DNA Restriction Fragment Patterns Research Articles

Genetic Characteristics and Clonal Dissemination of β-Lactamase-Negative Ampicillin-ResistantHaemophilus influenzaeStrains Isolated from the Upper Respiratory Tract of Patients in Japan

We evaluated the recent prevalence of antimicrobial-resistant Haemophilus influenzae isolated from the upper respiratory tracts (URT) of patients in Japan. Mutations in the ftsI gene, which encodes penicillin binding protein 3 (PBP3), and the clonal dissemination of the resistant strains were also investigated. A total of 264 H. influenzae isolates were collected from patients with URT infections. According to the criteria of the Clinical and Laboratory Standards Institute for the susceptibility of H. influenzae to ampicillin (AMP), the isolates were distributed as follows: 161 (61.0%) susceptible strains (MIC < or = 1 microg/ml), 37 (14.0%) intermediately resistant strains (MIC = 2 microg/ml), and 66 (25.0%) resistant strains (MIC > or = 4 microg/ml). According to PCR-based genotyping, 172 (65.1%) of the isolates had mutations in the ftsI gene and were negative for the beta-lactamase (bla) gene. These 172 isolates were thus defined as genetically beta-lactamase-negative ampicillin-resistant (gBLNAR) strains. The ftsI mutant group included 98 (37.1%) strains with group I/II mutations in the variable mutated region (group I/II gBLNAR) and 74 (28.0%) strains with group III mutations in the highly mutated region (group III gBLNAR). Eighty-seven (33.0%) of the isolates were genetically beta-lactamase-negative ampicillin-susceptible (gBLNAS) strains. The group III gBLNAR strains showed resistance to beta-lactams. Only five strains (1.9%) were positive for a bla gene encoding TEM-type beta-lactamase. The three clusters consisting of 16 strains found among the 61 BLNAR strains (MIC > or = 4 microg/ml and without the bla gene) showed identical or closely related DNA restriction fragment patterns. Those isolates were frequently identified among strains with a MIC to AMP of 16 microg/ml. The current study demonstrates the apparent dissemination and spread of a resistant clone of H. influenzae among medical centers in Japan. The gBLNAR strains show a remarkable prevalence among H. influenzae isolates, with the prevalence increasing with time. This fact should be taken into account when treating URT infections.

Expression of S-locus inhibitor gene (Sli) in various diploid potatoes

S-locus inhibitor gene (Sli), which can inhibit gametophytic self-incompatibility in diploid potatoes and alter self-incompatible to self-compatible plants, was introduced by crossing into 32 diploid genotypes as females and its expression in the F1 and S1 progenies was investigated. We found that the expression of self-compatibility in the F1 hybrid progeny depended largely upon the female genotypes and partly upon the male genotypes (=Sli gene donor clones). Successful females produced hybrid plants, in which 67.1% of self-pollinated plants set S1 seeds. By second selfing upon the S1 plants, an average of 44.2% of self-pollinated plants were self-compatible. Unsuccessful females produced hybrids, most of which were self-incompatible or male-sterile. Restriction fragment patterns of chloroplast DNA (ctDNA) were able to distinguish successful females (S- or A-type ctDNA) from unsuccessful females (W- or T-type ctDNA). A ctDNA high-resolution marker analysis using seven microsatellites and H3 marker supported a higher degree of differentiation between the two groups of ctDNA types and implied a possible interaction between the cytoplasm and Sli gene function. However, it has been known that the cytoplasm having T-type ctDNA and that derived from Solanum demissum (haplotype 26 of W-type ctDNA) cause male sterility, and the present case with unsuccessful females were likely caused by male sterility rather than the failure of Sli gene function.

