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PubMed1994

Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates.

Published 16S rDNA sequencing data for Aeromonas species were analysed and the validity of signature sequences derived from our investigations of these sequences was examined by sequencing the corresponding 16S rDNA regions of 67 environmental isolates from sewage effluents and receiving waters around Sydney and one clinical isolate, all previously classified as Aeromonas species. Species-specific probes for Aer. hydrophila and Aer. veronii were designed and tested in PCR assays and clearly discriminated these species from the other Aeromonas isolates as identified by 16S rDNA sequences.

Aeromonas↗

PubMedDate not supplied

Relationship between clinical and environmental isolates of Pseudomonas aeruginosa in a hospital setting.

BACKGROUND: Populations of Pseudomonas aeruginosa have been extensively studied, although there is no general agreement concerning their genetic structure. It has been proposed that P. aeruginosa is a very homogeneous species with 90% of individuals within the same clonal group; nonetheless, other results suggested that Pseudomonas populations are panmictic. Here we compared P. aeruginosa populations from clinical and environmental samples, both isolated from the Bellvitge Hospital of the University of Barcelona in Spain. METHODS: Antibiotic susceptibility determination as well as whole cell and outer membrane protein denaturing gel electrophoresis, pulsed-field electrophoresis, and random amplified polymorphic DNA analysis were performed. RESULTS: Environmental isolates were much more susceptible to antibiotics than those isolated from clinical specimens. The remainder of the analyses revealed high degree of diversity. CONCLUSIONS: Whole-cell proteins, outer-membrane proteins, and pulsed field electrophoresis did not support a close relationship between clinical and environmental isolates. Random amplified polymorphic DNA (RAPD) confirmed the distance between isolates from both sources. This suggests that the origin of hospital infections by P. aeruginosa is due mainly to growth of bacterial strains acquired by patients prior to hospital admission or from patient-to-patient through healthcare workers (HCWs).

Anti-Bacterial Agents↗

PubMed1994

Growth of Enterococcus, Lactococcus and Streptococcus strains and environmental isolates in liquid media and their reactions on BEEA.

Growth of known species of Enterococcus, Lactococcus and Streptococcus and Aerococcus viridans in selective and nonselective liquid media routinely used to enumerate faecal streptococci was measured optically at different temperatures. Growth of environmental isolates was measured in some of these media. Growth of the reference strains on Bile esculin azide agar at elevated incubation temperatures was tested. The results revealed only minor differences between media but strong influence of incubation temperature. Some media tended to yield higher cell densities than others. For many species the inoculum size affected maximum turbidity. To combine selective media with selective incubation temperatures seems to be necessary to achieve satisfactory reliability in traditional liquid enumeration methods for faecal streptococci. Because of the diversity of this group, optimal selectivity and recovery can hardly be achieved simultaneously.

Agar↗

PubMed2001

Genotypes associated with virulence in environmental isolates of Vibrio cholerae.

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.

Biomarkers↗

PubMed2006

Genetic variability in environmental isolates of Legionella pneumophila from Comunidad Valenciana (Spain).

Legionella pneumophila is associated to recurrent outbreaks in several Comunidad Valenciana (Spain) localities, especially in Alcoi, where social and climatic conditions seem to provide an excellent environment for bacterial growth. We have analysed the nucleotide sequences of three loci from 25 environmental isolates from Alcoi and nearby locations sampled over 3 years. The analysis of these isolates has revealed a substantial level of genetic variation, with consistent patterns of variability across loci, and comparable to that found in a large, European-wide sampling of clinical isolates. Among the tree loci studied, fliC showed the highest level of nucleotide diversity. The analysis of isolates sampled in different years revealed a clear differentiation, with samples from 2001 being significantly distinct from those obtained in 2002 and 2003. Furthermore, although linkage disequilibrium measures indicate a clonal nature for population structure in this sample, the presence of some recombination events cannot be ruled out.

Alleles↗

PubMed2005

ESAT-6 and CFP-10 in clinical versus environmental isolates of Mycobacterium kansasii.

