First isolation of blaVIM-2 in an environmental isolate of Pseudomonas pseudoalcaligenes.
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The ldh phylogeny for environmental isolates of Lactococcus lactis is consistent with rRNA genotypes but not with phenotypes.
Lactate dehydrogenase (ldh) gene sequences, levels of 16S rRNA group-specific probe binding, and phenotypic characteristics were compared for 45 environmental isolates and four commercial starter strains of Lactococcus lactis to identify evolutionary groups best suited to cheddar cheese manufacture, ldh sequences from the environmental isolates showed high similarity to those from two groups of L. lactis used for industrial fermentations, L. lactis subsp. cremoris and subsp. lactis. Within each phylogenetically defined subspecies, ldh sequence similarities were greater than 99.1%. Strains with phenotypic traits formerly diagnostic for both subspecies were found in each ldh similarity group, but only strains belonging to L. lactis subsp. cremoris by both the newer, genetic and the older, superseded phenotypic criteria were judged potentially suitable for the commercial production of cheddar cheese. Identical evolutionary relationships were inferred from ldh sequences and from binding of subspecies-specific, 16S rRNA-directed oligonucleotide probes. However, groups defined according to these chromosomal traits bore no relationship to patterns of arginine deamination, carbon substrate utilization, or bacteriophage sensitivity, which may be encoded by cryptic genes or sexually transmissible genetic elements. Fourteen new L. lactis subsp. cremoris isolates were identified as suitable candidates for cheddar cheese manufacture, and 10 of these were completely resistant to three different batteries of commercial bacteriophages known to reduce starter activity.
Bacterial Typing Techniques↗
Genomic profiles of clinical and environmental isolates of Vibrio cholerae O1 in cholera-endemic areas of Bangladesh.
Diversity, relatedness, and ecological interactions of toxigenic Vibrio cholerae O1 populations in two distinctive habitats, the human intestine and the aquatic environment, were analyzed. Twenty environmental isolates and 42 clinical isolates were selected for study by matching serotype, geographic location of isolation in Bangladesh, and season of isolation. Genetic profiling was done by enterobacterial repetitive intergenic consensus sequence-PCR, optimized for profiling by using the fully sequenced V. cholerae El Tor N16961 genome. Five significant clonal clusters of haplotypes were found from 57 electrophoretic types. Isolates from different areas or habitats intermingled in two of the five significant clusters. Frequencies of haplotypes differed significantly only between the environmental populations (exact test; P < 0.05). Analysis of molecular variance yielded a population genetic structure reflecting the differentiating effects of geographic area, habitat, and sampling time. Although a parameter confounding the latter differences explained 9% of the total molecular variance in the entire population (P < 0.01), the net effect of habitat and time could not be separated because of the small number of environmental isolates included in the study. Five subpopulations from a single area were determined, and from these we were able to estimate a relative differentiating effect of habitat, which was small compared with the effect of temporal change. In conclusion, the resulting population structure supports the hypothesis that spatial and temporal fluctuations in the composition of toxigenic V. cholerae populations in the aquatic environment can cause shifts in the dynamics of the disease.
Bangladesh↗
Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa.
The type III secretion system of Pseudomonas aeruginosa transports four known effector proteins: ExoS, ExoT, ExoU and ExoY. However, the prevalence of the type III secretion system genes or the effector-encoding genes in clinical and environmental isolates of P. aeruginosa has not been well studied. Southern hybridization analyses and PCR were performed on over 100 P. aeruginosa isolates to determine the distribution of these genes. Clinical isolates were obtained from urine, endotracheal, blood and wound specimens, from the sputum of cystic fibrosis (CF) patients, and from non-hospital environmental sites. The popB gene was used as a marker for the presence of the large chromosomal locus encoding the type III secretion machinery proteins. Each isolate contained the popB gene, indicating that at least a portion of this large chromosomal locus was present in all isolates. Likewise, each isolate contained exoT-like sequences. In contrast, the exoS, exoU and exoY genes were variable traits. Overall, 72% of examined isolates contained the exoS gene, 28% contained the exoU gene, and 89% contained the exoY gene. Interestingly, an inverse correlation was noted between the presence of the exoS and exoU genes in that all isolates except two contained either exoS or exoU but not both. No significant difference in exoS, exoU or exoY prevalence was observed between clinical and environmental isolates or between isolates cultured from different disease sites except for CF respiratory isolates. CF isolates harboured the exoU gene less frequently and the exoS gene more frequently than did isolates from some of the other sites of infection, including the respiratory tract of patients without CF. These results suggest that the P. aeruginosa type III secretion system is present in nearly all clinical and environmental isolates but that individual isolates and populations of isolates from distinct disease sites differ in their effector genotypes. The ubiquity of type III secretion genes in clinical isolates is consistent with an important role for this system in human disease.
