Comparison of clinical and environmental isolates of Legionella pneumophila obtained in the UK over 19 years

Between January 1980 and December 1998, 3458 cases of Legionnaires' disease were reported to the national surveillance scheme in England and Wales. Of these, 463 (13.4%) were reported as proven by culture and isolation of Legionella spp., with 96.3% being Legionella pneumophila. Serogroup (Sgp), monoclonal antibody (mAb) subgrouping and restriction fragment length polymorphism (RFLP) analysis data were obtained for 321 (69.3%) of these, of which 284 were classified as being unrelated to any other isolate in the study. Typing data were also available for 117 unrelated environmental isolates of L. pneumophila obtained from England and Wales, giving a total of 401 unrelated isolates in the study. Of the clinical isolates, 88.0% were Sgp1, compared with only 42.7% of environmental isolates (p <0.001); 79.6% of clinical isolates were subgrouped as mAb2+, compared with only 12.8% of environmental isolates (p <0.001). RFLP typing identified 67 types among the 401 isolates, with clinical isolates showing significantly less diversity than environmental isolates (index of diversity (IOD) 0.944 vs. 0.958; p <0.05), with three RFLP types (1, 5 and 14) accounting for 40.0% of all clinical isolates. Combining the phenotypic and genotypic data resulted in 173 distinct phenons, with clinical isolates showing significantly less diversity than environmental isolates (IOD 0.964 vs. 0.996; p <0.01). Three phenons accounted for 30% of all clinical isolates. These data strongly suggest that some strains of L. pneumophila are more likely to cause human infection than would be expected from their distribution in the environment.     

Mitochondrial DNA analysis of Sporothrix schenckii]

Restriction fragment length polymorphism (RFLP) in mitochondrial DNA (mtDNA) of clinical and environmental isolates of Sporothrix schenckii was investigated. Isolates of S. schenckii were classified into 24 mtDNA types (Types 1-24) based on mtDNA RFLP patterns with HaeIII and clustered into two major groups by phylogeny, Group A and Group B. Group A isolates are predominant in South Africa, North America, Central America and South America, while Group B isolates are predominant in Australia, Japan and China. Based on the mtDNA-RFLP patterns with HaeIII, most environmental isolates morphologically identified as S. schenckii were confirmed to be species distinct from S. schenckii and S. schenckii isolates were few, while all of more than 500 clinical isolates were confirmed as S. schenckii. Therefore, RFLP analysis of mtDNA is essential for the identification of environmental, but not clinical isolates of S. schenckii.     

Increased release of glucuronoxylomannan antigen and induced phenotypic changes in Trichosporon asahii by repeated passage in mice

Clinically important fungi such as Candida albicans and Cryptococcus neoformans are known to undergo phenotypic changes after repeated subculture or passages in vivo. However, there are no reports describing this phenomenon in Trichosporon species. This study investigated whether in-vivo passages of environmental isolates of Trichosporon asahii in mice changes their phenotype; three environmental isolates and 14 clinical isolates (from deep-seated infections) were used. The shape of the colony and cell type were observed, and the titre of glucuronoxylomannan (GXM) antigen and concentration of (1-->3)-beta-D-glucan were measured for each isolate. Changes in these features were also examined after three passages of the environmental isolates in mice. The shape of colonies and cell types were clearly different in environmental and clinical isolates. Furthermore, the clinical isolates released significantly higher levels of GXM antigen than environmental isolates (titre: log2 9.4 SD 0.7 versus log2 5.4 SD 1.4). The phenotype of passaged isolates was significantly different from the original environmental isolates with respect to the morphology of colonies and cell type and GXM release (titre: log2 10.0 SD 0.7 versus log2 5.4 SD 1.4). These results suggest that the phenotypic changes in T. asahii occur as a result of in-vivo passages. This process may allow a proportion of the fungal population to escape eradication by the host immune system, as GXM antigen is considered to protect the fungi against phagocytosis by polymorphonuclear leucocytes and monocytes in vivo.     

