Differentiation of Arthroderma spp. by random amplification of polymorphic DNA (RAPD) and Southern hybridization

To develop the molecular differentiation analysis of dermatophytes, we carried out RAPD and Southern hybridization analyses using genomic DNAs of six Arthroderma species, including A. fulvum, A. grubyi, A. gypseum, A. incurvatum, A. otae and A. racemosum. The RAPD analysis gave different band patterns specific to each of the six Arthroderma fungi. However, minor differences in the banding patterns were observed between the strains of plus (+) and minus (-) mating types of A. gypseum, A. fulvum and A. incurvatum. Southern blot analysis using a probe (1S) obtained from A. grubyi DNA gave specific bands only in the DNA samples of A. grubyi and A. incurvatum. On the other hand, Southern blot analysis using a probe (C3) obtained from A. otae DNA gave specific bands in all six Arthroderma species examined, and the size of the bands were specific to each species. These findings indicate that RAPD and Southern hybridization analyses are useful in the differentiation of these Arthroderma species.     

Phylogenetic analysis of 8 dermatophyte species using chitin synthase 1 gene sequences

Nucleotide sequences of chitin synthase 1 (CHS1) gene of eight species of dermatophytes, Arthroderma benhamiae, A. fulvum, A. grubyi, A. gypseum, A. incurvatum, A. otae, A. simii and A. vanbreuseghemii were obtained and analysed for their phylogenetic relationship. A 600-bp genomic DNA fragment of the CHS1 gene was amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these eight dermatophyte species showed more than 85% similarity between the species. The phylogenetic analysis of their sequences revealed three clusters, the first cluster consisting of A. benhamiae, A. simii and A. vanbreuseghemii, the second cluster consisting of A. fulvum, A. gypseum and A. incurvatum, and the third cluster consisting of A. grubyi and A. otae. The phylogenetic analysis of CHS1 gene in this study will provide useful information for classification and understanding the evolution of these dermatophyte species.     

Serotype determination of enteroviruses that cause hand-foot-mouth disease; identification of enterovirus 71 and coxsackievirus A16 from clinical specimens by using specific probe

Coxsackievirus A16 (CA16) and enterovirus 71 (EV71) are known to be major causative agents of hand-foot-and-mouth disease prevalent in summer in Japan. Discrimination and identification of these viruses were often hampered by a nonneutralizable or nontypable virus. Therefore, a Southern blot hybridization that utilizes mixed probes specific to serotype was developed. Firstly, an approximately 650 bases spanning 5'-noncoding region to one third of VP2 including entire VP4 was amplified with a set of primers containing enterovirus common sequences and a genomic RNA as template. Secondary, the nucleotide sequences were determined using seven CA16 and eighteen EV71 strains including the standard strains, and the deduced amino acid sequences of VP4 were searched to find residues which are conserved in the same serotypes but diverged among different serotypes. Candidate positions for the mixed probes were defined at the carboxyl terminus of VP4. Thirdly, Southern blot analyses were carried out using thirty-nine enterovirus standard strains, seven CA16 isolates and sixty-six EV71 isolates previously identified by the neutralization test. The results revealed that each mixed probe exclusively bound to the homologous DNAs but not to the heterologous ones. In an attempt to determine serotypes without virus isolation, clinical specimens from hand-foot-and-mouth disease were examined. Of 78 throat swabs and 15 vesicular fluids, 71 (91.0%) and 13 (86.7%) specimens were clearly identified, indicating that the method described here offer advantages over the traditional neutralization assay: It is rapid, specific and less labor-consuming.     

Random amplified polymorphic DNA typing versus pulsed-field gel electrophoresis for epidemiological typing of vancomycin-resistant enterococci

Sixty vancomycin-resistant vanA mutant Enterococcus faecium (VRE) isolates, collected during a 40-month period from 48 patients hospitalized in a French Cancer Referral Center, were typed by using random amplified polymorphic DNA (RAPD), and the results were compared with those previously obtained by typing with SmaI pulsed-field gel electrophoresis (PFGE), which is currently recognized as the "gold standard." The discriminating power of RAPD typing, with seven primers and 11 combinations of primers, was tested on 18 strains, and only the most discriminating combination was further tested on the whole collection. We compared the epidemiological usefulness of RAPD typing of 60 clinical VRE isolates with that of SmaI PFGE typing. With primers AP4 and ERIC1R, RAPD generated 30 patterns versus the 36 patterns generated by SmaI PFGE. However, this did not hamper the epidemiologically correct clustering of 15 related strains and the detection of multiple colonization in nine patients. We conclude that this simple RAPD technique is well suited to the epidemiological typing of VRE and the monitoring of its nosocomial spread.     