Mycobacterium kansasii consists of 5 genetically distinct groups, of which 2 are associated with human disease. Determinants of the differences in virulence are unknown. Potential genes of interest are esat-6 and cfp-10, which are associated with virulence of Mycobacterium tuberculosis and Mycobacterium bovis but are lacking in bacille Calmette-Guérin and in most environmental mycobacteria (M. kansasii is an exception). We investigated esat-6 and cfp-10 genes in 22 clinical and 14 environmental isolates of M. kansasii. Both were present in all isolates; each genetic group had its own characteristic Southern-blot pattern corresponding to a highly conserved fingerprint pattern. Nucleotide sequences of the genes differed 12.6% and 10.1%, respectively, from the M. tuberculosis homologues, but the deduced amino acid sequences were <5% different. In vitro, clinical and environmental genotypes of M. kansasii expressed CFP-10 and ESAT-6. Thus, virulence of M. kansasii is not directly related to esat-6 and cfp-10 genes or gene expression.

Amino Acid Sequence↗

PubMed2000

DNA sequence of the insertional hot spot of Tn916 in the Clostridium difficile genome and discovery of a Tn916-like element in an environmental isolate integrated in the same hot spot.

Tn916 is a broad host range tetracycline resistance conjugative transposon. In most bacteria, this element enters the bacterial genome at multiple sites. However, in Clostridium difficile, the element has a strong hot spot when introduced by filter mating from Bacillus subtilis. In this work, the DNA sequence of the preferred insertion site (att916) was obtained. An environmental isolate of C. difficile was also discovered which contained an element indistinguishable from Tn916, Tn916CD. Tn916CD was found integrated at att916.

Bacillus subtilis↗

PubMed1983

Colonization of the rabbit small intestine by clinical and environmental isolates of non-O1 Vibrio cholerae and Vibrio mimicus.

We examined the capability of 12 isolates of non-cholera toxin-producing O1 and non-O1 Vibrio cholerae to colonize the small intestine of adult rabbits and cause diarrhea. Using the removable intestinal tie-adult rabbit diarrhea model, we found that eight environmental isolates that showed no or marginal biological activity in other diarrhea models (rabbit ileal loop, infant rabbit, and suckling mouse) appeared to be incapable of attaching to and colonizing, even transiently, the small intestinal mucosa of animals with normal clearance mechanisms. In contrast, three clinical isolates attached, proliferated rapidly, and colonized mucosal surfaces of the entire small intestine within 8 h of challenge. This led to diarrhea with strikingly high rates of mortality compared with that of rabbits given similar challenges doses with strains of O1 V. cholerae that produce cholera toxin and Vibrio mimicus, which produces a toxin similar to cholera toxin. We have further demonstrated that multiple exposures to enteric infection by these strains elicited local and serum antibodies that reacted strongly with cell surface antigens of the homologous strain and showed a high degree of cross-reactivity against the cell surface antigens of the two heterologous strains. The enteric infections appeared to engender protection against subsequent infection as well, as evidenced by reduced incidence of diarrhea and duration of fecal shedding of the challenge organism upon subsequent challenges.

Animals↗

PubMedDate not supplied

[Environmental isolation of Vibrio cholerae 01 in continental waters of the Province of Seville].

BACKGROUND: Many bibliographical references suggest reserves of Vibrio cholerae in the aquatic world, strains of both the serovariety no 01 and 01 having been isolated in different parts of the globe in fresh and extensive salt waters. METHODS: Samples were taken monthly from January to May and from October to December, and every fortnight from June to September. The method of isolation applied is in accordance with the standard set down by the Institute of Health Carlos III, with some specific modifications. RESULTS: During the period 90/91, routine analyses carried out by the Andalusian Service of Health of waters from the area of the Continental Basin in the province of Seville showed the presence of Vibrio cholerae 01. In the same way, during the complete annual cycle Vibrio cholerae no 01 was also isolated. CONCLUSIONS: These isolations did not coincide with outbreaks or individual cases of cholera, so the authors, in agreement with what is described in the bibliography, consider that Vibrio cholerae no 01 forms a part of certain aquatic environments, which is in direct relation to environmental isolations of the serovariety 01.

Fresh Water↗

PubMed2003

Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp.

The distribution and phenotypic activity of the genes encoding for serine protease, glycerophospholipid-cholesterol acyltransferase, lipases, aerolysin/hemolysin and DNases were investigated in 234 isolates identified by 16S rDNA-RFLP representing all the species of Aeromonas. The former three genes were found to be highly conserved among the genus. Aerolysin/hemolysin and DNase genes and beta-hemolytic activity were significantly more frequent in clinical than in environmental isolates. Aerolysin/hemolysin and serine protease genes were present in all beta-hemolytic strains supporting serine protease as possibly important for the activation of the former gene. The high prevalence of virulence factors in clinical isolates indicates that they may play a role in the mechanisms of pathogenesis of these microorganisms.