Burkholderia cepacia complex bacteria from clinical and environmental sources in Italy: genomovar status and distribution of traits related to virulence and transmissibility

Sixty-eight Burkholderia cepacia complex isolates recovered from the sputum of 53 cystic fibrosis patients and 75 isolates collected from the maize rhizosphere were compared to each other to assess their genomovar status as well as some traits related to virulence such as antibiotic susceptibility, proteolytic and hemolytic activities, and transmissibility, in which transmissibility is determined by detection of the esmR and cblA genes. Among the clinical isolates, B. cepacia genomovar III comprised the majority of isolates examined and only a very few isolates were assigned to B. cepacia genomovar I, B. stabilis, and B. pyrrocinia; among the environmental isolates a prevalence of B. cepacia genomovar III and B. ambifaria was observed, whereas few environmental isolates belonging to B. cepacia genomovar I and B. pyrrocinia were found. Antibiotic resistance analysis revealed a certain degree of differentiation between clinical and environmental isolates. Proteolytic activity and onion tissue maceration ability were found to be spread equally among both clinical and environmental isolates, whereas larger percentages of environmental isolates than clinical isolates had hemolytic activity. The esmR gene was found exclusively among isolates belonging to B. cepacia genomovar III, with a marked prevalence in clinical isolates, whereas only one clinical isolate belonging to B. cepacia genomovar III was found to bear the cblA gene. In conclusion, the results of the present study show that the species compositions of the clinical and environmental B. cepacia complex populations examined are quite different and that some of the candidate determinants related to virulence and transmissibility are not confined solely to clinical isolates but are also spread among environmental isolates belonging to different species of the B. cepacia complex.     

Mitochondrial DNA fingerprinting of Acanthamoeba spp. isolated from clinical and environmental sources.

Restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA fingerprinting) was evaluated as an epidemiologic tool for identifying potential reservoirs of Acanthamoeba infection. Fingerprints for 15 clinical isolates recovered by our affiliated laboratories were compared with those for 25 environmental isolates from western Washington State and 10 American Type Culture Collection (ATCC) strains. Seven different fingerprint groups emerged from the analysis of clinical isolates with six selected restriction enzymes (BamHI, BglII, EcoRI, HindIII, KpnI, and SalI). Fourteen (56%) environmental and 4 (40%) ATCC isolates displayed fingerprints similar to those of clinical isolates. In all, five of the seven groups contained one or more environmental and/or ATCC isolates. Comparisons with published mtDNA fingerprints for Acanthamoeba isolates showed that two groups have counterparts in Europe and Japan and in Europe and Australia. The inclusion of environmental isolates demonstrated that the most common clinical isolates do have counterparts readily recoverable from the surrounding environment and that some of these counterparts appear to be geographically widespread.     

Genotypic characterisation of vancomycin-resistant Enterococcus faecium isolates from haemato-oncological patients at Olomouc University Hospital, Czech Republic

This study describes the first molecular characterisation of clinical isolates of vancomycin-resistant enterococci (VRE) in the Czech Republic. Of 2647 patient isolates of Enterococcus spp. from 1997-2002, 121 (4.6%) were identified as VRE. The most common isolates were VanA+ Enterococcus faecium (78%) and VanB+ Enterococcus faecalis (10%). In addition, five VanA+ E. faecium isolates were obtained from environmental and staff sampling. Macrorestriction analysis of SmaI restriction fragment length polymorphism was performed for 54 VanA+ E. faecium clinical isolates and the five VanA+ E. faecium environmental isolates. Thirty-two unique restriction endonuclease patterns were identified, including two predominant clonal types represented by five or more isolates. Two environmental VanA+ E. faecium isolates were closely related to two patient isolates, which had an identical SmaI macrorestriction pattern. The results indicated potential survival of strains in the hospital environment and possible subsequent transmission to hospitalised patients.     

Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis.

