Genetic diversity of Escherichia coli isolates in irrigation water and associated sediments: implications for source tracking

Identifying the sources of fecal contaminants in surface water bodies such as rivers and lakes is of significant importance for environmental quality, food safety and regulatory purposes. Current DNA library-based source tracking approaches rely on the comparison of the genetic relatedness among the fecal contaminants. The objective of this study was to determine the genetic relatedness of Escherichia coli isolated from irrigation water and associated sediments using pulse field gel electrophoresis (PFGE) and to evaluate the genetic stability of the E. coli PFGE patterns. The isolates were obtained over a 4-month period from specific locations within irrigation canals and sediments associated with the Rio Grande River along the Texas-Mexico border. Fifty E. coli isolates were genotyped using PFGE. Different E. coli genotypes were identified among samples collected in 11 different locations. Some isolates obtained over successive months showed similar genotypic patterns. In the laboratory experiment, the PFGE pattern of one E. coli strain changed during survival in irrigation water. The genetic relatedness of this strain changed from >95% to <83% over 8-week survival. These results imply that PFGE is of such extreme resolution that it may be a challenging task to rely solely on a PFGE-based source tracking DNA fingerprint library for large watersheds.     

Effects of the nuisance algae, Cladophora, on Escherichia coli at recreational beaches in Wisconsin

Recreational beaches constitute a large part of the 12 billion dollar per year tourism industry in Wisconsin. Beach closures due to microbial contamination are costly in terms of lost tourism revenue and adverse publicity for an area. Escherichia coli (E. coli), is used as an indicator of microbial contamination, as high concentrations of this organism should indicate a recent fecal contamination event that may contain other, more pathogenic, bacteria. An additional problem at many beaches in the state is the nuisance algae, Cladophora. It has been hypothesized that mats of Cladophora may harbor high concentrations of E. coli. Three beaches in Door County, WI were selected for study, based on tourist activity and amounts of algae present. Concentrations of E. coli were higher within Cladophora mats than in surrounding water. Beaches displayed an E. coli concentration gradient in water extending away from the Cladophora mats, although this was not statistically significant. Likewise, the amount of Cladophora observed on a beach did not correlate with E. coli concentrations found in routine beach monitoring samples. More work is needed to determine the impact of mats of Cladophora on beach water quality, as well as likely sources of E. coli found within the mats.     

Genotype diversity of Escherichia coli isolates in natural waters determined by PFGE and ERIC-PCR

Most library-dependent bacterial source tracking studies using Escherichia coli (E. coli) have focused on strain diversity of isolates obtained from known human and animal faecal sources for library development. In contrast, this study evaluated the genotype variation of E. coli isolated from natural surface water using pulsed field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) to better understand these naturally occurring populations. A total of 650 water samples were collected over a nine month period from eleven sampling stations from Lake Waco and Belton Lake in Central Texas. Of the 650 water samples collected, 412 were positive for E. coli, yielding a total of 631 E. coli isolates (1-12 isolates collected per sample). PFGE and ERIC-PCR patterns were successfully generated for 555 isolates and were compared using the curve-based Pearson's product-moment correlation coefficient. The 555 E. coli isolates represented 461 PFGE genotypes, with 84% (386/461) of the genotypes being represented by individual isolates. The remaining 75 genotypes were represented by 2-5 isolates each. Using ERIC-PCR, the 555 E. coli isolates represented 175 genotypes, with 63% (109/175) of the genotypes being represented by individual isolates. In contrast to the PFGE results, two ERIC-PCR genotypes represented 37% of the E. coli isolates, (83 and 124 isolates, respectively), and were found throughout the watersheds both spatially and temporally. Based on the PFGE genotype diversity of water isolates, there is little evidence that a small number of environmentally-adapted E. coli represent dominant populations in the studied waterbodies. However, with the lower discriminatory power technique ERIC-PCR, an opposing conclusion might have been drawn. These results emphasize the importance of considering the resolving power of the source tracking technique being used when assessing strain diversity and geographical stability.     