Bacterial Proteins↗
Physiological characteristics of environmental isolates of pathogenic dematiaceous fungi.
A total of 39 environmental isolates of pathogenic dematiaceous fungi (Fonsecaea pedrosoi: 14 isolates, Phialophora verrucosa: 6, Cladosporium carrionii: 9, Exophiala jeanselmei: 2, Ramichloridium subulatum: 6, Cladosporium tenuissimum: 1 and Phaeoisaria clematidis: 1) were evaluated for their various physiological characteristics including the ability to produce extracellular enzymes. Significant physiological characteristics included the ability of isolates of all the species except C. tenuissimum to decompose tyrosine, lyse human red blood cells, conversion of R. subulatum from filamentous to yeast form when cultured on Czapek-Dox agar and also on blood agar and the growth of C. tenuissimum on Sabouraud dextrose agar incorporating 3.6 M NaCl. Isolates of all the species except those of C. carrionii produced lipase, while only the isolates of P. verrucosa, E. jeanselmei and Ph. clematidis were positive for phospholipase.
Mitosporic Fungi↗
Molecular characterization of clinical and environmental isolates of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from a teaching hospital in Wales.
The present study describes the first molecular characterization of environmental and clinical isolates of vancomycin-resistant enterococci (VRE) in Wales. Over a 3-month period (May-July 2000), 134 isolates of VRE (89 Enterococcus faecium and 45 Enterococcus faecalis) were isolated from the patient environment of the University Hospital of Wales (UHW) in Cardiff, Wales, UK. In addition, over the same time-period, 24 clinical isolates of VRE (20 isolates of E. faecium and four isolates of E. faecalis) were obtained from 14 patients. All study isolates were subjected to PFGE typing and their van genotypes were determined by using multiplex PCR. The vanA PCR product (231 bp) was evident in 146 (92 %) of 158 VRE isolates; the remaining 12 isolates (8 %) were positive for the vanB gene. All isolates of E. faecalis were found to be vanA-positive. In total, 16 PFGE banding profiles (pulsotypes) were observed for environmental isolates of E. faecium, whilst eight pulsotypes were found for isolates of E. faecalis. Some of these pulsotypes were isolated from multiple sites, whereas others were more restricted in their distribution. Eleven pulsotypes were evident for clinical isolates and eight of these (representing 11 isolates) were also encountered in environmental isolates. Eleven clinical isolates of E. faecium (55 %) shared an identical pulsotype that was not detected in environmental isolates. These results demonstrate a heterogeneous environmental population of VRE and an association of certain strains with clinical isolates. Predominance of a single pulsotype (not detected in the environment) amongst clinical isolates suggests non-environmental transmission between patients.
Bacterial Proteins↗
Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in Goiânia city, Goiás, Brazil.
We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 microg/mL for amphotericin B, 0.125-8 microg/mL for fluconazole, 0.03-0.5 microg/mL for itraconazole and 0.03-0.25 microg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 microg/mL, 0.25-2.0 microg/mL, 0.007-0.125 microg/mL and 0.03-0.25 microg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.
Amphotericin B↗
Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water.
An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescence protein (GFP) isolated from Aequorea victoria. The hybrid transposon mini Tn5 gfp was transconjugated from E. coli to S. typhi, resulting in constitutive GFP production. The survival of S. typhi GFP155 introduced into groundwater and pond water microcosms was examined by GFP-based plate counts, total cell counts, and direct viable counts. A comparison between GFP-based direct viable counts and plate counts was a good method for verifying the viable, but non-culturable (VBNC), state of S. typhi. The entry into a VBNC state of S. typhi was shown in all microcosms. S. typhi survived longer in groundwater than in pond water as both a culturable and a VBNC state.