The largest recorded epidemic of sporotrichosis in the United States occurred in 1988 and involved a total of 84 cases in 15 states. All cases were associated with Wisconsin-grown sphagnum moss. Twenty-one clinical isolates of Sporothrix schenckii and 69 environmental isolates of Sporothrix spp. from the epidemic were characterized and compared. The environmental isolates were recovered from 102 samples of sphagnum moss and other material by using direct plating techniques. Characteristics examined included macroscopic and microscopic morphology, conversion to a yeast phase, exoantigen reactions, and virulence in mice. On the basis of these studies, eight environmental isolates were identified as S. schenckii, five were identified as Ophiostoma stenoceras, and the remainder were identified as Sporothrix species. The environmental isolates of S. schenckii were recovered from moss samples from one Pennsylvania nursery and from three New York State Soil and Water Conservation districts, but none were recovered from moss directly from the bogs in Wisconsin.     

Diversity among strains of Pseudomonas aeruginosa from manure and soil,evaluated by multiple locus variable number tandem repeat analysis and antibiotic resistance profiles

The results of a multiple locus variable number of tandem repeat (VNTR) analysis (MLVA)-based study designed to understand the genetic diversity of soil and manure-borne Pseudomonas aeruginosa isolates, and the relationship between these isolates and a set of clinical and environmental isolates, are hereby reported. Fifteen described VNTR markers were first selected, and 62 isolates recovered from agricultural and industrial soils in France and Burkina Faso, and from cattle and horse manure, along with 26 snake-related isolates and 17 environmental and clinical isolates from international collections, were genotyped. Following a comparison with previously published 9-marker MLVA schemes, an optimal 13-marker MLVA scheme (MLVA₁₃-Lyon) was identified that was found to be the most efficient, as it showed high typability (90%) and high discriminatory power (0.987). A comparison of MLVA with PFGE for typing of the snake-related isolates confirmed the MLVA₁₃-Lyon scheme to be a robust method for quickly discriminating and inferring genetic relatedness among environmental isolates. The 62 isolates displayed wide diversity, since 41 MLVA types (i.e. MTs) were observed, with 26 MTs clustered in 10 MLVA clonal complexes (MCs). Three and eight MCs were found among soil and manure isolates, respectively. Only one MC contained both soil and manure-borne isolates. No common MC was observed between soil and manure-borne isolates and the snake-related or environmental and clinical isolates. Antibiotic resistance profiles were performed to determine a potential link between resistance properties and the selective pressure that might be present in the various habitats. Except for four soil and manure isolates resistant to ticarcillin and ticarcillin/clavulanic acid and one isolate from a hydrocarbon-contaminated soil resistant to imipenem, all environmental isolates showed wild-type antibiotic profiles.     

Comparative evaluation of selective media for isolation of Pseudomonas cepacia from cystic fibrosis patients and environmental sources.

Pseudomonas cepacia has recently emerged as an important pathogen affecting cystic fibrosis (CF) patients. We evaluated three selective media to assess their comparative potential for identification of patients colonized with P. cepacia and for efficacy of detection of P. cepacia in environmental fluids. Test organisms included P. cepacia isolates from CF patients (10 each from two CF centers), non-CF patients (10 isolates), and environmental sources (10 isolates). Microbiologic assays were done by the membrane filter procedure; filters were placed on P. cepacia medium (PCM), OFPBL, TB-T, MacConkey agar (MAC), and blood agar (BA) or Standard Methods (SM) sugar, and colonies were counted after incubation at 30 or 35 degrees C for 72 h. Mean recovery efficiencies (MREs) (mean CFU/ml on selective media compared with CFU/ml on BA controls) for environmental and non-CF P. cepacia and patient isolates from one CF center showed a rank order of PCM greater than OFPBL greater than TB-T; for isolates from a second CF center, a rank order of PCM greater than TB-T greater than OFPBL was obtained. MREs for CF center isolates were generally lower than for non-CF patients or environmental isolates on P. cepacia-selective media. With MAC, the MREs for each group of CF isolates were extremely low (14 and 2%) compared with those for non-CF patient (47%) or environmental (84%) isolates. In laboratory and field studies, PCM and OFPBL showed good selectivity against bacteria commonly associated with CF patient respiratory secretions. These findings show that selective media should be used in clinical settings where P. cepacia is sought. With environmental fluids from CF centers, P. cepacia-selective media showed low selectivity against a variety of gram-negative water bacteria and appeared to afford little advantage over SM agar for isolating P. cepacia from environmental samples.     