Acyltransferases↗

PubMed2002

Comparative study of fluorogenic and chromogenic media for specific detection of environmental isolates of thermotolerant Escherichia coli.

In a field study 78 water samples were analysed employing Fluorocult Brilla Broth (BB) and its performance was compared with standard MPN procedure. Out of 78 water samples analysed 56 (71.7%) samples yielded positive reactions in BB whereas, 50 (64.1%) samples were positive by standard fecal coliform test. A comparative study of fluorogenic and chromogenic media containing substrate beta-D glucuronide for specific detection of environmental isolates of 313 thermotolerant E. coli has been undertaken. Five fluorogenic media were used: Fluorocult MacConkey agar (MCA), Fluorocult ECD agar (ECD), Fluorocult VRB agar (VRB), Fluorocult E. coli 0157:H7 agar (ECH7) and Fluorocult Brilla Broth (BB) and Chromogenic Chromocult agar (CCA). BB and CCA were found to be highly specific and sensitive media to detect E. coli as all E. coli yielded positive reaction on them. On ECH7 and ECD agar 67.5 and 64.9 of E. coli isolates gave positive reaction, respectively. Low sensitivity was observed in case of MCA and VRB agar in detecting E. coli. The performance of BB appears to be better when compared with standard MPN procedure employing MacConkey broth/Brilliant green bile broth in detecting E. coli in drinking water.

Adaptation, Physiological↗

PubMedDate not supplied

Molecular and physiological evaluation of subtropical environmental isolates of Acanthamoeba spp., causal agent of Acanthamoeba keratitis.

Previous molecular examination of Acanthamoeba spp. has resulted in the determination of distinct genotypes in this genus (designated T1-T12, T14). Genotype T4 has been responsible for the majority of cases of Acanthamoeba keratitis. Here we examine the relative abundance of environmental T4 isolates on beaches and ask whether they have temperature and salinity tolerances that could enhance pathogenicity. Twenty-four Acanthamoeba strains were isolated from beach sand (n = 20), soil (n = 3), and tap water (n = 1) in south Florida. Phylogenetic analysis identified 19 of 24 isolates as T4, the Acanthamoeba keratitis-associated genotype. The remaining isolates were genotype T5 (4) and T11 (1). Nearly all beach isolates were genotype T4, whereas the tap water and soil isolates were mostly T5. All amoebae grew at 0, 1.0, and 2.0% salt and 19 of 20 beach isolates also grew at 3.2%. No soil or tap-water acanthamoebae reproduced at 3.2%. All isolates grew at 37 degrees C and two (T5) at 42 degrees C. Little correlation existed between beach location, salt-tolerance, and genetic relatedness. Overall, the large majority of environmental isolates obtained were genotype T4, suggesting it may be the most common genotype in this environment and could be a potential source of Acanthamoeba keratitis infections.

Acanthamoeba↗

PubMed1999

Use of single-strand conformation polymorphism analysis to examine the variability of the rpoS sequence in environmental isolates of Salmonellae.

The natural environment places its resident microflora under stress, which may often result in adaptation by the microflora in order to increase the probability of survival. One such mechanism that has been postulated involves rpoS, which encodes a sigma factor that is known to enhance survival upon exposure to stress. The present work aimed to examine the genetic variability of rpoS in a selection of Salmonella enterica subspecies environmental isolates with an automated single-strand conformation polymorphism analysis technique. The results indicated that sequence variation does occur and that these changes are mainly located in two areas: at the center and near the end of the coding region. The variability was generally at the single-base level, although one strain (S. arizonae) did demonstrate significant differences in nucleotide sequence.

Animals↗

PubMed2005

Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity.

Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor approximately 40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MATalpha-specific SXI1alpha gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATalpha strains sequenced, the SXI1alpha gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations.

Alleles↗

PubMed2006

Genetic and phenotypic diversity of quorum-sensing systems in clinical and environmental isolates of Vibrio cholerae.