Intraspecies polymorphism of vsp genes and expression profiles of variable surface protein antigens (Vsps) in field isolates of Mycoplasma bovis

To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.     

Insertion possibility of 16S-23S space amplification and random amplified polymorphic DNA analysis for typing of methicillin-resistant Staphylococcus aureus strains in the context of nosocomial infections

Within the scope of the present study n = 183 MRSA isolates from the extended area of Düsseldorf and n = 93 international MRSA strains from seven different countries were typed by pulsed-field gel electrophoresis and two PCR methods (RAPD and 16S-23S-spacer amplification). The isolates could be subdivided into 30 different types by PFGE, into 21 by means of RAPD and 18 by 16S-23S-spacer amplification. PFGE had the highest discriminatory potential, however, a combined use of the three typing methods allows a more detailed differentiation even of those isolates with identical PFGE pattern. Both amplification procedures were rapid, easy in handling with reproductable results. For a temporary epidemiological analysis within 24 hours, both amplification methods could be combined. In case the investigated isolates were still suspected of showing a "clonal identity", they should be analysed by additional PFGE (lasting about four days). Although the international isolates were chosen by random selection, several MRSA strains with identical pattern could be found in different countries of the world. Some RAPD-, spacer- and PFGE pattern were constant over many years. This reflects a high genetic stability of single strains.     

Characterization of clinical isolates of Streptococcus agalactiae by random amplified polymorphic DNA using degenerate oligonucleotides

Epidemiological studies of Streptococcus agalactiae strains have been limited by the lack of sensitive and discriminatory methods for comparing clinical isolates. Serotyping, albeit a widely used methodology, has been shown to possess low capability to distinguish between epidemiologically related and unrelated isolates. We have employed here a random amplification of polymorphic DNA (RAPD) assay, using degenerate oligonucleotides as primers, to characterize S. agalactiae isolates from related or unrelated clinical samples. Epidemiologically-related isolates (mother-infant pairs) showed identical profiles by this methodology. On the contrary, 12 epidemiologically-unrelated isolates (classified into 5 different serotypes) resulted in 11 distinct RAPD patterns. This suggests that the proposed modified RAPD assay provides a highly discriminatory tool for the analysis of genomic diversity among isolates from pathogenic organisms.     

Lead intoxication in children with pervasive developmental disorders

Ten clinical isolated strains of Human cytomegalovirus (HCMV) were obtained from 73 urine specimens of different people. Isolations from urine were carried out in human embryolung fibroblasts. Viral isolates were passaged four times. HCMV DNAs of laboratory strain AD169 and 10 clinical isolated strains were extracted with Hirt method, digested with each of the restriction enzymes EcoRI, Hind II. Comparison of restriction fragment length polymorphism (RFLP) of AD169 and isolated strains were made by hybridizing digested DNA with 32P labeled with HCMV Hind II cloned subgenomic fragments as the probe (pCM1035, pCM1015). pCM1035 is located in the joining region between the long(L) and short (S) unique sequences of the virus (L-S junction) pCM1015 is located in the terminal sequences of the virus. The results showed the genomic high degree of homology existed among all strains and the variable restriction site was in the L-S junction and terminal portion. The RFLP patterns of the clinical isolates which did not have relation in epidemiology were different, but the patterns of clinical isolates related in epidemiology were quite similar. Polymorphism frequently occurred in this case of EcoRI digested fragment hybridized with the probe of pCM1035. Southern hybridization of HCMV isolations is useful to researches into the molecular epidemiology and pathogenesis of HCMV infection.     