Characterization of Mycobacterium bohemicum isolated from human, veterinary, and environmental sources

Chemotaxonomic and genetic properties were determined for 14 mycobacterial isolates identified as members of a newly described species Mycobacterium bohemicum. The isolates recovered from clinical, veterinary, and environmental sources were compared for lipid composition, biochemical test results, and sequencing of the 16S ribosomal DNA (rDNA) and the 16S-23S rDNA internal transcribed spacer (ITS) regions. The isolates had a lipid composition that was different from those of other known species. Though the isolates formed a distinct entity, some variations were detected in the features analyzed. Combined results of the phenotypic and genotypic analyses were used to group the isolates into three clusters. The major cluster (cluster A), very homogenous in all respects, comprised the M. bohemicum type strain, nine clinical and veterinary isolates, and two of the five environmental isolates. Three other environmental isolates displayed an insertion of 14 nucleotides in the ITS region; they also differed from cluster A in fatty alcohol composition and produced a positive result in the Tween 80 hydrolysis test. Among these three, two isolates were identical (cluster B), but one isolate (cluster C) had a unique high-performance liquid chromatography profile, and its gas liquid chromatography profile lacked 2-octadecanol, which was present in all other isolates analyzed. Thus, sequence variation in the 16S-23S ITS region was associated with interesting variations in lipid composition. Two of the isolates analyzed were regarded as potential inducers of human or veterinary infections. Each of the environmental isolates, all of which were unrelated to the cases presented, was cultured from the water of a different stream. Hence, natural waters are potential reservoirs of M. bohemicum.     

Pseudomonas aeruginosa enzyme profiling: predictor of potential invasiveness and use as an epidemiological tool

The enzymatic potential of 54 clinical and 22 environmental isolates of Pseudomonas aeruginosa from soil and water were evaluated by substrate plate assays. Clinical isolates produced substantial levels of 9 of the 11 enzymes assayed, whereas strains recovered from soil or water were relatively inert enzymatically. Elastase, deoxyribonuclease, and elevated protease activities were associated preferentially with clinical isolates of systemic origin; these activities were found twice as frequently in clinical isolates as in strains derived from sputum or the urogenital tract. Our data suggest that these factors may play an important role in the dissemination of P. aeruginosa from local or superficial sites. A comparison of the enzyme profiles of the environmental and clinical isolates indicated that colonization or infection by environmental strains of P. aeruginosa is a rare event and that environmental and clinical strains comprise separate biovars. Epidemiologically, enzyme profiles permitted the fingerprinting and differentiation of clinical strains from various sources.     

Use of a mouse model to evaluate clinical and environmental isolates of Sporothrix spp. from the largest U.S. epidemic of sporotrichosis.

Five clinical and 69 environmental isolates from the largest U.S. epidemic of sporotrichosis were evaluated in NYLAR male mice following intravenous injection of 5 x 10(6) to 2 x 10(8) conidia per mouse. The clinical isolates and eight environmental isolates produced 100% mortality in groups of three mice each between 12 and 24 days after injection. These virulent isolates grew at 37 degrees C, were dematiaceous by virtue of melanin (melanized) on permissive media (e.g., potato dextrose agar), produced ovoid conidia borne sympodially on lateral conidiophores and pleurogenously about the main hyphal axis, and were identified as Sporothrix schenckii. Two melanized environmental isolates that grew at 35 degrees C but not at 37 degrees C were not virulent and had subtle morphological differences from S. schenckii. The remaining environmental isolates were not melanized, were not virulent, and were not S. schenckii; five were identified as Ophiostoma stenoceras and the remainder were identified as Sporothrix spp. Quantitative organ cultures revealed that clinical isolates grew exponentially in livers and testes, in contrast to an isolate of O. stenoceras that was eliminated from liver, lung, and spleen but that persisted in the testes throughout the 14-day sample period. This model helped to confirm the identification of S. schenckii isolates obtained from the environment.     