Seasonal stability of Cladophora-associated Salmonella in Lake Michigan watersheds

The bacterial pathogens Shigella, Salmonella, Campylobacter, and shiga toxin-producing E. coli (STEC) were recently found to be associated with Cladophora growing in southern Lake Michigan. Preliminary results indicated that the Salmonella strains associated with Cladophora were genetically identical to each other. However, because of the small sample size (n = 37 isolates) and a lack of information on spatial-temporal relationships, the nature of the association between Cladophora and Salmonella remained speculative. In this study, we investigated the population structure and genetic relatedness of a large number of Cladophora-borne Salmonella isolates from Lake Michigan (n = 133), as well as those isolated from stream and lake water (n = 31), aquatic plants (n = 8), and beach sands and sediments (n = 8) from adjacent watersheds. Salmonella isolates were collected during 2005-2007 between May and August from Lake Michigan beachsheds in Wisconsin, Illinois, and Indiana. The genetic relatedness of Salmonella isolates was examined by using the horizontal, fluorophore-enhanced rep-PCR (HFERP) DNA fingerprinting technique. While the Salmonella isolates associated with Cladophora exhibited a high degree of genetic relatedness (≥92% similarity), the isolates were not all genetically identical. Spatial and temporal relationships were evident in the populations examined, with tight clustering of the isolates both by year and location. These findings suggest that the relationship between Salmonella and Cladophora is likely casual and is related to input sources (e.g. wastewater, runoff, birds) and the predominant Salmonella genotype surviving in the environment during a given season. Our studies indicate that Cladophora is likely an important reservoir for Salmonella and other enteric bacterial pathogens in Lake Michigan beachsheds, which in turn may influence nearshore water quality.     

Recovery of Escherichia coli in fresh water fish, Jenynsia multidentata and Bryconamericus iheringi

Escherichia coli concentration was determined in digestive tract and muscle of Jenynsia multidentata and Bryconamericus iheringi through bioassays. Field experiments were also conducted with J. multidentata collected in the Suquía River, Córdoba, Argentina. E. coli was quantified by the most probable number, using lauryl sulphate tryptose broth with 4-methylumbelliferyl-beta-D-glucuronide. For bioassays, E. coli concentrations 10(2), 10(3), 10(4), 10(5), 10(6)CFU/ml were introduced in aquarium water. E. coli was recovered from the digestive tracts of J. multidentata and B. iheringi in all the concentrations assayed. Bacterial critical load in water for the recovery of bacteria from muscle, was 10(3)CFU/ml for both species. The regression analysis between E. coli loads in water and those found in digestive tract and muscle showed a positive linear relationship for J. multidentata and B. iheringi. The same relation was observed between the concentration of bacteria in digestive tract and muscle in both species. In field experiments, E. coli was recovered from digestive tract and muscle of J. multidentata. The presence of E. coli in the studied fish suggests that they can carry bacteria to non-polluted waters. However, further studies are necessary to evaluate its significance for public and environmental health.     

Distribution of indicator bacteria in Canyon Lake, California

The spatial and temporal distributions of indicator bacteria in a small, multiple-use source drinking water reservoir in Southern California, USA were quantified over the period August 2001-July 2002. High levels of total and fecal coliform bacteria were present in Canyon Lake (annual geometric mean concentrations+/-SEM of 3.93+/-0.02 and 3.02+/-0.03 log cfu/100mL, respectively), while comparatively low levels of enterococci and E. coli were found (1.16+/-0.02 log cfu/100mL and 0.30+/-0.03 log MPN/100mL, respectively). As a result, these different indicator bacteria yielded quite divergent indices of water quality, with 72.1% of all surface samples (n=294) exceeding the USEPA single-sample limit of 400 cfu/100mL fecal coliform bacteria, while none (0%) of the samples exceeded the single-sample limit for E. coli (n=194). Regression analyses found a positive correlation between total and fecal coliform bacteria (R=0.50, significant at p<0.001) and between enterococci and E. coli (R=0.51, significant at p<0.001), but no correlation or inverse correlations were found between coliform concentrations and enterococci and E. coli levels. External sources responsible for the high total and fecal coliform bacteria were not identified, although laboratory studies demonstrated growth of the coliform bacteria in lake water samples. Enterococci and E. coli were not observed to grow, however. Bacteria concentrations varied relatively little laterally across the lake, although strong vertical gradients in fecal coliform and enterococcus bacteria concentrations were present during summer stratification, with concentrations about 10x higher above the thermocline when compared with surface concentrations. In contrast, total bacteria, total virus and total coliform bacteria levels were unchanged with depth. Seasonal trends in bacteria concentrations were also present. This study shows that the choice of indicator bacteria and sampling depth can both strongly affect the apparent microbial water quality of a lake or reservoir.     

