Biochemical Fingerprinting Research Articles

Phenotyping of E. coli serotypes associated to oedema disease.

BackgroundOedema disease is a severe disease, mainly affecting recently weaned pigs. It is caused by E. coli strains that express fimbriae F18 and produce verotoxin 2e, mainly belonging to serotype O138, O139 or O141. The aim of this study was to compare E. coli isolates within these serotypes with respect to diversity.MethodsFaecal E. coli strains belonging to serotypes O138, O139 and O141 isolated during the period 1994–1998 from Swedish pigs aged less than 12 weeks were compared using a biochemical fingerprinting system. Aiming to compare the results obtained over time, also strains isolated during 1964–67 and 1975–80 were included in the study. The study comprised 129, 263 and 95 isolates of E. coli serotype O138, O139 and O141, respectively.ResultsBiochemical phenotypes (BPTs) were defined. At each sampling occasion each herd could only contribute with one isolate per BPT. Consequently, all but one of identical BPTs identified at a specific sampling occasion was omitted. The final number of isolates from 1994–98 that was compared included 64, 182 and 41 isolates of serotypes O138, O139 and O141, respectively. Within each serotype, the dominating BPT included over 65% of the compared isolates, demonstrating a large dominance of one BPT per serotype. These dominating BPTs were also demonstrated in the material from the 1960ies and the 1970ies. Still, the presence of other common BPTs (especially within serotype O138 and O139) demonstrated a certain variation within serotype. In a herd severely affected by oedema disease, E. coli serotype O139 was easily demonstrated in diseased pigs but only rarely in apparently healthy weanersConclusionThe results obtained demonstrate the presence of dominating BPTs within the oedema disease inducing serotypes. A stability of these BPTs over time was observed, presumably at least partly due to a never-ending access to naïve pigs. Still, the presence of other common BPTs indicates a variation over time, which visualises the importance of monitoring for this. Such studies should focus on pigs affected by oedema disease, because oedema disease inducing strains of E. coli were only rarely demonstrated in healthy pigs in a herd affected by oedema disease.

Clonal heterogeneity of clinical isolates of vancomycin-resistant Enterococcus faecium with unique vanS

To examine the clonal diversity of vancomycin-resistant enterococci (VRE). A total of 900 clinical isolates of enterococci were obtained, and VRE isolates were subjected to antimicrobial susceptibility tests, biochemical fingerprinting with the PhPlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE) typing. Forty-nine of all enterococcal isolates were resistant to high levels of vancomycin (MIC >or= 128) and identified as Enterococcus faecium. Biochemical fingerprinting with PhP showed that the VRE isolates were highly diverse (diversity index, D(i) = 0.93) and belonged to 24 PhP-types. The VRE could be separated into 34 and 27 types with PFGE and ribotyping, giving diversity indices of 0.98 and 0.97, respectively. The PFGE method was more discriminatory than ribotyping and PhP system for E. faecium isolates. A combination of either of the two typing methods resulted in at least 44 types. Furthermore, sequencing analysis of vanS of Tn1546 showed one nucleotide mutation (C-->A) at position 5727 in comparison with the prototype BM4147, which was found to be unique in all Iranian VRE isolates. The isolated clinical VRE strains were highly diverse in Tehran.

Phenotypic variations of enterococci in surface waters: analysis of biochemical fingerprinting data from multi-catchments

The aim of this study was to identify the prevalence of environmentally adapted enterococci strains by analysing biochemical fingerprinting (BF) data of 3952 enterococci isolates collected over 5 years from the six catchments in Southeast Queensland, Australia. A BF method was used to type 3952 enterococci isolates from six catchments. The environmental isolates were compared with a large existing BF library comprised of 5803 enterococci isolates from 10 host groups. Environmental isolates belonged to 801 biochemical phenotypes (BPTs), of which, an average of 29.2% was specific to each catchment. When compared with the BF library, an average of 79.5% BPTs from each catchment was identical to those in the library (i.e. host-origin BPTs). The remaining 20.5% was regarded as non-host origin BPTs, as they were not in the library and constituted only 5.3% of the total isolates tested for each catchment. Our data suggest that less than 5% of studied environmental strains was not identical to those in the library and seemed to be of environmental origin. From a microbial source tracking context, such low level of environmentally adapted strains can have a minimal impact on the performance of the library-based methods if a large number of isolates were tested from both the host groups and environmental waters. These data shed light on the importance of the size and representativeness of library-based source-tracking methods and their implications for the identification of faecal pollution in environmental waters.

