Mycobacterium tuberculosis strains by random amplified polymorphic DNA analysis.

Investigations of the epidemiology of tuberculosis have been hampered by the lack of strain-specific markers that can be used to differentiate isolates of Mycobacterium tuberculosis. We report the development of a rapid protocol for random amplified polymorphic DNA analysis which included the use of a commercially available DNA extraction kit (GeneReleaser). This was applied to 14 strains of M. tuberculosis, including strains associated with temporal and geographical clusters of tuberculosis in the United Kingdom and those from India, Africa, and Saudi Arabia. Strains of M. tuberculosis could be discriminated in about 8 h by this method, which is therefore a rapid and simple alternative to restriction fragment length polymorphism analysis.     

Genomic variability of Haemophilus influenzae isolated from Mexican children determined by using enterobacterial repetitive intergenic consensus sequences and PCR

Genomic fingerprints from 92 capsulated and noncapsulated strains of Haemophilus influenzae from Mexican children with different diseases and healthy carriers were generated by PCR using the enterobacterial repetitive intergenic consensus (ERIC) sequences. A cluster analysis by the unweighted pair-group method with arithmetic averages based on the overall similarity as estimated from the characteristics of the genomic fingerprints, was conducted to group the strains. A total of 69 fingerprint patterns were detected in the H. influenzae strains. Isolates from patients with different diseases were represented by a variety of patterns, which clustered into two major groups. Of the 37 strains isolated from cases of meningitis, 24 shared patterns and were clustered into five groups within a similarity level of 1.0. One fragment of 1.25 kb was common to all meningitis strains. H. influenzae strains from healthy carriers presented fingerprint patterns different from those found in strains from sick children. Isolates from healthy individuals were more variable and were distributed differently from those from patients. The results show that ERIC-PCR provides a powerful tool for the determination of the distinctive pathogenicity potentials of H. influenzae strains and encourage its use for molecular epidemiology investigations.     

Enterobacterial repetitive intergenic consensus 1R PCR assay for detection of Raoultella sp. isolates among strains identified as Klebsiella oxytoca in the clinical laboratory

The enterobacterial repetitive intergenic consensus 1R PCR method, which provided recognizable profiles for reference strains of the three species of Raoultella and the two genetic groups of Klebsiella oxytoca, was applied to 19 clinical isolates identified as K. oxytoca. By this method, as confirmed by species-specific gene sequencing, two Raoultella ornithinolytica and two unclassifiable K. oxytoca isolates were identified.     

Molecular fingerprinting of fish-pathogenic Lactococcus garvieae strains by random amplified polymorphic DNA analysis

In this work, we used the random amplified polymorphic DNA (RAPD) technique to evaluate the genetic diversity in Lactococcus garvieae, an important pathogen for fish. Fifty-seven strains with different hosts and geographical origins, including Japan and several countries of the Mediterranean area such as Spain, Portugal, France, Italy, England, and Turkey, were analyzed. Two primers, oligonucleotides 5 and 6 (Pharmacia Biotech) were utilized; primer 5 was the most discriminative, since allowed us to differentiate 10 RAPD -types related to the origin of the strains. Regardless of the oligonucleotide primer employed, the 57 isolates of L. garvieae studied were separated into three genetic groups, composed of the Spanish, Portuguese, English, and Turkish strains (group A), the Italian and French strains (group B), and the Japanese strains (group C). The similarity of isolates within each group, estimated on the basis of the Dice coefficient, ranged from 75 to 100%. Our findings also indicate that RAPD profiling constitutes a useful tool for epidemiological studies of this fish pathogen.     

Typing and subtyping of 83 clinical isolates purified from surgically implanted silicone feeding tubes by random amplified polymorphic DNA amplification

In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 "buttons" and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of Bacillus brevis, 4 isolates of Bacillus licheniformis, 2 isolates of Bacillus pumilus, 3 isolates of Enterococcus durans, 19 isolates of Enterococcus faecalis, 8 isolates of Enterococcus faecium, 2 isolates of Enterococcus hirae, 7 isolates of Escherichia coli, 8 isolates of Lactobacillus plantarum, 19 isolates of Staphylococcus aureus, 2 isolates of Staphylococcus epidermidis, and 7 isolates of Staphylococcus saprophyticus. Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of E. coli, E. faecalis, and S. aureus in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes.     

