Seasonal metabolic analysis of marine sediments collected from Moreton Bay in South East Queensland, Australia, using a multi-omics-based approach.

Anthropogenic effects of urban density have altered natural ecosystems. Such changes include eutrophication of freshwater and adjoining coastal habitats, and increased levels of inorganic nutrients and pollutants into waterways. In Australia, these changes are intensified by large-scale ocean-atmospheric events, leading to considerable abiotic stress on the natural flora and fauna. Bacterial communities in marine sediments from Moreton Bay (South East Queensland, Australia) were examined in order to assess the impact of rainfall changes, chemical pollution, and subsequent abiotic stress on living organisms within a marine ecosystem. Sediments were collected during the wet and dry seasons and analyzed using bacterial metagenomics and community metabolomics techniques. Physicochemical data were also analyzed to account for biological variance that may be due to non-rainfall-based abiotic stresses. Wet-dry seasonality was the dominant control on bacterial community structure and metabolic function. Changes in the availability of nutrients, organic matter and light appeared to be the major seasonal stressors. In contrast, urban and industrial pollutants appeared to be minor stressors at the sites sampled. During the wet season, the bacterial community composition reflected organisms that utilize biogeochemical pathways with fast kinetics, such as aerobic metabolism, direct assimilation of inorganic compounds, and primary production. The transition to the dry season saw the bacterial community composition shift towards organisms that utilize more complex organic energy sources, such as carbohydrates and fatty acids, and anaerobic redox processes.

Comparisons of bacterial and archaeal communities in the rumen and a dual-flow continuous culture fermentation system using amplicon sequencing.

Dual-flow continuous culture (CC) fermenters are commonly used to study rumen fermentation in vitro. Research using culture-based and oligonucleotide techniques has shown that certain microbial populations within fermenters may be maintained at abundances similar to those observed in vivo. In this study, bacterial and archaeal communities in the rumen of dairy cattle and in a dual-flow CC fermentation system were compared using high-throughput amplicon sequencing targeting the V4 hypervariable region of 16S rRNA. We hypothesized that the in vitro system harbored a comparable bacterial and archaeal community to that observed in the rumen. Members of the Bacteroidetes and Firmicutes made up the 2 most abundant phyla in the rumen, inoculum, and fermenters and did not differ among sample types (P > 0.10). Similarly, Prevotellaceae, the most abundant family in all 3 sample types, did not differ based on source (P = 0.80). However, beta diversity analyses revealed that bacterial and archaeal communities differed between fermenters and rumen samples (P ≤ 0.001), but fermenter bacterial and archaeal communities stabilized by day 4 of each period. While the overall bacterial and archaeal community differs between natural rumens and those detected in in vitro fermenter systems, several prominent taxa were maintained at similar relative abundances suggesting that fermenters may provide a suitable environment in which to study shifts among the predominant members of the microbial community.

A multi-omics based ecological analysis of coastal marine sediments from Gladstone, in Australia's Central Queensland, and Heron Island, a nearby fringing platform reef.

The impact of anthropogenic factors arising from point and non-point pollution sources at a multi commodity marine port and its surrounding ecosystems were studied using sediment samples collected from a number of onshore (Gladstone Harbour and Facing Island) and offshore (Heron Island and Fitzroy Reefs) sites in Australia's Central Queensland. Sediment samples were analyzed for trace metals, organic carbon, polycyclic aromatic hydrocarbons (PAH), emerging chemicals of concern (ECC) and sterols. Similarly, the biological and biochemical interaction between the reef and its environment was analyzed by the multi-omic tools of next-generation sequencing characterization of the bacterial community and microbial community metabolic profiling. Overall, the trace elements were observed at the lower end of the Australian environmental guideline values at the offshore sites, while higher values were observed for the onshore locations Nickel and copper were observed above the high trigger value threshold at the onshore sites. The levels of PAH were below limits of detection across all sites. However, some of the ECC and sterols were observed at higher concentrations at both onshore and offshore locations, notably, the cholesterol family sterols and 17α-ethynylestradiol. Multi-omic analyses also indicated possible thermal and photo irradiation stressors on the bacterial communities at all the tested sites. The observed populations of γ-proteobacteria were found in combination with an increased pool of fatty acids that indicate fatty acid synthesis and utilisation of the intermediates of the shikimate pathways. This study demonstrates the value of applying a multi-omics approach for ecological assessments, in which a more detailed assessment of physical and chemical contaminants and their impact on the community bacterial biome is obtained.

