Use of repetitive DNA sequences and the PCR To differentiate Escherichia coli isolates from human and animal sources

The rep-PCR DNA fingerprint technique, which uses repetitive intergenic DNA sequences, was investigated as a way to differentiate between human and animal sources of fecal pollution. BOX and REP primers were used to generate DNA fingerprints from Escherichia coli strains isolated from human and animal sources (geese, ducks, cows, pigs, chickens, and sheep). Our initial studies revealed that the DNA fingerprints obtained with the BOX primer were more effective for grouping E. coli strains than the DNA fingerprints obtained with REP primers. The BOX primer DNA fingerprints of 154 E. coli isolates were analyzed by using the Jaccard band-matching algorithm. Jackknife analysis of the resulting similarity coefficients revealed that 100% of the chicken and cow isolates and between 78 and 90% of the human, goose, duck, pig, and sheep isolates were assigned to the correct source groups. A dendrogram constructed by using Jaccard similarity coefficients almost completely separated the human isolates from the nonhuman isolates. Multivariate analysis of variance, a form of discriminant analysis, successfully differentiated the isolates and placed them in the appropriate source groups. Taken together, our results indicate that rep-PCR performed with the BOX A1R primer may be a useful and effective tool for rapidly determining sources of fecal pollution.     

Differentiation of Fecal <Emphasis Type="Italic">Escherichia coli</Emphasis> from Human,Livestock, and Poultry Sources by rep-PCR DNA Fingerprinting on the Shellfish Culture Area of East China Sea

The rep-PCR DNA fingerprinting performed with REP, BOX A1R, and (GTG)₅ primers was investigated as a way to differentiate between human, livestock, and poultry sources of fecal pollution on the area of Xiangshan Bay, East China Sea. Of the three methods, the BOX-PCR DNA fingerprints analyzed by jack-knife algorithm were revealed high rate of correct classification (RCC) with 91.30, 80.39, 89.39, 86.14, 93.24, 87.72, and 89.28% of human, cattle, swine, chicken, duck, sheep, and goose E. coli isolates classified into the correct host source, respectively. The average rate of correct classification (ARCC) of REP-, BOX-, and (GTG)₅-PCR patterns was 79.88, 88.21, and 86.39%, respectively. Although the highest amount of bands in (GTG)₅-PCR fingerprints could be observed, the discriminatory efficacy of BOX-PCR was superior to both REP- and (GTG)₅-PCR. Moreover, the similarity of 459 isolates originated from shellfish and growing water was compared with fecal-obtained strains. The results showed that 92.4 and 96.2% E. coli strains isolated from midstream and downstream shellfish samples, respectively, had a ≥80% similarity with corresponding strains isolated from fecal samples. It was indicated that E. coli in feces could spread from human sewage or domestic farms to the surrounding shellfish culture water, and potentially affect the quality of shellfish. This work suggests that rep-PCR fingerprinting can be a promising genotypic tool applied in the shellfish growing water management on East China Sea for source identification of fecal pollution.     

Heterogeneity of<Emphasis Type="Italic">Escherichia coli</Emphasis> derived from artiodactyla animals analyzed with the use of rep-PCR fingerprinting

Genetic polymorphism of 83 isolates of E. coli, derived from 4 species of artiodactyla animals living in a relatively close contact on the grounds of a theme park ZOO Safarii Swierkocin (Poland) was determined using the rep-PCR fingerprinting method, which utilizes oligonucleotide primers matching interspersed repetitive DNA sequences in PCR reaction to yield DNA fingerprints of individual bacterial isolates based on repetitive extragenic palindrome (REP) primers. The fingerprint patterns demonstrated the essential polymorphism of distribution of REP sequences in genomes of the examined isolates. The arithmetic averages clustering algorithm (UPGMA) statistical analysis of fingerprints with the use of the Jaccard similarity coefficient differentiated E. coli isolates into three similarity groups containing various numbers of isolates. The groups comprised isolates derived from two, three and four species of the source animals. The isolates derived from each source segregated in the dendrogram in a different way, both within the similarity groups and among them, indicating an individual repertoire of E. coli in the examined species of animals. The similarity relations among E. coli derived from the same source, illustrated in a dendrogram with a number of subclusters of a low mutual similarity (< or = 20%), indicated an essential interstrain differentiation in terms of the distribution of REP sequences. Our results confirmed the hypothesis of the oligoclonal characters of populations obtained from particular sources. The rep-PCR fingerprinting method with REP primers is simple and highly differentiating and can be recommended for use in explorations of large groups of animals and monitoring the variability of strains.     