Assessing Microbial Community in Andisol Differing in Management Practices by Biochemical and Molecular Fingerprinting Methods

Assessing Microbial Community in Andisol Differing in Management Practices by Biochemical and Molecular Fingerprinting Methods

Dissemination of a single Vibrio cholerae clone in cholera outbreaks during 2005 in Iran

In this study, 50 Vibrio cholerae O1 serotype Inaba isolates were collected during several cholera outbreaks throughout Iran during the summer of 2005. The results of antibiotic susceptibility testing showed that 86, 84, 84 and 82 % of the isolates were resistant to streptomycin, chloramphenicol, co-trimoxazole and tetracycline, respectively. The strains were genotyped using randomly amplified polymorphic DNA (RAPD), PFGE and ribotyping techniques. PCR showed that 100, 98 and 98 % carried the ctx, zot and ace genes, respectively. Biochemical fingerprinting of the isolates using the PhenePlate (PhP) system showed a low diversity index level (0.755), suggesting that the strains were highly homogeneous. Among the strains, 100 and 96 % showed an identical ribotype and PFGE patterns, respectively. The two isolates showing different PFGE patterns also exhibited discrete PhP types. RAPD was able to discriminate the isolates into six distinct groups, suggesting some genetic dissimilarity was present among the strains. These ribotyping, PFGE and PhP techniques revealed the clonal dissemination of a single V. cholerae strain throughout Iran in 2005.

Application of biochemical fingerprinting and fatty acid methyl ester profiling to assess the effect of the pesticide Atradex on aquatic microbial communities

We investigated changes in biomass, biochemical fingerprints, fatty acid methyl ester (FAME) profile and functional status of the natural aquatic microbial communities upon impact of an Atradex pulse. The Atradex was applied to microcosm tanks at concentrations ranging from 24.5 μg L −1 to 245 mg L −1. The biomass of all microbial communities declined to a minimum level on day 4 with the effect being more pronounced in treated groups. Similarity between microbial communities also decreased on day 4 with the greatest change occurring at a concentration of 245 mg L −1 Atradex. After 8 days exposure to Atradex, microbial communities in all treated groups (except tanks spiked with 245 mg L −1 Atradex) recovered and showed similar metabolic fingerprints and FAME profiles to those of controls. Our results indicate that exposure to an Atradex pulse at concentration above 245 mg L −1, may irreversibly change the structure and functional status of aquatic microbial communities.

Phenotypic Diversity of Multiple Antibiotic Resistant Enterococci with Emphasis on Enterococcus gallinarum Carrying vanA and vanB Genes

The prevalence and diversity of antibiotic resistant enterococci populations in samples collected four times from urban sewage treatment plant in Tehran, Iran between June 2005 and July 2006 were studied. Filtered samples were grown on mEnterococci medium containing 4 μg/ml vancomycin after which the enterococci isolates were identified to the species level. All strains were then tested for their resistance against nine antibiotics. Of the 131 isolates, 98 (75%) isolates were identified as Enterococcus gallinarum, followed by 24 (18%) and 9 (7%) for E. faecium and E. casseliflavus, respectively. All E. gallinarum isolates carried vanC1 gene with 64 (65%) and 14 (14%) isolates concomitantly harboured either vanA or vanB gene, respectively. Some E. casseliflavus concomitantly harboured vanA and vanC2 or vanB and vanC2. Typing the total enterococci isolates with a high resolution biochemical fingerprinting method showed a high diversity (Di = 0.91). We have shown by biochemical fingerprinting the presence of highly diverse glycopeptide resistant E. gallinarum and E. casseliflavus that have captured vanA and vanB genetic determinants under natural conditions. To our knowledge this is the first report in this geographical region showing high frequency antibiotic resistant enterococcal populations in particular E. gallinarum carrying assorted vancomycin resistance genes.