Norepinephrine loss selectively enhances chronic nigrostriatal dopamine depletion in mice and rats

A total of 3,700 Pseudomonas aeruginosa isolates were collected from 17 general hospitals in Japan from 1992 to 1994. Of these isolates, 132 carbapenem-resistant strains were subjected to DNA hybridization analysis with the metallo-beta-lactamase gene (blaIMP)-specific probe. Fifteen strains carrying the metallo-beta-lactamase gene were identified in five hospitals in different geographical areas. Three strains of P. aeruginosa demonstrated high-level imipenem resistance (MIC, > or = 128 micrograms/ml), two strains exhibited low-level imipenem resistance (MIC, < or = 4 micrograms/ml), and the rest of the strains were in between. These results revealed that the acquisition of a metallo-beta-lactamase gene alone does not necessarily confer elevated resistance to carbapenems. In several strains, the metallo-beta-lactamase gene was carried by large plasmids, and carbapenem resistance was transferred from P. aeruginosa to Escherichia coli by electroporation in association with the acquisition of the large plasmid. Southern hybridization analysis and genomic DNA fingerprinting profiles revealed different genetic backgrounds for these 15 isolates, although considerable similarity was observed for the strains isolated from the same hospital. These findings suggest that the metallo-beta-lactamase-producing P. aeruginosa strains are not confined to a unique clonal lineage but proliferated multifocally by plasmid-mediated dissemination of the metallo-beta-lactamase gene in strains of different genetic backgrounds. Thus, further proliferation of metallo-beta-lactamase-producing strains with resistance to various beta-lactams may well be inevitable in the future, which emphasizes the need for early recognition of metallo-beta-lactamase-producing strains, rigorous infection control, and restricted clinical use of broad-spectrum beta-lactams including carbapenems.     

DNA restriction fragments length polymorphism of Mycobacterium tuberculosis isolates in Pisa, Italy

A total of 60 Mycobacterium tuberculosis strains isolated in the area of Pisa, Italy, over a period from April 1993 to December 1995, were analyzed for the IS6110-based restriction fragments length polymorphism (RFLP). Isolates were found to show a great heterogeneity and only few isolates shared identical DNA banding patterns. In particular, 55 distinct IS6110 patterns were found (average number of isolates per pattern: 1.09) and only 9 strains (15%) occurred in 4 clusters of 2-3 identical clones. Computer analysis of genetic similarities among the strains revealed a family of 17 isolates including the clustered clones implicated in recently acquired infections. No correlation was found between the RFLP DNA patterns of the isolates and drug susceptibility. Of the 5 isolates from immigrants only one showed abnormal DNA fingerprinting. Our data indicate that the patterns of M. tuberculosis isolates in Pisa area are comparable to those of countries with low-prevalence TB and that a low level of TB transmission occurs in this area.     

Genotypic characterization of seven strains of Mycoplasma fermentans isolated from synovial fluids of patients with arthritis

We performed a genotypic characterization of seven strains of Mycoplasma fermentans which have been isolated from the synovial fluid of patients with rheumatoid arthritis (n = 2), spondyloarthropathy (n = 1), and unclassified arthritis (n = 4). We compared them to three reference strains (strains PG18 and K7 and incognitus strain) and to a clinical isolate from the urethra of a patient with nongonococcal urethritis. The characterization methods included electrophoresis of native DNA, arbitrarily primed PCR, and restriction fragment length polymorphism analysis following conventional and pulsed-field gel electrophoresis. Southern blot analysis with a probe internal to an insertion sequence was performed with the restriction products produced by the last two techniques. No extrachromosomal DNA sequences were detected. The M. fermentans strains identified by these methods did not present a unique profile, but they could be separated into two main categories: four articular isolates were genetically related to PG18 and the three other isolates, the urethral isolate, and the incognitus strain were related to K7. We also looked for the presence of the bacteriophage MAV1 (associated with the arthritogenic property of Mycoplasma arthritidis in rodents) in the M. fermentans strains. MAV1 DNA was not detected in either the clinical isolates or the reference strains of M. fermentans.     

Kinins and the events influenced by an angiotensin-converting enzyme inhibitor during neointima formation in the rat carotid artery

Sixty-one strains of bacteria capable of growth on 4-methyl benzoic acid (29 isolates) or naphthalene (32 isolates) as the sole source of carbon and energy were isolated from sediments and water samples from the River Tyne, UK. Random amplification of polymorphic DNA from genomic DNA extracted from the different strains demonstrated that 14 of the 4-methyl benzoate-degrading isolates were unique and the remainder fell into seven groups containing two or three isolates that produced identical banding patterns. Thirteen of the naphthalene-degrading isolates were unique and nine groups with two or three identical representatives encompassed all other isolates. Screening of the bacterial strains for the presence of genes homologous to xylE, nahC and bphC by polymerase chain reaction and dot blot hybridization demonstrated that most strains harboured xylE- and/or nahC-like genes and only a single isolate was found that did not harbour any of these genes. None of the isolates harboured bphC-like genes. It was concluded that, while considerable diversity existed in host strains isolated using a single simple enrichment procedure, the extradiol dioxygenase genes involved in aromatic ring cleavage, present in these strains, were conserved to a considerable degree.     