Mycobacterium malmoense-specific nested PCR based on a conserved sequence detected in random amplified polymorphic DNA fingerprints

Mycobacterium malmoense is an opportunistic human pathogen of increasing clinical importance. Since it is difficult to detect and identify the organism by conventional techniques, it was decided to seek a nucleic acid amplification method specific for M. malmoense. The method was based on detection of a conserved band in random amplified polymorphic DNA (RAPD) fingerprints of 45 M. malmoense strains. This band was a 1,046-bp product which was proven to be M. malmoense specific in dot blot hybridization analysis with a panel of mycobacterial strains belonging to 39 other species. The fragment was sequenced, and oligonucleotide primers were synthesized to evaluate the specificity of the PCR. Two primer pairs were found to be specific and sensitive in the nested PCR that was developed. All 49 M. malmoense strains analyzed produced a PCR product of the expected size. In contrast, no strains belonging to the other mycobacterial species tested produced amplicons with these primers under specified reaction conditions. The results of the electrophoresis were confirmed by the hybridization with the M. malmoense-specific oligonucleotide probe. This method could be applied to the analysis of clinical or environmental samples, permitting the rapid detection of M. malmoense.     

Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis.

A random amplified polymorphic DNA (RAPD) fingerprinting method has been developed to differentiate Salmonella enteritidis isolates. A total of 65 arbitrary primers were screened with S. enteritidis isolates of different phage types. This allowed selection of a panel of primers capable of detecting DNA polymorphisms among S. enteritidis isolates. This panel was used to examine a panel of 29 isolates of S. enteritidis which had been previously characterized by other subtyping methods, including phage typing (PT) (n = 7), ribotyping (RT) (n = 13), and pulsed-field gel electrophoresis (PFGE). Applied collectively, these three methods resolved the collection into 20 different subtypes. However, by the RAPD fingerprinting method alone, 14 RAPD subtypes were revealed. Eight isolates of S. enteritidis phage type 8 that failed to be discriminated by other typing methods (PT, RT, and PFGE) were resolved into three different subtypes by RAPD analysis. In contrast, isolates that were derived from the same sources were not differentiated by any of the subtyping methods employed, including PT, RT, PFGE, and RAPD analysis. This RAPD approach to S. enteritidis subtyping provided more discriminatory power than did any of several other subtyping methods applied individually. Once the challenging step of primer identification was accomplished, determinations of the appropriate concentrations of arbitrary primer, DNA template, and MG2+ ion were also necessary for optimal discriminatory power. The bacterial DNA used in this RAPD protocol was obtained by boiling the bacterial sample. This simple procedure yielded DNA that produced fingerprint patterns as consistent as those obtained from phenol-chloroform-extracted DNA. Clearly, when appropriately constituted primer sets are identified and employed, RAPD analysis provides a simple, rapid, and powerful subtyping method for S. enteritidis.     

Randomly amplified polymorphic DNA PCR for comparison of Mycobacterium abscessus strains from nosocomial outbreaks.

Mycobacterium abscessus is an important cause of water-related nosocomial outbreaks or pseudo-outbreaks. Strain comparison has relied on pulsed-field gel electrophoresis (PFGE). Unfortunately, almost 50% of strains cannot be assessed by this method. We studied 118 strains of M. abscessus previously studied by PFGE by randomly amplified polymorphic DNA (RAPD) PCR, including isolates from eight nosocomial outbreaks. Ten random primers were evaluated by using DNA prepared by boiling or phenol-chloroform extraction. Both DNA preparations gave the same grouping of isolates for three outbreaks compared to the groupings obtained by PFGE. Five outbreaks due to M. abscessus which gave broken DNA by PFGE gave evaluable patterns when studied by RAPD-PCR, with isolate clustering being consistent with that from other laboratory and epidemiologic data. The patterns were highly method dependent, strain comparison required the use of multiple primers, and the method worked best with purified DNA and by using strains for comparison on the same gel. We propose categories of indistinguishable, different, and inconclusive when comparing strains by RAPD-PCR. This study demonstrates that RAPD-PCR can be used for genetic comparison of M. abscessus strains, including strains which cannot be compared by PFGE, but the potential for misinterpretation is greater than that by PFGE.     