Comparison of phenotypic and genotypic methods for the detection of environmental isolates of Pseudomonas aeruginosa

During remediation processes, biological monitoring should be generally required. Hydrocarbon contaminated soils may provide favorable conditions for several opportunistic pathogenic microorganisms, thereby increasing their populations over risky levels. Therefore, during remediation processes of the subsurface medium biological monitoring is of prime importance. The accuracy, time and cost efficiency of the relevant identification method are major factors while monitoring these microbes. During previous years (2002–05), we collected 68 soil samples from 17 different oil contaminated sites, such as petrol stations, airfields and pipeline-breaks. We compared frequently applied detection methods of Pseudomonas aeruginosa, both traditional microbiological and molecular biological techniques , on 43 environmental isolates originating from these sites. The following methods were subjected to comparative analysis: (i) the Hungarian Standard method; (ii) the method described in “The Prokaryotes” handbook; (iii) the API 20NE biochemical fingerprinting, as well as PCR protocols aimed to amplify; (iv) the exotoxin-A gene; and (v) the 16S rDNA variable regions V2 and V8. In five cases, phenotypic methods gave false-negative results. 16S rDNA sequence analysis was done to confirm the identity of these five strains, which verified the results of molecular methods. In addition, faults were found in the evaluation of the originally described ETA PCR protocol, which was corrected by us.

Characterisation of the Lactobacillus community in traditional Karst ewe's cheese

Ewe's cheese, produced in the Slovenian Karst region, was analysed for its lactobacilli population. Biochemical fingerprinting with PhenePlate™ LB grouped lactobacilli in 16 distinct PhP types. One representative from each PhP type was further analysed. Members of Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus curvatus, and Lactobacillus brevis species were determined phenotypically. The genus‐specific DNA band was amplified with all PhP representatives, whereas species‐specific polymerase chain reaction efficiently determined only L. paracasei and L. brevis. Lactobacilli were highly to moderate sensitive to six antibiotics, and resistant to vancomycin. From halo formation in the agar spot method, indicators exerted a promising level of sensitivity for antimicrobials, produced by test microorganisms. Lactobacilli from Karst ewe's cheese offer a remarkable reservoir of ‘natural’ microbes.

Prevalence and Antimicrobial Resistance of Enterococcal Species in Sewage Treatment Plants in Iran

We investigated for the first time the occurrence, stability and antibiotic resistance of 593 enterococci in six samples collected from three urban sewage treatment plants (STPs) located in the north, south and west part of Tehran, Iran between October 2004 and September of 2005. Isolates were typed with a biochemical fingerprinting method (the PhPlate system) and tested for their resistance to six antibiotics. The most prevalent species in all three STPs were E. faecium followed by E. hirae and E. faecalis accounting for 93% of the total isolates examined. In all, 317 (55%) isolates were susceptible to all six antibiotics tested and the remaining isolates were resistant to between 1 and 6 antibiotics. Biochemical fingerprinting with PhPlate system showed a high diversity for E. faecalis (Di = 0.95), E. hirae (Di = 0.93) and E. faecium (Di = 0.95) populations with an overall diversity of Di = 0.97 for the whole enterococcal populations found in all three STPs. Our data indicate a high degree of polyclonality among the enterococci populations of human origin. This study suggest that the municipal wastewaters might be an important source of dissemination of antibiotic-resistant enterococci in Iran.

Defining the sources of low-flow phosphorus transfers in complex catchments

Nutrient transfers from the land to rivers have the potential to cause persistent eutrophic impacts at low flows even though the transfers may constitute a minor percentage of total annual fluxes. In rural catchments, the contribution from agricultural soils during storm events can be particularly large and untangling the relative contributions from multiple sources that vary in time and space is especially problematic. In this study, the potential for domestic septic tank system pollution during low flows was investigated in 3 small catchments (3 to 5 km(2)) using an integrated series of methods. These included septic system surveys, continuous (10 min) total phosphorus (TP) monitoring at the outlet of each catchment, repeated low-flow water quality surveys in sub-catchments upstream of the catchment outlets and single day river-walk water quality surveys. A series of faecal matter and grey-water fingerprinting techniques were also employed. These included determining sterol ratios in stream sediments, monitoring the presence of proteins, E. coli and enterococci bacterial signatures and boron. The total density and density of poorly maintained septic systems mirrored the magnitude of frequent TP concentrations in the catchments although this relationship was less apparent in the nested sub-catchments. The exception was possibly related to the simple hydraulics in one particular catchment and indicated temporary effluent attenuation in the other catchments. Repeated low-flow and river-walk water quality surveys highlighted discrete areas and reaches where stepped changes in nutrient concentration occurred. Bio-chemical fingerprinting showed that between 7% and 27% of sediments were contaminated with human faecal material and correlation matrices indicated that, at least during low flows, P fractions were positively correlated with some markers of faecal and grey-water contamination.