Evaluation of repetitive extragenic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic- and wild-animals

The objective of this study was to investigate the potential of repetitive extragenic palindromic anchored polymerase chain reaction (rep-PCR) in differentiating fecal Escherichia coli isolates of human, domestic- and wild-animal origin that might be used as a molecular tool to identify the possible source(s) of fecal pollution of source water. A total of 625 fecal E. coli isolates of human, 3 domestic- (cow, dog and horse) and 7 wild-animal (black bear, coyote, elk, marmot, mule deer, raccoon and wolf) species were characterized by rep-PCR DNA fingerprinting technique coupled with BOX A1R primer and discriminant analysis. Discriminant analysis of rep-PCR DNA fingerprints of fecal E. coli isolates from 11 host sources revealed an average rate of correct classification of 79.89%, and 84.6%, 83.8%, 83.3%, 82.5%, 81.6%, 80.8%, 79.8%, 79.3%, 77.4%, 73.2% and 63.6% of elk, human, marmot, mule deer, cow, coyote, raccoon, horse, dog, wolf and black bear fecal E. coli isolates were assigned to the correct host source. These results suggest that rep-PCR DNA fingerprinting procedures can be used as a source tracking tool for detection of human- as well as animal-derived fecal contamination of water.     

Comparison of Ribotyping and Repetitive Extragenic Palindromic-PCR for Identification of Fecal Escherichia coli from Humans and Animals

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.     

Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals influence accuracy of determining sources of fecal pollution

A horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique (HFERP) was developed and evaluated as a means to differentiate human from animal sources of Escherichia coli. Box A1R primers and PCR were used to generate 2,466 rep-PCR and 1,531 HFERP DNA fingerprints from E. coli strains isolated from fecal material from known human and 12 animal sources: dogs, cats, horses, deer, geese, ducks, chickens, turkeys, cows, pigs, goats, and sheep. HFERP DNA fingerprinting reduced within-gel grouping of DNA fingerprints and improved alignment of DNA fingerprints between gels, relative to that achieved using rep-PCR DNA fingerprinting. Jackknife analysis of the complete rep-PCR DNA fingerprint library, done using Pearson's product-moment correlation coefficient, indicated that animal and human isolates were assigned to the correct source groups with an 82.2% average rate of correct classification. However, when only unique isolates were examined, isolates from a single animal having a unique DNA fingerprint, Jackknife analysis showed that isolates were assigned to the correct source groups with a 60.5% average rate of correct classification. The percentages of correctly classified isolates were about 15 and 17% greater for rep-PCR and HFERP, respectively, when analyses were done using the curve-based Pearson's product-moment correlation coefficient, rather than the band-based Jaccard algorithm. Rarefaction analysis indicated that, despite the relatively large size of the known-source database, genetic diversity in E. coli was very great and is most likely accounting for our inability to correctly classify many environmental E. coli isolates. Our data indicate that removal of duplicate genotypes within DNA fingerprint libraries, increased database size, proper methods of statistical analysis, and correct alignment of band data within and between gels improve the accuracy of microbial source tracking methods.     

Use of 16S-23S rRNA intergenic spacer region PCR and repetitive extragenic palindromic PCR analyses of Escherichia coli isolates to identify nonpoint fecal sources

Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28 degrees C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

rep-PCR fingerptinting as a tool for the analysis of genomic diversity in Escherichia coli strains isolated from an aqueous/freshwater environment

The rep-PCR fingerprinting method, with the support of ERIC and REP primers, was used to analyse the genomic diversity of 93 E. coli strains isolated from lake water samples drawn at two different depths. The applied UPGMA for DNA analysis did not reveale any genomic similarities between the 48 E. coli strains derived from the subsurface-zone water and the 43 of the bottom-zone water. The considerable genomic diversity of the E. coli of the surface zone was expressed as a dendrogram in the form of 8 similarity groups comprising strains isolated from samples drawn over one month. The bottom-zone strains, which display a lesser degree of genomic diversity (5 similarity groups), showed distinct common features in their DNA fingerprints. In the similarity dendrogram for the bottom-zone, strains derived in different months of sampling were segregated into the same similarity groups. Applying REP primers in rep-PCR generates more complex fingerprints increasing the discriminatory power of the analysis, whereas the ERIC primer generates less complex fingerprint patterns, and is thus clearer to interpret.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Genetic diversity among isolates of Paenibacillus larvae from Austria

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and αb). Genotypes aB and αb are new and have not been reported in other studies using the same techniques.     

Comparison of ribotyping and repetitive extragenic palindromic-PCR for identification of fecal Escherichia coli from humans and animals

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.     

