Longitudinal Characterization of Resistant Escherichia coli in Fecal Deposits from Cattle Fed Subtherapeutic Levels of Antimicrobials

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp^r) and tetracycline-resistant (Tet^r) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp^r and Tet^r E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet^r E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet^r and Amp^r E. coli. Except for Amp^r E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet^r E. coli numbers were detected in A44 and AS700 deposits [5.9 log₁₀ CFU (g dry matter)⁻¹ and 5.4 log₁₀ CFU (g dry matter)⁻¹, respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla_TEM1, tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.In North America antibiotics are widely used in beef cattle production as prophylactics or antimicrobial growth promoters (AGP). Used in this manner, antibiotics are generally administered in the diet either at times of high disease risk or on a continuous basis to improve feed efficiency. Employment of AGP in this manner may increase the prevalence of commensal antimicrobial-resistant (AR) bacteria (1, 41).There is evidence that resistant bacteria can be transferred from livestock to humans (5, 39, 55), and consequently the use of AGP has raised public health concerns. While the modes of transmission of AR bacteria and gene determinants are complex (33), survivability in agriculture-related matrices may be a critical factor in their dissemination (54). Most research on the persistence of AR bacteria in livestock waste has focused on large-scale management systems, such as land-applied manure (20, 44) or storage lagoons and pits (28, 49). In some instances, these systems have been shown to decrease the survival of select bacteria and the presence of AR genes (35, 51). Nevertheless, there is limited knowledge of the fate of AR bacteria or resistance determinants in feces shed from individuals, such as the fecal deposits that are formed in feedlot pens on intensively managed farms. Previous work has shown that fecal deposits provide an environment that is conducive to the growth and survival of pathogenic and commensal Escherichia coli (4, 48, 57). Because cattle fed AGP excrete antimicrobial residues in their feces (3), the residues may exert a selection pressure on bacteria after deposition of feces and potentially confer a growth advantage to resistant bacteria.The objective of the current study was to investigate both the longitudinal phenotypic and genotypic ecologies of AR E. coli in fecal deposits arising from groups of cattle administered either no AGP or one of two tetracycline-based AGP that are commonly used in the North American industry. We focused on ampicillin-resistant (Amp^r) and tetracycline-resistant (Tet^r) E. coli because both AGP treatments contained tetracycline, and we have previously found a link between the use of tetracycline-based AGP and Amp^r E. coli (1). We hypothesized that resistant E. coli would be more numerous in fecal deposits from animals previously fed AGP.     

Long-Term Persistence and Leaching of Escherichia coli in Temperate Maritime Soils