Microsatellite analysis of environmental and clinical isolates of the opportunist fungal pathogen Aspergillus fumigatus

Microsatellite analysis was used to examine the genetic relatedness of 111 clinical and environmental isolates of the opportunist human pathogenic fungus Aspergillus fumigatus from Ontario, Canada. Forty-three A. fumigatus isolates were from clinical sources and 68 from environmental sources. Phylogenetic analysis of the genotypes revealed that there were no geographical or temporal associations of clinical or environmental genotypes. In fact, several of the environmental and clinical isolates showed identical (clonal) genotypes from disparate geographical areas. However, a locus by locus examination revealed that there were several significant differences in allele frequencies between clinical and environmental isolates. There may be linkage of certain microsatellite loci with genes affecting virulence in A. fumigatus. A susceptible individual may be equally predisposed to infection by any isolate of A. fumigatus. However, under transient selection as a pathogen, genes encoding alleles for enhanced virulence may not assort independently from microsatellite loci. A dynamic equilibrium may exist between random recombination of loci in the natural environment and selection for virulence factors during host infection cycles.     

Restriction fragment length polymorphism analysis of Cryptococcus neoformans isolates from environmental (pigeon excreta) and clinical sources in New York City.

Restriction fragment length polymorphism analysis of environmental (pigeon excreta) and clinical Cryptococcus neoformans var. neoformans isolates in a limited geographic area distinguished 6 strains among 8 environmental isolates and 12 strains among 17 clinical isolates. Clusters of patients with three strains types accounted for 47% of clinical isolates. Despite this diversity, two strains were shared by environmental and clinical isolates.     

DNA typing of isolates associated with the 1988 sporotrichosis epidemic.

DNA typing techniques were used to examine selected clinical and environmental isolates of Sporothorix spp. recovered from the 1988 sporotrichosis epidemic in multiple states of the United States. Previous studies indicated that isolates in one of the six morphologically or physiologically distinct groups (group I) obtained from environmental sources were Sporothrix schenckii and were the possible etiologic agents responsible for the epidemic. To assess this hypothesis at the genetic level, whole-cell DNA was extracted from selected clinical isolates and representative members of each of the six environmental groups subjected to endonuclease digestion and then analyzed by gel electrophresis. DNA types were assigned on the basis of restriction fragment length polymorphism patterns. One DNA type was common to clinical and group I isolates but was dissimilar from the DNA types exhibited by groups II to VI. In contrast, a variety of DNA types were associated with isolates in groups II to VI. The latter groups appeared to make up a heterogeneous collection of fungi, with some members of the same group having different DNA types but with others from different groups possessing identical DNA types. Thus, DNA typing studies confirmed that group I environmental isolates are indistinguishable from clinical isolates and that group II to VI isolates represent a complex of related fungi with similar Sporothrix anamorphs.     

Lack of agreement between biochemical and genetic identification of Aeromonas spp

Biochemical and genetic identification by RFLP (restriction fragment length polymorphism) of the PCR-amplified 16S r-RNA sequence were compared for a selection of 171 clinical and environmental isolates of Aeromonas spp. The investigation revealed large differences between the two methods. The species phenotypic identification scheme and the genetic technique applied to the environmental strains gave divergent results for 96% of the strains tested. There was 46% discrepancy between the two methods for the clinical isolates. The distribution of species differed between clinical and environmental isolates. A. hydrophila, A. caviae, A. jandaei and A. veronii dominated the clinical material (81% of isolates by RFLP), whilst only 21% of the environmental isolates belonged to those four species. From the environmental group A. salmonicida, A. bestiarum, A. sobria, A. media, and A. encheleia contributed 72% of the strains tested. The poor parity between the biochemical and the genetic identification of the environmental isolates, and to a lesser extent for the clinical isolates, underlines the fact that our current biochemical methods cannot adequately differentiate Aeromonas spp. This work also shows that the biochemical schemes derived from clinical isolates are incomplete for the identification of environmental strains.     