Sourcing faecal pollution: A combination of library-dependent and library-independent methods to identify human faecal pollution in non-sewered catchments

Library-dependent (LD) (biochemical fingerprinting of Escherichia coli and enterococci) and library-independent (LI) (PCR detection of human-specific biomarkers) methods were used to detect human faecal pollution in three non-sewered catchments. In all, 550 E. coli isolates and 700 enterococci isolates were biochemically fingerprinted from 18 water samples and compared with metabolic fingerprint libraries of 4508 E. coli and 4833 enterococci isolates. E. coli fingerprints identified human unique biochemical phenotypes (BPTs) in nine out of 18 water samples; similarly, enterococci fingerprints identified human faecal pollution in 10 water samples. Seven samples were tested by PCR for the detection of biomarkers. Human-specific HF134 Bacteroides and enterococci surface protein ( esp) biomarkers were detected in five samples. Four samples were also positive for HF183 Bacteroides biomarker. The combination of biomarkers detected human faecal pollution in six out of seven water samples. Of the seven samples analysed for both the indicators/markers, at least one indicator/marker was detected in every sample. Four of the seven PCR-positive samples were also positive for one of the human-specific E. coli or enterococci BPTs. The results indicated human faecal pollution in the studied sub-catchments after storm events. LD and LI methods used in this study complimented each other and provided additional information regarding the polluting sources when one method failed to detect human faecal pollution. Therefore, it is recommended that a combination of methods should be used to identify the source(s) of faecal pollution where possible.

Detection of virulence genes in Escherichia coli of an existing metabolic fingerprint database to predict the sources of pathogenic E. coli in surface waters

A collection of 366 Escherichia coli strains from 10 host groups and surface waters were tested for the presence of 15 virulence genes associated with strains causing intestinal and extra-intestinal infections. The virulence genes included eaeA, VT1, 2 and 2e, LT1, ST1 and 2, Einv gene, EAgg gene, CNF1 and 2, papC, O111 and O157 side chain LPS. Of the 262 strains obtained from nine different hosts, 39 (15%) carried one or more of these virulence genes. These included six strains from humans, two from horses, eight from dogs, two from ducks, five from cattle, seven from chickens, four from pigs, two from sheep and three from deer. Of the remaining 104 strains obtained from water samples, 10 (10%) also carried one or more of the tested virulence genes. Of these, six had identical biochemical phenotypes (BPTs) to strains isolated from humans (two strains), dogs (two strains), chickens (one strain) and sheep (one strain) with 4 BPTs also carrying same virulence genes. Our results indicate that the sources of clinically important E. coli strains found in surface waters due to faecal contamination can be predicted by using a combination of biochemical fingerprinting method and the detection of virulence genes. From the public health point of view this information will be of great importance for evaluating the risk associated with public use of the catchment.

Characterization of enterotoxigenic Escherichia coli from diarrhoeal patients in Bangladesh using phenotyping and genetic profiling

A total of 99 isolates out of 370 colonization factor (CF)-positive, well-characterized enterotoxigenic Escherichia coli (ETEC) strains belonging to 13 different CF types isolated from diarrhoeal patients admitted to the hospital of the International Centre for Diarrhoeal Disease Research, Bangladesh, were tested. The isolates were selected at random based on expression of the major CFs prevailing in Dhaka, Bangladesh, from 1996 to 1998. These isolates were characterized by O-antigenic serotyping, randomly amplified polymorphic DNA (RAPD) analysis and biochemical fingerprinting using the PhenePlate (PhP) system. The 99 ETEC isolates belonged to 10 O serogroups, the predominant ones being O6 (n=28), O115 (n=20) and O128 (n=20). Most isolates of serogroup O6 (CS1+CS3, 11/14; CS2+CS3, 5/8) belonged to the same PhP/RAPD type (H/f), whereas other isolates of serogroup O6 (n=12) belonged to different PhP/RAPD types (Si/f and F/c). Eleven serogroup O128 (CFA/I) isolates belonged to the same PhP/RAPD type (E/b), whereas the other O128 isolates formed different PhP/RAPD types. Fifteen (75%) serogroup O115 isolates (together with fourteen isolates from serogroups O25, O114, O142 and O159) demonstrated two closely related common groups by PhP typing (A and A1) and belonged to the same PhP/RAPD type (A/a). Three major clonal groups were identified among the ETEC strains in this study, largely based on O-antigenic type, CF expression pattern and toxin profile.