Enteropathogen contamination of groundwater, including potable water sources, is a global concern. The spreading on land of animal slurries and manures, which can contain a broad range of pathogenic microorganisms, is considered a major contributor to this contamination. Some of the pathogenic microorganisms applied to soil have been observed to leach through the soil into groundwater, which poses a risk to public health. There is a critical need, therefore, for characterization of pathogen movement through the vadose zone for assessment of the risk to groundwater quality due to agricultural activities. A lysimeter experiment was performed to investigate the effect of soil type and condition on the fate and transport of potential bacterial pathogens, using Escherichia coli as a marker, in four Irish soils (n = 9). Cattle slurry (34 tonnes per ha) was spread on intact soil monoliths (depth, 1 m; diameter, 0.6 m) in the spring and summer. No effect of treatment or the initial soil moisture on the E. coli that leached from the soil was observed. Leaching of E. coli was observed predominantly from one soil type (average, 1.11 ± 0.77 CFU ml⁻¹), a poorly drained Luvic Stagnosol, under natural rainfall conditions, and preferential flow was an important transport mechanism. E. coli was found to have persisted in control soils for more than 9 years, indicating that autochthonous E. coli populations are capable of becoming naturalized in the low-temperature environments of temperate maritime soils and that they can move through soil. This may compromise the use of E. coli as an indicator of fecal pollution of waters in these regions.The contamination of groundwater, including potable water supplies, with microbial pathogens continues to be a global concern (52, 59). Of particular importance in developed countries are the high levels of contamination associated with small-scale and very-small-scale drinking water supplies (5, 19, 57), often groundwater, which serve an estimated 10% of the total population in the European Union (13). The high numbers of these water supplies found to be contaminated with fecal bacteria and thus considered to be unfit for human consumption are worrying because the water from them is often untreated or inadequately treated prior to consumption. Microbial pathogens are known to survive for considerable periods of time in groundwater (29), which increases the health risk due to utilization of contaminated supplies. There are various sources of contamination, but evidence suggests that contamination from the spreading of animal slurries and manures on land can be a significant contributor (3, 33, 53). Spreading of agricultural slurries and manures on land is used by the agricultural sector as a means of nutrient recycling. The health risks associated with the spreading of animal and human wastes containing enteric pathogens have been recognized for a long time (10, 18). Animal manure and wastewaters may contain a broad range of pathogenic microorganisms, including Escherichia coli O157:H7, Campylobacter, Cryptosporidium, Salmonella spp., and pathogenic viruses, which are released into the environment during spreading (15, 22, 55). The levels and incidence of pathogens present in animal manures and slurries are influenced by a number of factors, including herd health, age demographics, stress factors, diet, season, and manure management and storage (37, 39).Soils (and subsoils) often act as a zone for mitigating microbial contamination of groundwater associated with the spreading of animal slurries and manures on land. Some of the pathogenic microorganisms applied to agricultural soils have, however, been observed to leach through the soil into groundwater, which can affect drinking water quality and pose a risk to public health (16, 26, 28, 42, 50), confirming that soil is not always a sufficient obstruction for protection of groundwater (16, 53). Consequently, characterization of the movement of pathogens through the unsaturated soil and subsoil zone (vadose zone) has become critical for assessment of the risk to groundwater posed by agricultural activities (8, 14, 42). The soil and subsoil type is believed to be a major factor influencing the potential transfer of pathogens through soil to groundwater (3, 34, 41, 50). The preapplication moisture status of a soil, which may be influenced by the season, also impacts pathogen survival, fate, and transport (2, 11, 43, 54).E. coli is widely used as an indicator of fecal contamination of water, and certain strains are known to be pathogenic (12). Thus, characterizing this organism''s transport through soil is important because of the health risk posed by the organism itself and with regard to its validity as an indicator of the fate of enteropathogens in the environment. E. coli strains have diverse properties and capabilities that affect their survival and transport in soils (9, 36, 56, 60). Consequently, data obtained by using total E. coli rather than individual surrogate strains can be more representative of the fate and transport of E. coli present in animal slurries. E. coli O157 die-off in soils has been reported to be the same as or quicker than total E. coli die-off, suggesting that data for total E. coli provide a conservative estimate of the survival potential (38, 56). Although many field and laboratory studies have investigated E. coli transport through soil columns (4, 6, 16, 43, 46, 47, 50, 51), most studies have investigated transport through soil to a depth of less than 30 cm. For assessment of the risk of transport to groundwater, such studies may not take into account the variation in soil physical and chemical characteristics with depth (e.g., the frequency and continuity of macropores, organic matter, and moisture contents) that affect bacterial transport. Furthermore, rainfall was often simulated in previous studies, which allows experimental conditions to be controlled but may not be representative of the risk due to variable natural rainfall events over time. In this study, we used intact soil monoliths that were 1 m deep to assess the risk of leaching of total E. coli in four representative Irish soil types under natural rainfall and environmental conditions.The objective of this study was to quantitatively investigate the impact of soil type and season (soil moisture content) on the fate and transport of E. coli spread on four different temperate maritime soil types under natural rainfall conditions. We hypothesized that there would be a greater microbial risk to underlying groundwater with better-drained soil types than with relatively poorly drained soil types following the application of animal slurry. In addition, we hypothesized that E. coli cells spread on wetter spring soils would be transported in greater numbers than E. coli cells spread on drier soils in the summer.     

Absence of Escherichia coli Phylogenetic Group B2 Strains in Humans and Domesticated Animals from Jeonnam Province,Republic of Korea