Diversity of Puccinia boroniae assessed by teliospore morphology and restriction fragment patterns of ribosomal DNA

Preliminary investigations into the diversity of Puccinia boroniae, collected from across southern Western Australia, were conducted by morphological comparison of the teliospores derived from host species Boronia heterophylla and Boronia megastigma, and by restriction enzyme profiling of the internal transcribed spacer (ITS) region of the nuclear rRNA (rRNA) genes of 21 isolates from a variety of different Boronia species and cultivars. Assessment of teliospore morphology provided significant evidence of two distinct rust groups, separated on the basis of their host plant origin. In general, teliospores from Boronia heterophylla had wider apical and basal cell widths, thinner cell walls and apical papilla, than those isolated from Boronia megastigrna. Restriction analysis of the ITS region provided no evidence of intraspecific variation among the 21 isolates; however, one isolate, distinct from the screened population both geographically and by host origin, was shown to have a length variation (insertion) within the ITS region amplified by PCR. The results indicate that subgroups of P. boroniae in Western Australia may be present, with further evidence required to define the level of separation in the population.

Evidence of rapid evolution and incipient speciation in Vicia sativa species complex based on nuclear and organellar RFLPs and PCR analysis

Seventy three accessions of the sevenVicia species belonging to Sativa speciescomplex were screened for nuclear and organellar restriction fragmentlength polymorphic (RFLP), and random amplified polymorphicDNA (RAPD) markers. Total genomic DNAs of 73 accessions wasrestricted with three enzymes, and the restriction fragments werehybridized to the wheat rDNA probe pTa71 (containing 18S, 5.8Sand 25S rDNA genes, and spacers), and faba bean probes Ver6-5 (entire intergenic spacer flanked by small part of25S and 18S fragments) and Ver 18-6 (part of thecoding region of the gene and internal transcribed spacers). InXbaI digests, 16 repeat unit length classes in24 combinations were identified. Digestion withEcoRI and DraI gave2–4 and 1–3 fragments, respectively, with detectablehybridization to the probes, indicating the existence of internalXbaI sites. All the accessions produced 3.5 EcoRI fragment arising fromcoding region of the repeat unit. Four hundred and eighteen RAPDmarkers among 45 accessions were identified with 14 arbitrary10-base primers. The percentage of polymorphic bands withinspecies ranged from 20 in V.angustifolia to 98% inV. nigra. Both RFLP andRAPD markers were unable to assess the relationships betweenaccessions within species as there was often much closer resemblancesbetween certain accessions of different species rather than betweenaccessions within each taxon. This analysis supports the view basedon morphological, cytogenetical and crossability data that it is notpossible to classify Sativa species complex into a small finitenumber of taxa which are clearly circumscribed, and that the complexrepresents a unique case of rapid evolution and incipient speciation.A study of chloroplast and mitochondrial RFLPs was undertaken toanalyze phylogeny through maternal lineage. Chloroplast DNArestriction fragment patterns, using 13 restriction endonucleases,revealed 92.6 to 99% homology between the seven species.Twelve enzyme-probe combinations yielded identical fragmentpatterns for all the seven species. The molecular sizes of thechloroplast DNAs obtained were similar (121.5–123.5), indicating that they had all lost one of theinverted repeats. Total DNAs digested with three restriction enzymesand hybridized to six heterologous probes of mitochondrial originyielded monomorphic bands in five enzyme—probe combinationsacross all the 73 accessions. In other combinations as well,40–66 accessions yielded monomorphic profiles. The smallvariation in the remaining accessions was not species-specificsince the same profiles were present in more than one species. Theseresults i) strongly suggest that the seven species within thecomplex share a common ancestor, or direct lineage and, ii)indicate that these species should be relegated to a rank, perhaps ofsubspecies, within V.sativa species complex.

Restriction Enzyme Analysis of PCR Amplified rDNA as a Taxonomic Tool in Yeast Identification

A method has been developed to simplify the identification of yeast strains. We used the restriction fragment patterns of PCR-amplified 18S rRNA-coding DNA with the neighbouring ITS1 region for differentiation and identification of 169 yeast strains representing 128 species associated mainly with food, wine, beer, and soft drinks. The amplicons were digested with four different four-base-cutting restriction enzymes. To construct a database of restriction fragment patterns, the gels have been scanned and analyzed using the Molecular Analyst Fingerprint 2.0 software. The use of four enzymes proved to be sufficient for strain identification.

Molecular epidemiology of Vibrio cholerae O1 isolated from sporadic cholera cases in Okinawa, Japan.