Transport of Escherichia coli in saturated porous media: dual mode deposition and intra-population heterogeneity

Because of heterogeneity among members of a bacteria population, deposition rates of bacteria may decrease upon the distance bacteria are transported in an aquifer. Such deposition rate decreases may result in retained bacteria concentrations, which decrease hyper-exponentially as a function of transport distance, and may therefore significantly affect the transport of colloids in aquifers. We investigated the occurrence of hyper-exponential deposition of Escherichia coli, an important indicator for fecal contamination, and the causes for such behavior. In a series of column experiments with glass beads of various sizes, we found that attachment of E. coli decreased hyper-exponentially, or, on logarithmic scale in a bimodal way, as a function of the transported distance from the column inlet. From data fitting of the retained bacteria concentration profiles, the sticking efficiency of 40% of the E. coli population was high (alpha=1), while the sticking efficiency of 60% was low (alpha=0.01). From the E. coli total population, an E. coli subpopulation consisting of slow attachers could be isolated by means of column passage. In subsequent column experiments this subpopulation attached less than the E. coli total population, consisting of both slow and fast attachers. We concluded that the main driver for the observed dual mode deposition was heterogeneity among members of the bacteria population. Intra-population may result in some microbes traveling surprisingly high distances in the subsurface. Extending the colloid filtration theory with intra-population variability may provide a valuable framework for assessing the transport of bacteria in aquifers.     

Multiple lines of evidence to identify the sources of fecal pollution at a freshwater beach in Hamilton Harbour, Lake Ontario

Multiple microbial source-tracking methods were investigated to determine the source of elevated Escherichia coli levels at Bayfront Park Beach in Hamilton Harbour, Lake Ontario. E. coli concentrations were highest in wet foreshore sand (114,000 CFU/g dry sand) and ankle-depth water (177,000 CFU/100mL), declining rapidly in deeper waters. Many gull and geese droppings were enumerated each week on the foreshore sand within 2m of the waterline. Both antimicrobial resistance analysis and rep-PCR DNA fingerprinting of E. coli collected at the beach and nearby fecal pollution sources indicated that E. coli in sand and water samples were predominantly from bird droppings rather than from pet droppings or municipal wastewater. Both methods indicated a trend of decreasing bird contamination, and increasing wastewater contamination, moving offshore from the beach. When foreshore sand was treated as a reservoir and secondary source of E. coli, waterborne E. coli were found to be more similar to sand isolates than bird or wastewater isolates out to 150 m offshore. Multiple lines of evidence indicated the importance of bird droppings and foreshore sand as primary and secondary sources of E. coli contamination in beach water at Bayfront Park.     

Reactivation and growth of non-culturable indicator bacteria in anaerobically digested biosolids after centrifuge dewatering

Recent literature has reported that high concentrations of indicator bacteria such as fecal coliforms (FCs) were measured in anaerobically digested sludges immediately after dewatering even though low concentrations were measured prior to dewatering. This research hypothesized that the indicator bacteria can enter a non-culturable state during digestion, and are reactivated during centrifuge dewatering. Reactivation is defined as restoration of culturability. To examine this hypothesis, a quantitative polymerase chain reaction (qPCR) method was developed to enumerate Escherichia coli, a member of the FC group, during different phases of digestion and dewatering. For thermophilic digestion, the density of E. coli measured by qPCR could be five orders of magnitude greater than the density measured by standard culturing methods (SCMs), which is indicative of non-culturable bacteria. For mesophilic digestion, qPCR enumerated up to about one order of magnitude more E. coli than the SCMs. After centrifuge dewatering, the non-culturable organisms could be reactivated such that they are enumerated by SCMs, and the conditions in the cake allowed rapid growth of FCs and E. coli during cake storage.     