AM1 theoretical study, synthesis and biological evaluation of some benzofuran analogues of anti-inflammatory arylalkanoic acids

To evaluate different methods for strain differentiation, 10 isolates of Aspergillus terreus from Germany and two epidemiologically unrelated strains were investigated. The sources of the isolates were patients with cystic fibrosis (4), immunosuppression (2), otitis externa (2), sinusitis (1) and endocarditis (1). Environmental isolates were obtained from a contaminated cell culture and from soil. The isolates did not differ in their macroscopic and microscopic morphology, in their protein patterns analysed by SDS-PAGE and in their susceptibility to amphotericin B and itraconazole. The RFLP analysis of total genomic DNA digested by EcoRI resulted in patterns that were too faint for interpretation. However, after hybridisation of the digested DNA with a short DNA probe of repetitive sequence, six different patterns were found. Based on the patterns of the randomly amplified polymorphic DNA (RAPD) with three primers, nine different genotypes were discriminate. RAPD patterns discriminated the epidemiologically unrelated reference strains (endocarditis isolate from Thailand, soil isolate from the USA) and the isolates from Germany. It is concluded that, in contrast to the phenotypic methods, the analysis of RAPD patterns is useful for strain differentiation of A. terreus.     

Molecular characterization of Vibrio cholerae O1 strains isolated during cholera outbreaks in Guinea-Bissau

In the present study, 19 strains of Vibrio cholerae O1 biotype El Tor isolated during outbreaks of cholera in Guinea-Bissau in 1987, 1994, and 1995 were characterized to investigate a possible epidemiological relationship among the isolates. On the basis of ribotyping with the restriction enzyme BglI, 5 strains isolated in 1987 showed two closely related ribotypes, while 14 strains isolated in 1994 and 1995 showed the same ribotype that was distinct from the ribotypes of strains isolated in 1987. Southern blot hybridization of BglI-digested genomic DNA with a cholera toxin probe demonstrated that the strains isolated in 1987 showed an identical cholera toxin genotype, whereas O1 strains isolated in 1994 and 1995 showed the same genotype that was distinct from the genotype of strains isolated in 1987. These results were supported by the results of antibiotic susceptibility testing, in which strains isolated in 1987 showed resistance to polymyxin B only, while each of the strains from 1994 and 1995 showed resistance to polymyxin B, trimethoprim-sulfamethoxazole, and the vibriostatic agent O/129. Although our results are based on a limited number of V. cholerae O1 strains, they suggest that the epidemic in Guinea-Bissau in 1994 and 1995 was due to the introduction of a new strain to the country.     

Characterization of the rpoB gene mutations in clinical isolates of rifampicin-resistant Mycobacterium tuberculosis

OBJECTIVES: Characterization and frequency of the rpoB gene mutations associated with rifampin resistance in Mycobacterium tuberculosis clinical isolates in Sevilla. METHODS: Characterization of rpoB mutations in 21 rifampicin-resistant strains of M. tuberculosis isolated during a three-year period (1994-1996) by three different molecular methods: a nonradioactive Single-strand conformation polymorphism (SSCP) analysis, DNA sequence analysis and a commercial method the line probe assay InnoLiPA. RESULTS: Five distinct rpoB mutations were identified. Ser531-->Leu mutation was detected in 14 strains (66.7%), H526-->Asp in 3 strains (14.3%), Ans512-->Ser in 1 strain (4.8%), Glu513-->Leu in 1 strain (4.8%). A nine nucleotide deletion (codon 510-513) was found in one strain (4.8%) while in the remaining resistant strain (4.8%) no mutation was detected. CONCLUSIONS: The frequency of the different mutations found in the rpoB gene, associated with rifampicin resistance in Mycobacterium tuberculosis clinical isolates in Seville, are similar to those previously reported. However, two new mutations has been detected: a nine nucleotide deletion (codon 510-513), and the Asn512-->Ser point mutation. The characterization of the mutations in the rpoB gene could serve as epidemiological marker for the rifampicin resistant clinical isolates of M. tuberculosis.     