In July 1994, 6 cholera cases due to Vibrio cholerae O1 El Tor Ogawa sporadically appeared in Okinawa. All 6 patients had no history of traveling abroad. In the period of this cholera outbreak, a strain of V. cholerae O1 El Tor Ogawa was detected from an imported fish at the Naha port quarantine station. The isolates were characterized to clarify whether or not, they belonged to a common clone. Phenotypes were identical except that one strain revealed cured Celebes and the others were original Celebes in kappa phage typing. The restriction fragment patterns of DNA of the isolates hybridized with an enzyme-labeled oligonucleotide probe for cholera toxin gene (ctx) were identical. Randomly amplified polymorphic DNA of the isolates were identical when a primer was used, but 2 patterns were seen when another primer was used. Pulsed-field gel electrophoresis of the chromosomal DNA digested with NotI restriction enzyme showed 3 patterns. The DNA fragment pattern of the strain isolated from the imported fish was different from the clinical isolates. These results suggested that there was no epidemiological relation among the strains of V. cholerae O1 isolated during this period.

Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis.

Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.

An unusual mitochondrial DNA polymorphism in the Chorthippus biguttulus species group (Orthoptera: Acrididae)

AbstractMitochondrial DNA restriction fragment patterns have been used to investigate the Chorthippus biguttulus group: a group of morphologically similar grasshopper species with very distinct male calling songs. Ten haplotypes were identified in 21 populations, representing six species. Haplotype diversity was as great within species, and in some case within populations, as it was among species and no clear geographical patterns of haplotype distribution were revealed. In the light of grasshopper range changes since the last glaciation it is unlikely that these data can be explained entirely by the retention of ancestral polymorphism. This suggests that hybridization has occurred during periods of grasshopper range expansion since the last glacial maximum, allowing mtDNA transfer between populations that have previously diverged in allopatry.

Genetic analysis of fructan-hyperproducing strains of Streptococcus mutans.

Fructan polymer, synthesized from sucrose by the extracellular fructosyltransferase of Streptococcus mutans, is thought to contribute to the progression of dental caries. It may serve as an extracellular storage polysaccharide facilitating survival and acid production. It may also have a role in adherence or accumulation of bacterial cells on the tooth surface. A number of clinical isolates of S. mutans which produce large, mucoid colonies on sucrose-containing agar as a result of increased production of fructan have been discovered. By using eight independent isolates, we sought to determine if such fructan-hyperproducing strains represented a genetically homogeneous group of organisms. Restriction fragment patterns of total cellular DNA were examined by using pulsed-field and conventional gel electrophoresis. Four genetic types which appeared to correlate with the serotype of the organism and the geographic site of isolation were evident. Southern blot analysis of several genetic loci for extracellular enzymes revealed some minor differences between the strains, but the basic genomic organizations of these loci were similar. To evaluate whether the excess fructan produced by these strains enhanced the virulence of these organisms in the oral cavity, it was of interest to create mutants deficient in fructosidase (FruA), the extracellular enzyme which degrades this polymer. The fruA gene was inactivated by allelic exchange in two fructan-hyperproducing strains as well as in S. mutans GS5, a strain which does not hyperproduce fructan. All of the fruA mutant strains were devoid of fructan hydrolase activity when levan was used as a substrate. However, the fructan-hyperproducing strains retained the ability to hydrolyze inulin, suggesting the presence of a second fructosidase with specificity for inulin in these strains.

Mycobacterium abscessus pseudoinfection traced to an automated endoscope washer: utility of epidemiologic and laboratory investigation.

After 15 patients had positive cultures for Mycobacterium abscessus without evidence of infection (i.e., pseudoinfection) following endoscopy, retrospective cohort studies of patients undergoing endoscopy and microbiologic sampling of the environment were done to examine potential nosocomial transmission and to identify the source and risk factors for M. abscessus pseudoinfection. In the epidemic period, M. abscessus-positive cultures were significantly more likely to be obtained during bronchoscopy than gastroendoscopy (16/149 vs. 1/860, P < .001) and during procedures using bronchoscopes disinfected in an automated washer rather than by other methods (16/54 vs. 0/95, P < .001). M. abscessus was recovered from the automated washer, the inlet water feeding the washer, and a flexible bronchoscope. Environmental and case-patient isolates had identical large restriction fragment (LRF) patterns of genomic DNA separated by pulsed-field gel electrophoresis. Molecular typing using LRF analysis supported the epidemiologic results and demonstrate the utility of combined epidemiologic and laboratory investigations in nosocomial outbreaks of nontuberculous mycobacteria.

Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units

During a 14-month period, a unique strain of Corynebacterium striatum that produces a diffusible brown pigment was isolated from purulent sputa of nine patients and from nonrespiratory sites of two additional patients. Seven nonpigmented clinical isolates from the same period and three reference strains of C. striatum were compared with the brown isolates. Most patients had multiple sputum cultures with no coryneforms before the brown strain emerged, suggesting that the organism was hospital acquired. DNA restriction fragment patterns and Southern hybridization with the att site probe of Corynebacterium diphtheriae indicated that the brown isolates were a single strain which was distinct from the heterogeneous nonpigmented strains. A common source for the brown C. striatum was not recognized, although all of these patients were located in two adjoining intensive care units. All of the brown isolates, three of the nonpigmented clinical isolates, and two reference strains had positive CAMP reactions with Staphylococcus aureus, which has not been reported for C. striatum prior to this study.

Relatedness and Spatial Distribution ofArmillariaGenets Infecting Red Pine Seedlings

Genetic similarity among Armillaria genets responsible for root disease foci in a northern Michigan red pine plantation was investigated. The study plantation was established in 1984 in a clear-cut portion of a 60-yr-old hardwood forest. Armillaria isolates obtained from 87 moribund seedlings were examined. All 87 isolates were identified as A. ostoyae on the basis of mitochondrial DNA (mtDNA) restriction fragment patterns and/or mating interactions with voucher isolates. Independent assays of nuclear DNA restriction fragment patterns and somatic incompatibility groups agreed completely in distinguishing genets at the study site. Given knowledge of the stand history and previous estimates of mycelial growth rates, we conclude that several genets occupying large territories must have been established long before the 1984 stand conversion [...]

Use of small fragment restriction endonuclease analysis (SF-REA) for epidemiological fingerprinting of Klebsiella oxytoca

A group of infections caused by Klebsiella oxytoca was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of unique antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of one single strain existed. All 4 K. oxytoca isolates from the NICU and, in addition, 50 epidemiologically non-related strains were fingerprinted by small fragment restriction endonuclease analysis (SF-REA). From an analysis of silver-strained EcoR I-generated restriction fragment patterns of whole-cell DNA in polyacrylamide gels, it was evident that the 4 NICU isolates were closely related, whereas all epidemiologically unrelated strains exhibited different patterns. From the results of this study, DNA fingerprinting by SF-REA is proposed as a useful tool for investigating the epidemiological relatedness of K. oxytoca strains of clinical and environmental origin.

Chloroplast segregation in somatic hybrids of Nicotiana plumbaginifolia and N. sylvestris having different ratios of parental nuclear genomes

Fusion of mesophyll protoplasts of haploid Nicotiana plumbaginifolia (P) and N. sylvestris (S) resulted in the production of somatic hybrid plants of various ploidy levels. Analysis of the restriction fragment patterns of chloroplast DNA from 118 plants belonging to genome constitutions PS, PPS, PSS, and PPSS revealed that two had a pattern corresponding to a mixture of parental DNA while all the others had the pattern of either N. plumbaginifolia or N. sylvestris. In the latter case, the ratio of the two parental types fits 1∶1 in all the four genome constitutions studied. Since the protoplasts used in the fusion experiment were physiologically similar and the hybrid cells were not deliberately selected, these results suggest that chloroplast segregation in the somatic hybrids is independent of the chloroplast input of the fusion partners and the nuclear background of the fusion products.

Molecular analysis of cytoplasmic male sterility in chives (Allium schoenoprasum L.)