Environmental Escherichia coli: ecology and public health implications-a review.

Escherichia coli is classified as a rod-shaped, Gram-negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm-blooded animals, including humans, and is often discharged into the environment through faeces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent faecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extraintestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a faecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long-term survival. Large-scale studies of population genetics revealed the diversity and complexity of E. coli strains in various environments, which are affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments with regard to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.

Amplicon-based taxonomic characterization of bacteria in urban and peri-urban roof-harvested rainwater stored in tanks.

Overall, 26% of Australian households use rainwater tanks as a source of potable and nonpotable water. Limited information is available on the total bacterial communities in tank water. Therefore, identification of dominant bacterial communities, diversity, and their distribution is important in understanding the microbial quality of tank water. In this study, the abundance and diversity of bacterial communities in 88 tank water samples collected from the urban areas of Brisbane (n=44) and the peri-urban center of Currumbin (n=44) in Southeast Queensland, Australia were determined using amplicon-based Illumina next-generation sequencing. In addition, the SourceTracker program was used to identify the sources of fecal contamination in tank water samples. Sequence reads were also analyzed to detect potential bacterial pathogenic genera in the tank water samples collected. Differences in sample coverage, alpha diversity, and richness did not differ significantly between the Brisbane and Currumbin tank water samples. Comamonadaceae and Planctomycetaceae were the most abundant families in all tank water samples. Curvibacter was the most abundant genus in all tank water samples. SourceTracker revealed that around 34% (Brisbane) and 43% (Currumbin) of tank water samples had a signature for bird fecal contamination. The potential opportunistic pathogenic genera including Burkholderia, Chromobacterium, Clostridium, Legionella, Mycobacterium, Nocardia, and Pseudomonas were most prevalent in tank water samples. Next-generation sequencing can be used as an initial screening tool to identify a wide array of potential pathogenic genera in tank water samples followed by quantifying specific pathogen(s) of interest using more sensitive molecular assays such as quantitative PCR (qPCR).

Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water.

In this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

Predominant populations of indigenous soybean-nodulating Bradyrhizobium japonicum strains obtained from organic farming systems in Minnesota.

AIMS: Bradyrhizobium from organic fields in Minnesota were isolated and genotyped to assess diversity of soybean-bradyrhizobia in organic farming systems that can be used to improve soybean productivity. METHODS AND RESULTS: Soil samples were collected from 25 organic fields in Minnesota during May to July 2012. Soybean (cv. Lambert) was used as a host to trap indigenous bradyrhizobia in each sample. Genetic diversity of Bradyrhizobium strains (n=733) was determined using the horizontal, fluorophore-enhanced, repetitive extragenic palindromic-PCR (HFERP) DNA fingerprinting technique and the soybean-bradyrhizobia were classified into 79 different genotypes. Of these, 15 dominant genotypes were found and were highly similar (>92% fingerprint similarity) to serotypes USDA 127 (40.4%), USDA 4 (31.8%) and USDA 123 (15.5%), which were the three main populations of soybean-bradyrhizobia in organic fields. CONCLUSIONS: Bradyrhizobium japonicum serogroup USDA 4 strains were found to make up a previously unrecognized, predominant rhizobial population in the organic farming soils examined. The relative abundance of strain USDA 4 was negatively correlated with that of USDA 127 and this relationship may be influenced by the levels of NO3 -N and other soil edaphic factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The local community of bradyrhizobia can be affected by applying inoculant bacteria to organic fields. Based on these results, soybean production in organic farms may be improved by displacing strains similar to USDA 4 with those better at nitrogen fixation and competitive ability than indigenous strains.

Application of Illumina next-generation sequencing to characterize the bacterial community of the Upper Mississippi River.