Linking Species Richness, Biodiversity and Ecosystem Function in Soil Systems

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Population similarity of enterococci and Escherichia coli in surface waters: A predictive tool to trace the sources of fecal contamination

A biochemical fingerprinting method (the PhPlate system) was used to compare similarities between Escherichia coli and enterococci populations from surface water samples with those found in different animal species during the wet and the dry seasons in order to predict the dominant source(s) of fecal contamination in a local creek. A significant increase in the number and diversity of enterococci was observed in the creek during the wet season. Enterococci population from water samples also showed a higher population similarity with animal species than did E. coli. A higher population similarity was found between both indicator bacteria and animal species during the wet season with highest population similarities found in dogs, horses, cows and kangaroos. In contrast, a low population similarity was found for both fecal indicator bacteria from humans with water samples during the wet and the dry seasons, indicating that humans are not a major source of contamination in the studied creek. The results also indicate that the population similarity analysis of enterococci population has an advantage over E. coli in tracing the possible source(s) of contamination in the studied creek and that population similarity analysis as used in this study can be used to predict the source(s) of fecal contamination in surface waters.

Microbial Source Tracking of Escherichia coli in a Constructed Wetland

Little information has been gathered on the effect of avian species on the microbial water quality in constructed wetlands. To address this concern, fecal pollution from nonpoint and point sources was evaluated in a constructed wetland in Tolleson, Arizona. Antibiotic resistance profiling and biochemical fingerprinting were performed on 325 Escherichia coli isolates, collected from key points in the wetlands. Multivariate statistical analysis was used to interpret the data for samples collected on October 3 and December 12, 2000, and January 16, 2001. It was found that the passerine population was the major source of the Escherichia coli in the water samples collected in the wetlands on October 3 and December 12, 2000, whereas the regrowth in the treated municipal wastewater was the main source on January 16, 2001. This information is useful in providing data for operators in the monitoring of wetlands created for wastewater treatment and wildlife habitat.

Biochemical fingerprinting of Vibrio parahaemolyticus by the PhenePlate system: comparison between pandemic and non-pandemic serotypes

During recent years a pandemic clone of Vibrio parahaemolyticus has emerged. Isolates of this clone are distributed among several serotypes, but are genotypically related. In the present study, a phenotyping method (biochemical fingerprinting) was used to characterize pandemic and non-pandemic isolates belonging to V. parahaemolyticus. It was found that the pandemic isolates showed a high level of phenotypic homogeneity and a majority of the pandemic isolates belonged to the same biochemical phenotype, whereas non-pandemic V. parahemolyticus isolates were more heterogeneous. In conclusion, biochemical fingerprinting of V. parahaemolyticus can be used as a first screening method to differentiate between pandemic and non-pandemic isolates of V. parahaemolyticus.

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.

Phene Plate (PhP) biochemical fingerprinting

Pulsed-field gel electrophoresis (PFGE) is currently considered the gold standard for genotyping of enterococci. However, PFGE is both expensive and time-consuming. The purpose of this study was to investigate whether the PhP system can be used as a reliable clinical screening method for detection of genetically related isolates of enterococci. If so, it should be possible to minimize the number of isolates subjected to PFGE typing, which would save time and money. Ninety-nine clinical enterococcal isolates were analysed by PhP (similarity levels 0.90-0.975) and PFGE (similarity levels < or =3 and < or =6 bands) and all possible pairs of isolates were cross-classified as matched or mismatched. We found that the probability that a pair of isolates (A and B) belonging to the same type according to PhP also belong to the same cluster according to PFGE, i.e. p(A(PFGE)=B(PFGE) * A(PhP)=B(PhP)), and the probability that a pair of isolates of different types according to PhP also belong to different clusters according to PFGE, i.e. p(A(PFGE) not equalB(PFGE) * A(PhP) not equalB(PhP)), was relatively high for E. faecalis (0.86 and 0.96, respectively), but was lower for E. faecium (0.51 and 0.77, respectively). The concordance which shows the probability that PhP and PFGE agree on match or mismatch was 86%-93% for E. faecalis and 54%-66% for E. faecium, which indicates that the PhP method may be useful for epidemiological typing of E. faecalis in the current settings but not for E. faecium.