The mitochondria of chive plants with normal N or male-sterile S cytoplasms have been examined by restriction fragment analysis and Southern hybridizations of mitochondrial DNA (mtDNA) and in organello protein biosynthesis. Restriction fragment patterns of the mtDNA differed extensively between N-and S-cytoplasms. The percentage of fragments with different mobility varied between 44-48% depending on the restriction enzyme used. In contrast to mtDNA, the restriction fragment patterns of the chloropolast DNA from N- and S-cytoplasms were identical. The organization of the analyzed mitochondrial genes coxII, coxIII, nad1 and nad3 was different in N- and S-cytoplasms. Comparison of mitochondrial proteins analyzed by in organello translation revealed an 18-kDa protein present only in S-cytoplasm. The restorer gene X suppressed the synthesis of that protein in S-cytoplasm. Thus, the 18-kDa protein seems to be associated with the cytoplasmic male-sterile phenotype.

Restriction fragment patterns of chloroplast and mitochondrial DNA of Dasypyvum villosum (L.) candargy and wheats

To elucidate the phylogenetic relationships and cytoplasmic types, restriction endonuclease fragment patterns of chloroplast (cp) and mitochondrial (mt) DNAs isolated from two different accessions of Dasypyrum villosum (L.) candargy were compared with those of tetraploid wheat (Triticum durum Desf., PI265007), hexaploid wheat (Triticum aestivum L., cv Chinese Spring), Aegilops longissimum (S. and M., in Muschli) Bowden and Hordeum vulgare L. T. aestivum and T. durum had identical restriction patterns for their cp and mtDNAs in digestions with four different enzymes. Likewise, no differences were found between the restriction fragment patterns of two accessions of D. villosum. But, there were distinct differences in chloroplast and mitochondrial DNA restriction fragment patterns between D. villosum and tetraploid and hexaploid wheats. A. longissimum (G609) showed a similar pattern to those wheats for PstI digestion of cpDNA. Organellar DNA from Hordeum vulgare (cv Himalaya) showed a distinctly different restriction pattern from those of wheat and D. villosum. These results suggest that D. villosum is unlikely to be the donor of cytoplasm to wheats, and its cytoplasmic organelles were also different from those of A. longissimum.

Screening for Sulfolobales, their Plasmids and their Viruses in Icelandic Solfataras

We have sampled acidic springs, water and mud holes of 14 major solfataric fields of Iceland in order to isolate both heterotrophic and autotrophic members of the order Sulfolobales and to find affiliated genetic elements e.g. plasmids and viruses. The diverstiy of 120 isolates was analysed by comparing DNA restriction fragment patterns. The 44 heterotrophic isolates belonged to only two types, the solfataricus (S) type and the islandicus (I) type as judged by the restriction patterns of their DNAs. None of the heterotrophic isolates was able to grow autotrophically. The more than 70 autotrophic isolates belonged to only three types, the Desulfurolobus (D) type, the closely related A type and, rarely, the B type. None of the autotrophic isolates was able to grow heterotrophically. Primary heterotrophic colonies often gave rise to obligately autotrophic isolates when submitted to autotrophic selection, probably because they constituted synthrophic associatons. Inversely, primary autotrophic colonies sometimes yielded obligately heterotrophic isolates when submitted to heterotrophic selection. Cell-free filtrates of the field samples precipitated with PEG 6000 yielded different types of viruslike particles as visualized by electron microscopy. Some of these were probably Thermoproteus viruses. No infectious or even lytic virus was obtained from these samples. Four different multicopy plasmids, three, pRN1, pRN2 and pHE7 from heterotrophic hosts and one, pDL10, originally found in Desulfurolobus ambivalens DSM 3772, occurring in all but two autotrophs of the D type, were characterized and used for developing cloning vectors. All 18 representatives of the heterotrophic S type and none of the I type were lysogenic for the virus SSV1 originally discovered in S. shibatae . Different lysogens exhibited different ratios of free circular and linearly integrated viral DNA. The I type isolate KVEM10H1 multiplied SSV 1 but did not integrate its genome into the chromosome. One heterotrophic I type and one autotrophic D type isolate carried doublestranded DNA viruses, SIRV and DAFV. SIRV is a stiff rod, 0.95 μm long and 0.026 μm in diameter, consisting of 33 kbpairs of linear double stranded DNA, a strongly basic DNA binding protein and terminal tentacles involved in attachment to thin filaments, most probably pili. It lacks a membrane or hydrophobic coat and represents a novel virus tpye. The flexible filamentous DAFV, 2.3 μm long and 0.027 μm in diameter, containing 56 kbpairs of linear double-stranded DNA, appears to be enwrapped in a membrane. It resembles representatives of the lipotrixviridae, most closely the virus TTV2.