AIMS: A next-generation, Illumina-based sequencing approach was used to characterize the bacterial community at ten sites along the Upper Mississippi River to evaluate shifts in the community potentially resulting from upstream inputs and land use changes. Furthermore, methodological parameters including filter size, sample volume and sample reproducibility were evaluated to determine the best sampling practices for community characterization. METHODS AND RESULTS: Community structure and diversity in the river was determined using Illumina next-generation sequencing technology and the V6 hypervariable region of 16S rDNA. A total of 16,400 operational taxonomic units (OTUs) were observed (4594 ± 824 OTUs per sample). Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Verrucomicrobia accounted for 93.6 ± 1.3% of all sequence reads, and 90.5 ± 2.5% belonged to OTUs shared among all sites (n = 552). Among nonshared sequence reads at each site, 33-49% were associated with potentially anthropogenic impacts upstream of the second sampling site. Alpha diversity decreased with distance from the pristine headwaters, while rainfall and pH were positively correlated with diversity. Replication and smaller filter pore sizes minimally influenced the characterization of community structure. CONCLUSIONS: Shifts in community structure are related to changes in the relative abundance, rather than presence/absence of OTUs, suggesting a 'core bacterial community' is present throughout the Upper Mississippi River. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is among the first to characterize a large riverine bacterial community using a next-generation-sequencing approach and demonstrates that upstream influences and potentially anthropogenic impacts can influence the presence and relative abundance of OTUs downstream resulting in significant variation in community structure.

Gene expression profiling of Escherichia coli in response to interactions with the lettuce rhizosphere.

AIMS: The objective of this study was to examine transcriptional changes in Escherichia coli when the bacterium was growing in the lettuce rhizoshpere. METHODS AND RESULTS: A combination of microarray analyses, colonization assays and confocal microscopy was used to gain a more complete understanding of bacterial genes involved in the colonization and growth of E. coli K12 in the lettuce root rhizosphere using a novel hydroponic assay system. After 3 days of interaction with lettuce roots, E. coli genes involved in protein synthesis, stress responses and attachment were up-regulated. Mutants in curli production (crl, csgA) and flagella synthesis (fliN) had a reduced capacity to attach to roots as determined by bacterial counts and by confocal laser scanning microscopy. CONCLUSIONS: This study indicates that E. coli K12 has the capability to colonize lettuce roots by using attachment genes and can readily adapt to the rhizosphere of lettuce plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study show curli production and biofilm modulation genes are important for rhizosphere colonization and may provide useful targets to disrupt this process. Further studies using pathogenic strains will provide additional information about lettuce-E. coli interactions.

Therapeutic transplantation of the distal gut microbiota.

Although it is generally accepted that the distal gut microbiota are relatively stable in healthy adult individuals, a collapse of the microbial community structure resulting from antibiotic therapy or pathogen presence can lead to gut dysfunction. However, recent findings demonstrate that it is possible to engraft new microbiota from a donor source, resulting in the restoration of gut functionality and improvement in health. This builds upon decades of case reports and series in which fecal transfers were used to successfully treat refractory and recurrent Clostridium difficile infection. As fecal transplantation becomes part of mainstream medicine, it will likely provide a unique opportunity to study the interactions of humans with their attendant microbiota and allow greater insights into their synergistic functionality.

Identification of a Brevibacterium marker gene specific to poultry litter and development of a quantitative PCR assay.

AIM: To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. METHODS AND RESULTS: Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571-bp product was detected in 76% of poultry-associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 10(7) -10(9) gene copies per gram in soiled litter, up to 10(5) gene copies per gram in spread-site soils, and 10(7) gene copies per litre in field run-off water. Results were corroborated by a blinded study conducted by a second laboratory. CONCLUSION: The poultry-specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land-applied poultry litter. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline.

AIMS: The tet(X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet(X) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet(X) gene. METHODS AND RESULTS: Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet(X) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet(X) gene revealed a mobilizable transposon-like element (Tn6031) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet(X) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet(X) gene using three different recipient strains and numerous experimental conditions. CONCLUSIONS: This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet(X) gene. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria.

Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies.

s-Triazine ring compounds are common industrial chemicals: pesticides, resin intermediates, dyes, and explosives. The fate of these compounds in the environment is directly correlated with the ability of microbes to metabolize them. Microbes metabolize melamine and the triazine herbicides such as atrazine via enzyme-catalyzed hydrolysis reactions. Hydrolytic removal of substituents on the s-triazine ring is catalyzed by enzymes from the amidohydrolase superfamily and yields cyanuric acid as an intermediate. Cyanuric acid is hydrolytically processed to yield 3 mol each of ammonia and carbon dioxide. In those cases studied, the genes underlying the hydrolytic reactions are localized to large catabolic plasmids. One such plasmid, pADP-1 from Pseudomonas sp. ADP, has been completely sequenced and contains the genes for atrazine catabolism. Insertion sequence elements play a role in constructing different atrazine catabolic plasmids in different bacteria. Atrazine chlorohydrolase has been purified to homogeneity from two sources. Recombinant Escherichia coli strains expressing atrazine chlorohydrolase have been constructed and chemically cross-linked to generate catalytic particles used for atrazine remediation in soil. The method was used for cleaning up a spill of 1,000 pounds of atrazine to attain a level of herbicide acceptable to regulatory agencies.

Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP.

The complete 108,845-nucleotide sequence of catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP was determined. Plasmid pADP-1 was previously shown to encode AtzA, AtzB, and AtzC, which catalyze the sequential hydrolytic removal of s-triazine ring substituents from the herbicide atrazine to yield cyanuric acid. Computational analyses indicated that pADP-1 encodes 104 putative open reading frames (ORFs), which are predicted to function in catabolism, transposition, and plasmid maintenance, transfer, and replication. Regions encoding transfer and replication functions of pADP-1 had 80 to 100% amino acid sequence identity to pR751, an IncPbeta plasmid previously isolated from Enterobacter aerogenes. pADP-1 was shown to contain a functional mercury resistance operon with 99% identity to Tn5053. Complete copies of transposases with 99% amino acid sequence identity to TnpA from IS1071 and TnpA from Pseudomonas pseudoalcaligenes were identified and flank each of the atzA, atzB, and atzC genes, forming structures resembling nested catabolic transposons. Functional analyses identified three new catabolic genes, atzD, atzE, and atzF, which participate in atrazine catabolism. Crude extracts from Escherichia coli expressing AtzD hydrolyzed cyanuric acid to biuret. AtzD showed 58% amino acid sequence identity to TrzD, a cyanuric acid amidohydrolase, from Pseudomonas sp. strain NRRLB-12227. Two other genes encoding the further catabolism of cyanuric acid, atzE and atzF, reside in a contiguous cluster adjacent to a potential LysR-type transcriptional regulator. E. coli strains bearing atzE and atzF were shown to encode a biuret hydrolase and allophanate hydrolase, respectively. atzDEF are cotranscribed. AtzE and AtzF are members of a common amidase protein family. These data reveal the complete structure of a catabolic plasmid and show that the atrazine catabolic genes are dispersed on three disparate regions of the plasmid. These results begin to provide insight into how plasmids are structured, and thus evolve, to encode the catabolism of compounds recently added to the biosphere.

A marker-dense physical map of the Bradyrhizobium japonicum genome.

Bacterial artificial chromosome (BAC) clones are effective mapping and sequencing reagents for use with a wide variety of small and large genomes. This report describes the development of a physical framework for the genome of Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybean. A BAC library for B. japonicum was constructed that provides a 77-fold genome coverage based on an estimated genome size of 8.7 Mb. The library contains 4608 clones with an average insert size of 146 kb. To generate a physical map, the entire library was fingerprinted with HindIII, and the fingerprinted clones were assembled into contigs using the software (; Sanger Centre, UK). The analysis placed 3410 clones in six large contigs. The ends of 1152 BAC inserts were sequenced to generate a sequence-tagged connector (STC) framework. To join and orient the contigs, high-density BAC colony filters were probed with 41 known gene probes and 17 end sequences from contig boundaries. STC sequences were searched against the public databases using and algorithms. Query results allowed the identification of 113 high probability matches with putative functional identities that were placed on the physical map. Combined with the hybridization data, a high-resolution physical map with 194 positioned markers represented in two large contigs was developed, providing a marker every 45 kb. Of these markers, 177 are known or putative B. japonicum genes. Additionally, 1338 significant results (E < 10(-4)) were manually sorted by function to produce a functionally categorized database of relevant B. japonicum STC sequences that can also be traced to specific locations in the physical map.

Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different.

The gene encoding melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively.

Substrate specificity of atrazine chlorohydrolase and atrazine-catabolizing bacteria.

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.

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.