Vibrio cholerae is the causative agent of cholera, a severe and devastating diarrheal disease. V. cholerae lives naturally in various aquatic habitats during interepidemic periods. Recent studies reveal that quorum-sensing systems, which exist in many bacteria and help them monitor their population densities and regulate various cellular functions, control V. cholerae pathogenesis, biofilm formation, and protease production. In this study we surveyed quorum-sensing systems in 16 geographically diverse V. cholerae strains from epidemic-causing O1 and O139 strains as well as non-O1/non-O139 and environmental isolates and discovered an unexpectedly high rate of dysfunctional components. We also found that a functional quorum-sensing system conferred a survival advantage on bacteria in biofilms when the bacteria were exposed to seawater, though quorum sensing was less important to survival in a planktonic state under the same conditions. These findings suggest that variations in quorum-sensing systems are due to environmental selective pressures and might be beneficial to V. cholerae's fitness under certain conditions found in its natural reservoirs.

Amino Acid Sequence↗

PubMed2006

Serological cross-reactivity of environmental isolates of Enterobacter, Escherichia, Stenotrophomonas, and Aerococcus with Shigella spp.-specific antisera.

Using protocols designed for the isolation of Shigella from environmental freshwater samples from different regions of Bangladesh, 11 bacterial strains giving rise to Shigella-like colonies on selective agar plates and showing serological cross-reaction with Shigella-specific antisera were isolated. Phylogenetic analyses revealed that three of the isolates were most closely related to Escherichia coli, four to Enterobacter sp., two to Stenotrophomonas, and two isolates belonged to the Gram-positive genus Aerococcus. The isolates cross-reacted with six different serotypes of Shigella and were, in each case, highly type-specific. Two of the isolates belonging to the Enterobacter and Escherichia genera gave extremely strong cross-reactivity with Shigella dysenteriae and Shigella boydii antisera, respectively. The Aerococcus isolates gave relatively weak but significant cross-reactions with S. dysenteriae. Western blot analysis revealed that a number of antigens from the isolates cross-react with Shigella spp. The results indicate that important Shigella spp. surface antigens are shared by a number of environmental bacteria, which have implications for the use of serological methods in attempts for the detection and recovery of Shigella from aquatic environments.

Antigens, Bacterial↗

PubMed1997

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.

Aspergillus fumigatus↗

PubMed2005

Macro- and nanoscale observations of adhesive behavior for several E. coli strains (O157:H7 and environmental isolates) on mineral surfaces.

Subsurface biobarriers can be conceived to attenuate the migration of pathogens by adhesion to mineral surfaces. Candidate biobarrier materials of varied surface characteristics (dolomite, alpha-alumina, silica, pyrophyllite, and Pyrax (a composite form of pyrophyllite, mica, and silica)) were tested for Escherichia coli adhesive capacity in macroscale continuous-flow columns. Atomic force microscopy (AFM) was used to determine nanoscale interaction energies. Predicted attractive interaction energies correlated well with macroscale adhesive behavior for tested E. coli strains. AFM measurements confirmed ExDLVO model predictions of attachment in the primary minima for E. coli O157:H7 and two environmental isolates E. coli (UCFL339 and UCFL-348) with MOPS conditioned Pyrax. In macroscale column experiments, pyrophyllite and Pyrax demonstrated significantly higher bacterial retention, higher deposition coefficients and lower initial cell breakthrough values for E. coli O157:H7 than did alpha-alumina, silica, or dolomite (pyrophyllite, 0.93, 3.56 h(-1), 3.2% ODo; Pyrax, 0.95, 3.73 h(-1), 2.8% ODo; alpha-alumina, 0.74, 1.60 h(-1), 33% ODo; silica, 0.63, 0.43 h(-1), 73% ODo; and dolomite, 0.33, 0.17 h(-1), 89% ODo, respectively). Bacterial hydrophilicity impacted cell retention in Pyrax columns with the relatively hydrophobic E. coli isolate UCFL-339 (0.99, 6.13 h(-1), 0.4% ODo) retained better than the more hydrophilic E. coli isolate UCFL348 (0.94, 3.70 h(-1), 3.6% ODo). The strong adhesive behavior of Pyrax was attributed to the hydrophobic (deltaGiwi = -32.4 mJ/m2) pyrophyllite component of the mineral. Vicinal water appears poised between the bacterial and the mineral surface during initial attachment. Overall, observed behavior of the various E. coli strains and the selected mineral surfaces was consistent with surface analyses, conducted at both the macro- and nanoscale.

Aluminum Oxide↗