Identification and DNA fingerprinting of Legionella strains by randomly amplified polymorphic DNA analysis.

The randomly amplified polymorphic DNA (RAPD) technique was used in the development of a fingerprinting (typing) and identification protocol for Legionella strains. Twenty decamer random oligonucleotide primers were screened for their discriminatory abilities. Two candidate primers were selected. By using a combination of these primers, RAPD analysis allowed for the differentiation between all different species, between the serogroups, and further differentiation between subtypes of the same serogroup. The usefulness of RAPD analysis was also evaluated with outbreak-related clinical and environmental isolates previously typed by the restriction fragment length polymorphism technique. RAPD analysis proved to be as accurate as other genotypic methods, reproducible, and highly discriminatory and is a valuable new alternative to traditional fingerprinting and identification of Legionella species and strains.     

Use of pulsed-field gel electrophoresis, enterobacterial repetitive intergenic consensus typing, and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae

We compared 75 nontypeable (NT) Haemophilus influenzae isolates by pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and automated ribotyping. PFGE was the most discriminatory of the techniques. ERIC-PCR provides a useful screen but should not replace other techniques as the sole method to group NT H. influenzae strains.     

Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis.

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.     

Typing of nonencapsulated haemophilus strains by repetitive-element sequence-based PCR using intergenic dyad sequences

Intergenic dyad sequences (IDS) are short repeated elements that have been described for several Haemophilus genomes and for only two other bacterial genera. We developed a repetitive-element sequence-based PCR using an IDS-specific primer as a typing method (IDS-PCR) for nonencapsulated Haemophilus strains and compared this technique with pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. IDS-PCR was rapid, easy to perform, and reproducible, with a high discriminatory capacity for nontypeable Haemophilus influenzae (NTHI) strains. The 69 NTHI strains tested generated 65 different banding patterns. Epidemiologically related strains gave similar or identical fingerprints, and all of the unrelated strains except two showed different patterns. These results were in agreement with those obtained by PFGE. For 20 genital strains usually identified as being biotype IV NTHI and belonging to a cryptic genospecies of Haemophilus with remarkable genetic homogeneity, four bands were significantly present and six bands were significantly absent from the fingerprints. The 20 strains were gathered in 11 closely related profiles, whereas PFGE provided no band when DNA was treated with SmaI. IDS-PCR improved the differentiation previously obtained within this species by ribotyping and multilocus enzyme electrophoresis. Our findings suggest that IDS-PCR is a rapid, reliable, and discriminatory method for typing NTHI strains and is currently the most efficient method for distinguishing strains within the cryptic genospecies of HAEMOPHILUS:     

Characterization of Streptococcus agalactiae strains by randomly amplified polymorphic DNA analysis.

A collection of 54 unrelated Streptococcus agalactiae strains isolated from cerebrospinal fluid samples from neonates and 60 unrelated strains isolated from carriers that had been previously studied by multilocus enzyme electrophoresis (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995) were characterized by randomly amplified polymorphic DNA (RAPD) assay. Four primers, 5'AGGGGGTTCC3', 5'AACGCGCAAC3', 5'GCATCAATCT3', and 5'AGTCGGGTGG3', named OPS16, AP42, A4, and OPS11, respectively, were selected from 29 primers tested. This investigation identified 71 RAPD types. The three families of strains defined by multilocus enzyme electrophoresis analysis, which contain most of the cerebrospinal fluid isolates, were also identified by clustering analysis of RAPD data. Each of these three groups exhibits specific RAPD patterns or fragments. The discriminatory power of the RAPD typing method was also evaluated. The simplest typing scheme was obtained by the combination of RAPD typing done with primers AP42 and OPS11 and serotyping (index of discrimination, 0.97).     