Diversity of DNA sequences among enterohemorrhagic Escherichia coli O157:H7 detected by PCR-based DNA fingerprinting

A random amplified polymorphic DNA(RAPD) fingerprinting method has been developed to differentiate Enterohemorrhagic Escherichia coli(EHEC) O157:H7 isolates. This method uses an oligonucleotide of arbitrarily chosen sequence to prime DNA synthesis from pairs of sites to which it is matched or practically matched and results in strain-specific arrays of DNA products. By the RAPD analysis using A07(5'-TGCCTCGCACCA-3'), EHEC strains tested in this study were found to be divided into 5 groups. The RAPD arrays among 5EHEC clinical isolates from a single outbreak were identical from each other, and different from other origin. The data indicate that the RAPD method is feasible for investigating the source of an outbreak associated with EHEC O157:H7 infection.     

Genetic diversity among clinical and environmental isolates of Aspergillus fumigatus

To determine if cases of invasive aspergillosis (IA) were caused by strains of Aspergillus fumigatus with unique characteristics, strains from immunosuppressed patients with IA were compared to strains obtained from sputa of patients with cystic fibrosis and to strains from the environment. An extremely high genomic diversity was observed among the 879 strains typed by Southern blotting with a retrotransposon-like element from A. fumigatus (C. Neuvéglise, J. Sarfati, J. P. Latgé, and S. Paris, Nucleic Acids Res. 24:1428-1434, 1996). Analysis of Southern blot hybridization patterns showed the absence of clustering between environmental isolates and clinical isolates from patients with IA or cystic fibrosis. In addition, strains could not be clustered depending on their geographical location. This study implies that practically any strain of A. fumigatus is potentially pathogenic and can provoke a case of IA when it encounters a favorable environment in an immunosuppressed host.     

Multilocus evolutionarily stable strategy models: additive effects

Forty illness associated phage-type (PT) 4 and PT 8 strains of Salmonella enteritidis were analyzed by the pulsed-field technique of clamped homogeneous electric fields (CHEF) electrophoresis. Using NotI and XbaI, the 40 strains were subdivided by each enzyme into seven restriction endonuclease digestion profiles (REDP). The 35 PT 4 isolates from Austria were subdivided into six NotI and five XbaI REDP, while the five PT 8 isolates from the United States displayed a single NotI and two XbaI REDP. When highly-concentrated, uncleaved genomic DNA was subjected to CHEF electrophoresis, plasmid DNA in the size range of 350 kb relative to a linear DNA standard was discernible in 38 of the 40 strains. Subsequent isolation and restriction analyses of plasmid DNA from one strain (E40) revealed a single plasmid (pE40; ca. 54 kb) with one XbaI and two NotI cleavage sites that was similar in size to the S. enteritidis virulence plasmid pRQ29. Hybridization of the PE40 probe with S. enteritidis genomic DNAs identified a 54 kb fragment within the XbaI REDP and two fragments, 20 and 34 kb, in NotI REDP of plasmid-positive strains. It was not possible to identify plasmid-specific bands in NotI REDP without hybridization due to comigrating chromosomal and plasmid DNA fragments. Regardless of PT, all 40 S. enteritidis strains showed highly related REDP. The similarity between PT 4 and PT 8 strains as further revealed by Dice similarity coefficients was 90% to 95% for NotI REDP and 79% to 93% for XbaI REDP. These results support the hypothesis that the pandemic observed today is the result of the efficient spread of a single clone, or clusters of closely related clones, of S. enteritidis.     

Isozyme variation within and among populations of Microsporum species

Isozyme variation among 54 isolates of Microsporum canis, 18 Microsporum cookei isolates and two Diheterospora isolates were studied using starch gel electrophoresis. Of eight enzymes examined, four were polymorphic (EST, G6P, MDH and PEP), having from two to four electrophoretic forms. Within each species, consistent and reproducible isozyme patterns of the eight enzyme systems were obtained. Phenotypic diversity (H) in M. canis was higher than in M. cookei (H = 0.459 and H = 0.408 respectively), but phenotypic differentiation of M. canis isolates from different geographical regions (Auckland, Wellington and Palmerston North, New Zealand) was low, with a proportion of total diversity (Gst) of 0.151 found among the localities. The results suggest that the isolates of M. canis from different geographical regions are closely related, supporting the theory of a common lineage.