Bacteriological Techniques↗
Outer membranes of environmental isolates of Pseudomonas aeruginosa.
The outer membrane composition of 30 environmental isolates of Pseudomonas aeruginosa was examined. Other than variations in the amounts of lipoprotein H2, there were no major differences in the outer membrane protein or lipopolysaccharide patterns when compared with those of previously studied clinical isolates.
Bacterial Outer Membrane Proteins↗
Detection of tdh and trh genes in a urea-hydrolysing environmental isolate of Vibrio parahaemolyticus from the Andamans.
Co-existence of trh gene and urea-hydrolysing property in one of 44 marine water isolates of Vibrio parahaemolyticus correlates strongly with both genotypic and phenotypic characteristics of the bacterium. Thus, urease-producing phenotype can be considered a marker of virulence for the production of thermostable direct haemolysin-related haemolysin (TRH) (i.e. possession of trh gene). The same isolate also possessed the tdh gene. An environmental isolate possessing all the characteristics of a pathogenic V. parahaemolyticus in this marine environment suggest that there is a likelihood of the occurrence of clinical cases of gastroenteritis caused by V. parahaemolyticus in the Andamans.
Bacterial Proteins↗
Clinical and environmental isolates of Legionella pneumophila serogroup 1 cannot be distinguished by sequence analysis of two surface protein genes and three housekeeping genes.
We used gene sequencing to determine whether clinical (sporadic, epidemic, and endemic) and environmental isolates of Legionella pneumophila serogroup (sg) 1 belong to specific lineages. A total of 178 clinical and environmental L. pneumophila sg 1 isolates, defined by pulsed-field gel electrophoresis and epidemiological data as sporadic, epidemic, or endemic, were analyzed for polymorphisms in five gene fragments. The fragments belonged to three housekeeping genes (coding for aconitase [acn], aspartate-beta-semialdehyde dehydrogenase [asd], and RNA polymerase beta subunit [rpoB]) and two surface protein genes (coding for the macrophage infectivity potentiator [mip] and the major outer membrane protein [mompS]). The phylogenetic tree inferred from sequence polymorphisms of the five genes identified two large clusters, one consisting of 133 poorly differentiated strains and containing two smaller clusters (10 and 2 strains) unrelated to each other and the other consisting of 42 strains. Clinical and environmental isolates could not be distinguished on this basis, and no link between genetic background and epidemiological type was found, suggesting that other factors are responsible for differences in pathogenicity.
Bacterial Proteins↗
Plasmid DNA profiles as epidemiological markers for clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum.
Plasmid DNA was isolated, and profiles of a variety of clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (MAIS) were compared to determine whether plasmid DNA content would be useful as an epidemiological marker for these environmental pathogens. Since plasmids are common in clinical isolates and are stable during culture and exposure to NaOH, plasmid DNA analysis appears to be a suitable epidemiological tool. Based on the high frequency of plasmids in only clinical (56%) and aerosol (75%) isolates and low frequencies in soil (5%), dust (7%), sediment (less than 6%), and water (25%) isolates, the data suggest that MAIS-laden aerosols generated over waters of the southeastern United States are a likely source of human infection.
Aerosols↗
In vivo evaluation of pathogenicity of clinical and environmental isolates of Vibrio cholerae.
Thirty-three minimally passaged clinical and environmental isolates of Vibrio cholerae were examined for ability to survive and multiply in the upper bowel of infant mice and to elicit diarrhea. All of 21 smooth O-1 V. cholerae isolates from stool were able to multiply and elicit diarrhea. Three rough strains isolated from stool were unable to multiply or to elicit diarrhea. Two smooth O-1 isolates associated with cholera cases (from a sewer and a septic tank) also were able to cause disease. However, four O-1 strains and one non-O-1 strain from sources not associated with cholera cases did not cause mouse disease. A human gall bladder isolate was also avirulent, whereas a Louisiana shrimp isolated showed low mouse virulence. We conclude that smooth human diarrheal isolates of V. cholerae of serogroup O-1 are virulent for infant mice. Examination of sequential isolates from single patients showed that some strains isolated later in infection had a reduced ability to induce diarrhea. Comparison of epidemiologically related strains showed that an isolate from crab had a low ability to induce disease in infant mice, whereas the isolates from patients showed the expected ability to multiply and elicit diarrhea in mice.