Inactivation of Escherichia coli O157:H7 during thermophilic anaerobic digestion of manure from dairy cattle

Inactivation of the pathogenic Escherichia coli serotype O157:H7 and a non-pathogenic E. coli strain isolated from dairy cattle manure was evaluated with batch tests at 50 and 55 degrees C in biosolids from a thermophilic anaerobic digester treating the manure. Using differential-selective plating on sorbitol-MacConkey (SMAC) agar to quantify E. coli, the decline in concentrations of both the sorbitol-negative (putative E. coli O157:H7) and sorbitol-positive (putative non-pathogenic E. coli) organisms followed a model that assumed there was a heat-sensitive fraction and a heat-resistant fraction. Inactivation rates of the heat-sensitive fractions were similar for both colony types at each temperature, suggesting that wild-type E. coli can be used as an indicator of inactivation of serotype O157:H7. The decimal reduction time for the heat-sensitive fractions was in the order of 10min at 55 degrees C and ranged from approximately 1-3h at 50 degrees C. Concentrations of heat-resistant organisms at 55 degrees C were 1.4-1.7log(10)cfu/mL. Confirmatory analyses conducted on 30 randomly selected colonies of heat-resistant sorbitol-negative cells from treatment at 55 degrees C indicated that none were serotype O157:H7, nor were they E. coli. Similar analyses on 10 sorbitol-negative isolates from untreated manure indicated that none were serotype O157:H7, although 16S rRNA gene sequence analysis indicated that eight were E. coli or closely related enteric bacteria. These findings suggest that plating on differential-selective media to quantify E. coli, including serotype O157:H7, in effluent samples from thermophilic anaerobic digestion can lead to false positive results. Therefore, more specific methods should be used to evaluate the extent of thermal inactivation of both pathogenic and non-pathogenic E. coli in manure treatment systems.     

Relationships between Bacteroides 16S rRNA genetic markers and presence of bacterial enteric pathogens and conventional fecal indicators

Occurrence and prevalence of different bacterial enteric pathogens as well as their relationships with conventional (total and fecal coliforms) and alternative fecal indicators (host-specific Bacteroides 16S rRNA genetic markers) were investigated for various water samples taken from different sites with different degrees of fecal contamination. The results showed that a wide range of bacterial pathogens could be detected in both municipal wastewater treatment plant samples and in surface water samples. Logistic regression analysis revealed that total and human-specific Bacteroides 16S rRNA genetic markers showed significant predictive values for the presence of Escheriachia coli O-157, Salmonella, heat-labile enterotoxin (LT) of enterotoxigenic E. coli (ETEC), and heat-stable enterotoxin for human (STh) of ETEC. The probability of occurrence of these pathogenic bacteria became significantly high when the concentrations of human-specific and total Bacteroides 16S rRNA genetic markers exceeded 10(3) and 10(4) copies/100 mL. In contrast, Clostridium perfringens was detected at high frequency regardless of sampling sites and levels of Bacteroides 16S rRNA genetic markers. No genes related to Shigella spp., Staphylococcus aureus and Vibrio cholerae were detected in any samples analyzed in this study. Conventional indicator microorganisms had low levels of correlation with the presence of pathogens as compared with the alternative fecal indicators. These results suggested that real-time PCR-based measurement of alternative Bacteroides 16S rRNA genetic markers was a rapid and sensitive tool to identify host-specific fecal pollution and probably associated bacterial pathogens. However, since one fecal indicator might not represent the relative abundance of all pathogenic bacteria, viruses and protozoa, combined application of alternative indicators with conventional ones could provide more comprehensive pictures of fecal contamination, its source and association with pathogenic microorganisms.     

Confirmation of E. coli among other thermotolerant coliform bacteria in paper mill effluents, wood chips screening rejects and paper sludges