Genetic Characterization of Escherichia coli Populations from Host Sources of Fecal Pollution by Using DNA Fingerprinting

Escherichia coli isolates were obtained from common host sources of fecal pollution and characterized by using repetitive extragenic palindromic (REP) PCR fingerprinting. The genetic relationship of strains within each host group was assessed as was the relationship of strains among different host groups. Multiple isolates from a single host animal (gull, human, or dog) were found to be identical; however, in some of the animals, additional strains occurred at a lower frequency. REP PCR fingerprint patterns of isolates from sewage (n = 180), gulls (n = 133), and dairy cattle (n = 121) were diverse; within a host group, pairwise comparison similarity indices ranged from 98% to as low as 15%. A composite dendrogram of E. coli fingerprint patterns did not cluster the isolates into distinct host groups but rather produced numerous subclusters (approximately >80% similarity scores calculated with the cosine coefficient) that were nearly exclusive for a host group. Approximately 65% of the isolates analyzed were arranged into host-specific groups. Comparable results were obtained by using enterobacterial repetitive intergenic consensus PCR and pulsed-field gel electrophoresis (PFGE), where PFGE gave a higher differentiation of closely related strains than both PCR techniques. These results demonstrate that environmental studies with genetic comparisons to detect sources of E. coli contamination will require extensive isolation of strains to encompass E. coli strain diversity found in host sources of contamination. These findings will assist in the development of approaches to determine sources of fecal pollution, an effort important for protecting water resources and public health.     

Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

Determination of genetic differences between fluid and nonfluid variants of<Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subsp.<Emphasis Type="Italic">sepedonicus</Emphasis> using rep-PCR technique

Testing of 23 isolates of Clavibacter michiganensis subsp. sepedonicus for analysis by rep-PCR (using BOX, ERIC, REP primer sets) was used for the purpose of localization of genetic markers for fluid and/or nonfluid strains. None of the primer sets was successful in detecting genetic differences between the isolates and no polymorphism was generated.     

Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.     

Temporal Dynamics and Impact of Manure Storage on Antibiotic Resistance Patterns and Population Structure of Escherichia coli Isolates from a Commercial Swine Farm

Many confined-livestock farms store their wastes for several months prior to use as a fertilizer. Storing manure for extended periods could significantly bias the composition of enteric bacterial populations subsequently released into the environment. Here, we compared populations of Escherichia coli isolated from fresh feces and from the manure-holding tank (stored manure) of a commercial swine farm, each sampled monthly for 6 months. The 4,668 confirmed E. coli isolates were evaluated for resistance to amikacin, ampicillin, cephalothin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and trimethoprim plus sulfamethoxazole. A subset of 1,687 isolates was fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) with the BOXA1R primer to evaluate the diversity and the population structure of the collection. The population in the stored manure was generally more diverse than that in the fresh feces. Half of the genotypes detected in the stored manure were never detected in the fresh fecal material, and only 16% were detected only in the fresh feces. But the majority of the isolates (84%) were assigned to the 34% of genotypes shared between the two environments. The structure of the E. coli population showed important monthly variations both in the extent and distribution of the diversity of the observed genotypes. The frequency of detection of resistance to specific antibiotics was not significantly different between the two collections and varied importantly between monthly samples. Resistance to multiple antibiotics was much more temporally dynamic in the fresh feces than in the stored manure. There was no relationship between the distribution of rep-PCR fingerprints and the distribution of antibiotic resistance profiles, suggesting that specific antibiotic resistance determinants were dynamically distributed within the population.     

Presence and Sources of Fecal Coliform Bacteria in Epilithic Periphyton Communities of Lake Superior

Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior from June 2004 to August 2005 to determine if fecal coliforms and Escherichia coli were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 × 10⁵ CFU cm⁻² by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the E. coli bacteria isolated from these periphyton communities could be identified when compared with a library of E. coli fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton E. coli strains that could be identified. Several periphyton E. coli isolates were genotypically identical (≥92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified E. coli strains from periphyton were added to the known source library, then 57% to 81% of E. coli strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated E. coli rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that E. coli from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution.     

Genetic and phenotypic microdiversity of Ochrobactrum spp

The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.     

Methods To Increase Fidelity of Repetitive Extragenic Palindromic PCR Fingerprint-Based Bacterial Source Tracking Efforts

The goal of the study was to determine which similarity coefficient and statistical method to use to produce the highest rate of correct assignment (RCA) in repetitive extragenic palindromic PCR-based bacterial source tracking. In addition, the use of standards for deciding whether to accept or reject source assignments was investigated. The use of curve-based coefficients Cosine Coefficient and Pearson's Product Moment Correlation yielded higher RCAs than the use of band-based coefficients Jaccard, Dice, Jeffrey's x, and Ochiai. When enterococcal and Escherichia coli isolates from known sources were used in a blind test, the use of maximum similarity produced consistently higher RCAs than the use of average similarity. We also found that the use of a similarity value threshold and/or a quality factor threshold (the ratio of the average fingerprint similarity within a source to the average similarity of this source's isolates to an unknown) to decide whether to accept source assignments of unknowns increases the reliability of source assignments. Applying a similarity value threshold improved the overall RCA (ORCA) by 15 to 27% when enterococcal fingerprints were used and 8 to 29% when E. coli fingerprints were used. Applying the quality factor threshold resulted in a 22 to 32% improvement in the ORCA, depending on the fingerprinting technique used. This increase in reliability was, however, achieved at the expense of decreased numbers of isolates that were assigned a source.