Animals↗
Occurrence of high-level aminoglycoside resistance in environmental isolates of enterococci.
High-level resistance to aminoglycosides was observed in environmental isolates of enterococci. Various aquatic habitats, including agricultural runoff, creeks, rivers, wastewater, and wells, were analyzed. Strains of Enterococcus faecalis, E. faecium, E. gallinarum, and other Enterococcus spp. demonstrated multiple antibiotic resistance patterns to aminoglycosides.
Aminoglycosides↗
Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus.
Twenty clinical isolates of Vibrio vulnificus were compared with 10 environmental strains by using electron microscopy and agglutination assays with human erythrocytes, guinea pig erythrocytes, and Saccharomyces cerevisiae. In addition, the isolates were tested for ability to adhere to the human epithelial cell lines HEp-2 and A549. When examined by electron microscopy, 16 (80%) of the 20 clinical isolates demonstrated the presence of piluslike structures; the composition of the bacterial populations ranged from 0 to 68% piliated cells. In contrast, only 3 (30%) of the 10 environmental isolates were piliated, with a range from 0 to 16% piliated cells. A significant association between the presence of piliated cells and the isolate source was found (P less than 0.05). None of the 30 strains agglutinated erythrocytes or yeast cells. V. vulnificus adherence results obtained with HEp-2 cells showed 10 (50%) of 20 clinical isolates and 0 (0%) of 10 environmental isolates with averages of greater than 10 adherent bacteria per cell, demonstrating a correlation between attachment and the isolate source (P less than 0.05). Selected strains were tested to determine whether methyl alpha-D-mannopyranoside, fructose, or alpha-L-(-)-fucose would inhibit bacterial adherence to HEp-2 cells. Multiple patterns of adherence inhibition were observed. Adherence to A549 cells showed 8 (40%) of 20 clinical isolates and 0 (0%) of 10 environmental strains with averages of greater than 10 adherent bacteria per cell. A statistical association between attachment and the isolate source was demonstrated (P less than 0.05). These data suggest that the presence of piluslike structures and the ability to adhere to human epithelial cell lines may be more closely associated with V. vulnificus isolates from clinical specimens than with environmental strains.
Agglutination Tests↗
Comparison of molecular methods for subtyping patients and epidemiologically linked environmental isolates of Legionella pneumophila.
We used the molecular techniques of monoclonal antibody typing, plasmid analysis, and outer membrane protein profiling to subtype 159 patients' and environmental (water distribution system) isolates of Legionella pneumophila serogroup 1 from 18 institutions. The ability of these techniques to match patients' and epidemiologically linked environmental isolates from outbreaks of Legionnaires' disease at seven institutions was also compared. Two different panels of monoclonal antibodies (I and II) identified nine subtypes (one new disease-causing subtype) and six subtypes, respectively. The Bellingham 1 subtype type was the most common among environmental isolates, and the Philadelphia 1 subtype predominated among patients' isolates from all institutions except the Veterans Administration Medical Center in Pittsburgh, Pennsylvania. The source of an isolate (patient vs. environment) and its monoclonal antibody subtype were significantly associated (P less than .01). With use of the molecular techniques tested, the subtypes of patients' isolates were identical to those of epidemiologically linked environmental isolates from the same hospital.
Antibodies, Monoclonal↗
Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus.
To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.
Aspergillosis↗
Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens.
A total of 525 Bacillus thuringiensis environmental isolates, belonging to the five flagellar (H) serovars (alesti, sotto, kenyae, aizawai, and morrisoni), were serotyped by extracellular heat-stable somatic antigens (HSSAs). The isolates belonging to a given H serovar were assigned to a single HSSA serogroup at a high frequency, 87-100%. This indicates that the extent of HSSA variation within a single H serovar is small in the field populations of these B. thuringiensis serovars.
Antigenic Variation↗