Paper sludges are solid wastes material generated from the paper production, which have been characterized for their chemical contents. Some are rich in wood fiber and are a good carbon source, for example the primary and de-inking paper sludges. Others are made rich in nitrogen and phosphorus by pressing the activated sludge, resulting from the biological water treatments, with the primary sludge, yielding the combined paper sludge. Still, in the absence of sanitary effluents very few studies have addressed the characterization of their coliform microflora. Therefore, this study investigated the thermotolerant coliform population of one paper mill effluent and two paper mill sludges and wood chips screening rejects using chromogenic media. For the first series of analyses, the medium used was Colilert broth and positive tubes were selected to isolate bacteria in pure culture on MacConkey agar. In a second series of analyses, double selective media, based on ss-galactosidase and ss-glucuronidase activities, were used to isolate bacteria. First, the presence of thermotolerant coliforms was detected in low numbers in most water effluents, but showed that the entrance of the thermotolerant coliforms was early in the industrial process. Also, large numbers of thermotolerant coliforms, i.e., 7,000,000 MPN/g sludge (dry weight; d.w.), were found in combined sludges. From this first series of isolations, bacteria were purified on MacConkey medium and identified as Citrobacter freundii, Enterobacter sp, E. sakazakii, E. cloacae, Escherichia coli, K. pneumoniae, K. pneumoniae subsp. rhinoscleromatis, K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. pneumoniae, Pantoea sp, Raoultella terrigena, R. planticola. Second, the presence of thermotolerant coliforms was measured at more than 3,700-6,000 MPN/g (d.w) sludge, whereas E. coli was detected from 730 to more than 3,300 MPN/g (d.w.) sludge. The presence of thermotolerant coliform bacteria and E. coli was sometimes detected from wood chips screening rejects in large quantities. Also, indigenous E. coli were able to multiply into the combined sludge, and inoculated E. coli isolates were often able to multiply in wood chips and combined sludge media. In this second series of isolations, API20E and Biolog identified most isolates as E. coli, but others remained unidentified. The sequences of the 16S rDNA confirmed that most isolates were likely E. coli, few Burkholderia spp, but 10% of the isolates remained unidentified. This study points out that the coliform bacteria are introduced by the wood chips in the water effluents, where they can survive throught the primary clarifier and regrow in combined sludges.     

Comparison of bacterial indicator analysis methods in stormwater-affected coastal waters

Membrane filtration (MF) and multiple tube fermentation (MTF) have been used for decades to measure indicator bacteria levels in beach water samples, but new methods based on chromogenic substrate (CS) technology are becoming increasingly popular. Only a few studies have compared results among these methods and they have generally been based on samples collected from a limited number of sites during dry weather. In this study, samples were collected from 108 sites the day after a major rainstorm, and three indicator bacteria (total coliforms (TCs), fecal coliforms (FCs) or E. coli, and enterococci (EC)) were each measured using MF, MTF, and CS. Sampling sites were selected using a stratified random design, stratified by open sandy beach, rocky shoreline, and beach areas near urban runoff outlets. The CS results were found to be highly correlated with both MF and MTF for all three indicators regardless of whether the samples were taken along open shoreline or near a runoff outlet. While correlated, TC values were higher using the CS method, consistent with other studies that have demonstrated false positives with this method. FC values were 12% lower with CS, reflecting the specificity of the CS method for E. coli rather than for the entire FC group. No significant differences were observed for EC, although some differences were observed within specific laboratories. Differences for all of these indicators were small enough that, when assessed categorically, there was more than 90% agreement between CS methods and either MF or MTF methods as to whether State of California Beach Water Quality Standards were met or exceeded.     

DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method

Accurate enumeration of indicator organisms such as Escherichia coli is important for assessing the safety of water and wastewater samples. Recent research has shown that E. coli can enter a viable but non-culturable state; therefore, traditional cultivation methods could potentially underestimate the quantities of the organisms. The goals of the research were to develop and verify a DNA extraction protocol and a quantitative polymerase chained reaction (PCR) method for E. coli enumeration in digested biosolids. A solvent-based DNA extraction protocol with extensive cell lysis recovered approximately 78-84% of spiked DNA. In comparison, a commercial kit only recovered 28-42% of DNA, likely from inefficient cell lysis. The developed competitive touchdown PCR (cPCR) method for E. coli enumeration was comparable to both real-time PCR (rt-PCR) and cultivation methods with sensitivity of approximately 50,000-500,000 E. coli per gram dry solids (DS), which is suitable for Class B biosolids monitoring in the US and "conventional" biosolids in the European Union. The cPCR protocol provides a less expensive alternative than the rt-PCR as a culturing independent method for enumerating E. coli.     