Comparative analysis of environmental and clinical populations of Cryptococcus neoformans

Cryptococcus neoformans is a major, global cause of meningoencephalitis in immunocompromised patients. Despite advances in the molecular epidemiology of C. neoformans, its population structure and mode of reproduction are not well understood. In the environment, it is associated with avian guano or vegetation. We collected nearly 800 environmental isolates from three locations in the United States (viz., North Carolina, California, and Texas) and compared them with one another and with clinical isolates from North Carolina. As expected, they consisted of the most prevalent serotypes, serotypes A and D, as well as serotype AD hybrids. The majority of environmental isolates were obtained from pigeon excreta. All environmental and clinical isolates of serotype A or D had the MATalpha mating-type allele. However, the AD hybrids included MATa alleles typical of serotypes A and D. Using an amplified fragment length polymorphism fingerprinting technique with two primer pairs, we identified 12 genotypes among the isolates of serotype A. Six of these genotypes were present in both the clinical and the environmental populations. However, one of the most prevalent environmental genotypes was absent from the clinical samples, and two other genotypes were isolated only from patients. The combined molecular data suggest that this environmental population of C. neoformans is predominantly clonal, although there was evidence for recent or past recombination.     

Most human isolates of Mycobacterium avium Mav-A and Mav-B are strong producers of hemolysin, a putative virulence factor

Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.     

Presence of CTX gene cluster in environmental non-O1/O139 Vibrio cholerae and its potential clinical significance

Purpose: The aim of this study was to understand the epidemiological linkage of clinical and environmental isolates of Vibrio cholerae and to determine their genotypes and virulence genes content. Materials and Methods: A total of 60 V. cholerae strains obtained from clinical specimens (n = 40) and surface waters (n = 20) were subjected to genotyping using PFGE and determination of their virulence-associated gene clusters. Result: PCR analysis showed the presence of chromosomally located hly and RTX genetic elements in 100% and 90% of the environmental isolates, respectively. The phage-mediated genetic elements such as CTX, TLC and VPI were detected in 5% of the environmental isolates suggesting that the environmental isolates cannot acquire certain mobile gene clusters. A total of 4 and 18 pulsotypes were obtained among the clinical and environmental V. cholerae isolates, respectively. Non-pathogenic environmentally isolated V. cholerae constituted a distinct cluster with one single non-O1, non-O139 strain (EP6) carrying the virulence genes similar to the epidemic strains. This may suggest the possible potential of conversion of non-pathogenic to a pathogenic environmental strain. Conclusions: The emergence of a single environmental isolate in our study containing the pathogenicity genes amongst the diverse non-pathogenic environmental isolates needs to be further studied in the context of V. cholerae pathogenicity sero-coversion.     

Multilocus sequence typing of historical Burkholderia pseudomallei isolates collected in Southeast Asia from 1964 to 1967 provides insight into the epidemiology of melioidosis

A collection of 207 historically relevant Burkholderia pseudomallei isolates was analyzed by multilocus sequence typing (MLST). The strain collection contains environmental isolates obtained from a geographical distribution survey of B. pseudomallei isolates in Thailand (1964 to 1967), as well as stock cultures and colony variants from the U.S. Army Medical Research Unit (Malaysia), the Walter Reed Army Institute for Research, and the Pasteur Institute (Vietnam). The 207 isolates of the collection were resolved into 80 sequence types (STs); 56 of these were novel. eBURST diagrams predict that the historical-collection STs segregate into three complexes when analyzed separately. When added to the 760 isolates and 365 STs of the B. pseudomallei MLST database, the historical-collection STs cluster significantly within the main complex of the eBURST diagram in an ancestral pattern and alter the B. pseudomallei "population snapshot." Differences in colony morphology among reference isolates were found not to affect the STs assigned, which were consistent with the original isolates. Australian ST84 is likely characteristic of B. pseudomallei isolates of Southeast Asia rather than Australia, since multiple environmental isolates from Thailand and Malaysia share this ST with the single Australian clinical isolate in the MLST database. Phylogenetic evidence is also provided suggesting that Australian isolates may not be distinct from those of Thailand, since ST60 is common to environmental isolates from both countries. MLST and eBURST are useful tools for the study of population biology and epidemiology, since they provide methods to elucidate new genetic relationships among bacterial isolates.