Molecular fingerprinting of Porphyromonas gingivalis by PCR of repetitive extragenic palindromic (REP) sequences and comparison with other fingerprinting methods.

Knowledge of the genetic structure of populations of potentially pathogenic bacteria is important in understanding the epidemiology of diseases. Porphyromonas gingivalis is thought to be an important aetiological agent in periodontal diseases and several methods have been used for typing strains of this species. Here, PCR with primers to repetitive extragenic palindromic sequences (REP-PCR) was compared with three other widely used molecular fingerprinting techniques -- restriction endonuclease analysis (REA), ribotyping and PCR with arbitrary primers (AP-PCR) -- to type P. gingivalis isolates from healthy and diseased periodontal sites. The data obtained with all four methods were in broad agreement and, with one exception, each subject harboured a single unique genotype of P. gingivalis. REP-PCR of P. gingivalis resulted in the production of 5-10 amplicons, which gave unique electrophoretic patterns in each individual (10 REP-PCR types in 10 patients) and similar results were obtained with AP-PCR. Two isolates from one subject appeared identical by REP-PCR and AP-PCR, but could be differentiated by ribotyping, although there was only minor polymorphism. Thus, ribotyping and REA were the most discriminating methods; however, these are time-consuming and expensive relative to the PCR-based techniques. REP-PCR has the advantage that the same pair of primers is used for all species, whereas AP-PCR needs to be optimised by screening a range of primers. These results show that REP-PCR is a useful and rapid technique for typing P. gingivalis.     

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR fingerprinting.

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.     

Genetic differentiation of pathogenic and nonpathogenic strains ofEntamoeba histolytica by random amplified polymorphic DNA polymerase chain reaction

The random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) method was used to compare pathogenic and nonpathogenic strains ofEntamoeba histolytica. DNA polymorphisms were detected among the different strains and dendrograms were constructed by PHYLIP and PAUP analyses to study the relationship of the strains. Both analyses resulted in identical results, which indicated that pathogenic strains ofE. histolytica are closely related and clearly separated from the nonpathogenic strains. The results of this study agree with classification of the strains based on isoenzyme analyses. This suggests that RAPD-PCR is a valuable method in differentiating between strains of this parasite, and the results are consistent with the concept that pathogenic and nonpathogenicEntamoeba represent two different species.Abbreviations DNA Deoxyribonucleic acid - PAUP phylogenetic analysis using parsimony - RAPD random amplified polymorphic DNA - PCR polymerase chain reaction - PHYLIP phylogeny inference package - UPGMA unweighted pair-group method with arithmetic mean     

Characterization of Hafnia alvei by biochemical tests, random amplified polymorphic DNA PCR, and partial sequencing of 16S rRNA gene.

Hafnia alvei strains which possess the attachment-effacement gene (eaeA) may have clinical importance as new diarrhea-causing pathogens and should therefore be differentiated from other H. alvei strains. We characterized diarrheal H. alvei strains, which were positive in the PCR test for the eaeA gene, using biochemical tests not routinely used for identification of members of the family Enterobacteriaceae, and compared them with eaeA-negative strains isolated from different clinical and nonclinical sources to find characteristics useful for identification. Random amplified polymorphic DNA (RAPD)-PCR and partial sequencing of the 16S rRNA gene were utilized to study the genetic diversity of the isolates. The eaeA-positive strains were found to have many characteristic biochemical properties. Negative reactions in the 2-ketogluconate and histidine assimilation tests and a positive reaction in the 3-hydroxybenzoate assimilation test may be useful in routine diagnostics. Nearly identical RAPD-PCR profiles and identical 353-bp fragments of the 16S rRNA genes indicated little genetic diversity among the eaeA-positive strains. The low level of homology (92%) in the partial 16S rRNA genes of eaeA-positive and -negative H. alvei strains raises questions about the taxonomic positioning of eaeA-positive H. alvei.