Optimization of PCR-based methods for rapid detection of Campylobacter jejuni, Campylobacter coli and Yersinia enterocolitica serovar 0:3 in wastewater samples

PCR-based methods were evaluated for their adequacy to assess the removal of pathogens from wastewater samples. For the development and optimization of the methods, samples were taken at two different sites from two different constructed wetlands. Campylobacter jejuni/coli and Yersinia enterocolitica serogroup 0:3 were selected as model pathogens and Enterococcus faecalis as a standard microbiological indicator. The chosen PCR protocols were optimized for wastewater DNA extracts in order to obtain high sensitivity and reproducibility independently of the background flora. All PCR protocols were successfully performed and reproducible with a background of up to 10(10) nontarget cells per reaction. Five cells of Y. enterocolitica, 50 cells of C. jejuni/coli, and 500 cells of E. faecalis per 100ml treated water could be detected. The method detection limit in the settled wastewater was higher: 200 cells per 100ml for Y. enterocolitica, 2000 cells per 100ml for C. jejuni/coli, and 20,000 cells per 100ml for E. faecalis. C. jejuni/coli and Y. enterocolitica PCRs were adapted to municipal wastewater, with higher loads of potential PCR inhibitors. Sensitivity was lower for this type of wastewater: 200 cells of Y. enterocolitica and 2000 cells of C. jejuni/coli were detected per 100ml treated wastewater, 2500 cells of Y. enterocolitica and 25,000 cells of C. jejuni/coli per 100ml settled wastewater. The developed PCR methods enable the detection of C. jejuni/coli, Y. enterocolitica serogroup 0:3 and E. faecalis within 12h. They show specificity, reproducibility and low detection limits for the investigated pathogens.     

Seasonal variation in accurate identification of Escherichia coli within a constructed wetland receiving tertiary-treated municipal effluent

As the reuse of municipal wastewater escalates worldwide as a means to extend increasingly limited water supplies, accurate monitoring of water quality parameters, including Escherichia coli (E. coli), increases in importance. Chromogenic media are often used for detection of E. coli in environmental samples, but the presence of unique levels of organic and inorganic compounds alters reclaimed water chemistry, potentially hindering E. coli detection using enzyme-based chromogenic technology. Over seven months, we monitored E. coli levels using m-Coli Blue 24((R)) broth in a constructed wetland filled with tertiary-treated municipal effluent. No E. coli were isolated in the wetland source waters, but E. coli, total coliforms, and heterotrophic bacteria increased dramatically within the wetland on all sampling dates, most probably due to fecal inputs from resident wildlife populations. Confirmatory testing of isolates presumptive for E. coli revealed a 41% rate of false-positive identification using m-Coli Blue 24((R)) broth over seven months. Seasonal differences were evident, as false-positive rates averaged 35% in summer, but rose sharply to 75% in the late fall and winter. Corrected E. coli levels were significantly correlated with electrical conductivity, indicating that water chemistry may be controlling bacterial survival within the wetland. This is the first study to report that accuracy of chromogenic media for microbial enumeration in reclaimed water may show strong seasonal differences, and highlights the importance of validation of microbiological results from chromogenic media for accurate analysis of reclaimed water quality.     

Deer diet affects ribotype diversity of Escherichia coli for bacterial source tracking

Ribotyping is one of a number of genotypic methods for bacterial source tracking. This method requires a host origin database of one bacterial species be established in order to identify environmental isolates. Researchers establishing these databases have observed considerable ribotype diversity within a specific bacterial species. One source of this diversity may be diet. We determined the effect of diet on ribotype diversity for Escherichia coli in penned and wild deer (Odocoileus virginianus) in a 13-ha forested watershed. A total of 298 E. coli isolates was obtained, 100 from penned deer, 100 from wild deer, and 98 from the stream in the watershed to which all deer had access. The wild deer had significantly more ribotypes (35) than the penned deer (11 ribotypes, p = 0.05). This result suggests that diet affected ribotype diversity, and that a host origin database for bacterial source tracking should contain bacterial isolates from wild rather than from captive animals. Also, 42 of 98 (42.9%) environmental isolates matched penned and wild deer ribotypes. If bacterial source tracking determines that fecal contamination is predominantly from wildlife, then it may be unnecessary to monitor these watersheds because control over wildlife is difficult.     