Epidemiological study on nosocomial infection of Pseudomonas cepacia by plasmid analyses

In order to clarify the usefulness of plasmid analyses in epidemiological study, plasmid DNA profiles, restriction fragment patterns of chromosomal DNA analysed by pulsed-field gel electrophoresis (PFGE), and patterns of extracellular products were performed in twenty-four nosocomial strains of Pseudomonas cepacia isolated at the Kagawa Medical School Hospital. Fifteen strains were obtained from 15 patients admitted in A ward, three strains were obtained from nebulizer devices used in A ward, 5 strains were obtained from 5 patients admitted in B ward, and 1 strain was obtained from a patient admitted in C ward. Three different patterns which differed from ward to ward were clearly distinguished by PFGE patterns and patterns of extracellular products. On the other hand, plasmid DNA profiles were different in some strains obtained from B ward. These results suggested that plasmid analyses combined with other typing methods are useful in more precise epidemiological analysis on nosocomial infection of P. cepacia.

A comparison of morphology, pathogenicity and restriction fragment patterns of mitochondrial DNA among isolates of Phytophthora porri Foister

Thirteen strains ofPhytophthora porri from five different hosts were compared with respect to their morphology, cardinal temperatures for growth, pathogenicity to leek and cabbage and restriction fragment patterns of mitochondrial DNA. Morphology of vegetative growth was rather similar in most isolates. Those characters which differed among isolates showed overlapping variability and could not be used to distinguish groups, with the exception of production of oogonia and sporangia and the antheridium type. Considerable differences were found in restriction patterns of mitochondrial DNA, isolates from the same host mostly showing identical patterns. Isolates from differentAllium species showed relatively similar restriction patterns if compared to the other isolates. Isolates fromBrassica oleracea proved to be a homogeneous group, quite different from the others with respect to restriction patterns, production of sporangia, production of oogonia, antheridium type and pathogenicity. One isolate, CBS 366.59, isolated from and pathogenic toA. porrum, deviated in many characters from the other isolates. It showed the restriction patterns ofPhytophthora nicotianae and also the high cardinal temperatures for growth typical for this species. The sporangia, however, were distinctly non-papillate and the majority of antheridia was of the paragynous type.

Large restriction fragment patterns of genomic Mycobacterium fortuitum DNA as strain-specific markers and their use in epidemiologic investigation of four nosocomial outbreaks.

Pulsed-field gel electrophoresis and restriction endonucleases with rare recognition sites were used to generate large restriction fragment (LRF) patterns of genomic DNA from 48 isolates of Mycobacterium fortuitum biovariant fortuitum. Epidemiologically unrelated isolates gave highly diverse patterns when AsnI, HpaI, AflII, DraI, NdeI, XbaI, SpeI, or SspI was used. Epidemiologically related isolates produced identical or minimally different LRF patterns. Minor variations in LRF patterns were seen in two epidemic isolates digested with XbaI, suggesting that genetic alteration had occurred. LRF patterns were used to study three cardiac surgery wound infection outbreaks and one respiratory disease nosocomial outbreak. In two outbreaks, LRF patterns confirmed the reported clustering of isolates on the basis of multiple phenotyping methods. In the remaining two outbreaks, isolates which could not be separated by prior typing methods were easily distinguished by LRF pattern analysis. Environmental water isolates from two outbreaks had LRF patterns identical to those of the disease-producing strains, confirming that the local environment was the source of infection. Pulsed-field gel electrophoresis of LRFs of genomic DNA offers great promise as an epidemiologic tool for the study of M. fortuitum.