High diversity and abundance of antibiotic-resistant Escherichia coli isolated from humans and farm animal hosts in Jeonnam Province, South Korea

The spread of antibiotics resistance among bacteria is a threat to human health. Since South Korea uses approximately 1.5 times more antibiotics than do other OECD countries, this is likely to impact the numbers and types of antibiotic-resistant bacteria found in the environment. In this study we examined feces from domesticated animals and humans for the diversity and abundance of antibiotic-resistant Escherichia coli. Abundant antibiotic-resistant E. coli were isolated from all the tested animals and humans and were examined by horizontal, fluorophore-enhanced, rep-PCR (HFERP) DNA fingerprint analysis. A total of 793 unique, non-clonal, E. coli isolates were obtained from the 513 human and animal hosts examined. Antibiotic resistance analysis, done using 14 antibiotics, indicated that 72.3% of the isolates (573 of 793) were found resistant to more than one antibiotic. The E. coli isolated from swine were resistant to the greatest number of antibiotics. Tetracycline resistant E. coli were routinely isolated from all animal hosts (36 to 77% per host), except for dairy cattle (9.3%). Twenty nine E. coli isolates from all hosts, except for duck, were resistant to more than 10 antibiotics. Gene transfer and southern hybridization studies revealed that resistance to 13 of the antibiotics was self-transmissible, and likely mediated by plasmids and integrons. Since genetically diverse and numerically abundant antibiotic-resistant E. coli were consistently recovered from chicken, swine and other domesticated animals in South Korea, our results suggest that the use of sub-therapeutic levels of antibiotics for disease prophylaxis and growth promotion should be curtailed.     

Hydroxyapatite-supported Ag-TiO2 as Escherichia coli disinfection photocatalyst

A series of hydroxyapatite (HAP), 1wt% Ag-TiO(2) (AT1), 1wt% Ag-HAP and 5wt% AT1/HAP composite catalysts were prepared by incipient wetness and mechanical mixing methods. They were characterized by X-ray diffraction (XRD), FT-IR, SEM and ESCA analyses and their photocatalytic bactericidal activities were measured in suspension using Escherichia coli (E. coli), a water pollutant indicator. The surface analysis revealed that the Ag/Ti ratio is found to be ca. 0.0273 and also it indicated that the titania is present in the form of Ti(4+) and Ag is present as metallic silver. Both the XRD and ESCA analyses confirmed the phase of metallic Ag particles, which played a significant role on the bactericidal activity of the Ag doped TiO(2) catalysts. The FT-IR analysis of HAP revealed that the peak intensity is due to the absorbance of surface PO(4)(3-) group centered at wave number 1030cm(-1) and is drastically decreased upon exposure to UV for 1h. The HAP displayed high amount of bacteria adsorption, ca. 80% during the dark experiments compared to other catalytic systems tested. The cumulative photocatalytic properties of AT1/HAP catalytic system resulted in 100% E. coli bacteria reduction within 2min.     

Antibiotic resistant enterococci and staphylococci isolated from flies collected near confined poultry feeding operations

Use of antibiotics as feed additives in poultry production has been linked to the presence of antibiotic resistant bacteria in farm workers, consumer poultry products and the environs of confined poultry operations. There are concerns that these resistant bacteria may be transferred to communities near these operations; however, environmental pathways of exposure are not well documented. We assessed the prevalence of antibiotic resistant enterococci and staphylococci in stored poultry litter and flies collected near broiler chicken houses. Drug resistant enterococci and staphylococci were isolated from flies caught near confined poultry feeding operations in the summer of 2006. Susceptibility testing was conducted on isolates using antibiotics selected on the basis of their importance to human medicine and use in poultry production. Resistant isolates were then screened for genetic determinants of antibiotic resistance. A total of 142 enterococcal isolates and 144 staphylococcal isolates from both fly and poultry litter samples were identified. Resistance genes erm(B), erm(A), msr(C), msr(A/B) and mobile genetic elements associated with the conjugative transposon Tn916, were found in isolates recovered from both poultry litter and flies. Erm(B) was the most common resistance gene in enterococci, while erm(A) was the most common in staphylococci. We report that flies collected near broiler poultry operations may be involved in the spread of drug resistant bacteria from these operations and may increase the potential for human exposure to drug resistant bacteria.