Multiplex PCR analyses of DNAs from genotypically unique Escherichia coli strains isolated from the feces of 138 humans and 376 domesticated animals from Jeonnam Province, South Korea, performed using primers specific for the chuA and yjaA genes and an unknown DNA fragment, TSPE4.C2, indicated that none of the strains belonged to E. coli phylogenetic group B2. In contrast, phylogenetic group B2 strains were detected in about 17% (8 of 48) of isolates from feces of 24 wild geese and in 3% (3 of 96) of isolates obtained from the Yeongsan River in Jeonnam Province, South Korea. The distribution of E. coli strains in phylogenetic groups A, B1, and D varied depending on the host examined, and there was no apparent seasonal variation in the distribution of strains in phylogenetic groups among the Yeongsan River isolates. The distribution of four virulence genes (eaeA, hlyA, stx₁, and stx₂) in isolates was also examined by using multiplex PCR. Virulence genes were detected in about 5% (38 of 707) of the total group of unique strains examined, with 24, 13, 13, and 9 strains containing hlyA, eaeA, stx₂, and stx₁, respectively. The virulence genes were most frequently present in phylogenetic group B1 strains isolated from beef cattle. Taken together, results of these studies indicate that E. coli strains in phylogenetic group B2 were rarely found in humans and domesticated animals in Jeonnam Province, South Korea, and that the majority of strains containing virulence genes belonged to phylogenetic group B1 and were isolated from beef cattle. Results of this study also suggest that the relationship between the presence and types of virulence genes and phylogenetic groupings may differ among geographically distinct E. coli populations.Escherichia coli is a normal inhabitant of the lower intestinal tract of warm-blooded animals and humans. While the majority of E. coli strains are commensals, some are known to be pathogenic, causing intestinal and extraintestinal diseases, such as diarrhea and urinary tract infections (42). Phylogenetic studies done using multilocus enzyme electrophoresis and 72 E. coli strains in the E. coli reference collection showed that E. coli strains can be divided into four phylogenetic groups (A, B1, B2, and D) (20, 41, 48). Recently, a potential fifth group (E) has also been proposed (11). Since multiplex PCR was developed for analysis of phylogenetic groups (6), a number of studies have analyzed a variety of E. coli strains for their phylogenetic group association (10, 12, 17, 18, 23, 54). Duriez et al. (10) reported the possible influence of geographic conditions, dietary factors, use of antibiotics, and/or host genetic factors on the distribution of phylogenetic groups among 168 commensal E. coli strains isolated from human stools from three geographically distinct populations in France, Croatia, and Mali. Random-amplified polymorphic DNA analysis of the intraspecies distribution of E. coli in pregnant women and neonates indicated that there was a correlation between the distribution of phylogenetic groups, random-amplified polymorphic DNA groups, and virulence factors (54). Moreover, based on comparisons of the distribution of E. coli phylogenetic groups among humans of different sexes and ages, it has been suggested that E. coli genotypes are likely influenced by morphological, physiological, and dietary differences (18). In addition, climate has also been proposed to influence the distribution of strains within E. coli phylogenetic groups (12). There are now several reports indicating that there is a potential relationship between E. coli phylogenetic groups, age, and disease. For example, E. coli isolates belonging to phylogenetic group B2 have been shown to predominate in infants with neonatal bacterial meningitis (27) and among urinary tract and rectal isolates (55). Also, Nowrouzian et al. (39) and Moreno et al. (37) reported that strains belonging to phylogenetic group B2 persisted among the intestinal microflora of infants and were more likely to cause clinical symptoms.Boyd and Hartl (2) reported that among the E. coli strains in the E. coli reference and the diarrheagenic E. coli collections, strains in phylogenetic group B2 carry the greatest number of virulence factors, followed by those in group D. Virulence factors carried by group B2 strains are thought to contribute to their strong colonizing capacity; a greater number of virulence genes have been detected in resident strains than in transient ones (38). Moreover, a mouse model of extraintestinal virulence showed that phylogenetic group B2 strains killed mice at greater frequency and possessed more virulence determinants than strains in other phylogenetic groups, suggesting a link between phylogeny and virulence genes in E. coli extraintestinal infection (45). In contrast, Johnson and Kuskowski (25) suggested that a group B2 ancestral strain might have simply acquired virulence genes by chance and that these genes were vertically inherited by group members during clonal expansion. However, numerous studies published to date suggest that there is a relationship between the genomic background of phylogenetic group B2 and its association with virulence factors (12, 28, 35, 39, 45).Both enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively) strains are among the most important food-borne pathogens worldwide, often causing severe gastrointestinal disease and fatal infections (13). While EPEC strains cause diarrhea and generally do not produce enterotoxin, they possess an adherence factor which is controlled by the chromosomal gene eaeA, encoding intimin (8). Unlike the EPEC strains, however, the EHEC strains typically contain the hlyA, stx₁, and stx₂ virulence genes, encoding hemolysins and Shiga-like type 1 and 2 toxins, respectively, and eaeA. The ability to detect EHEC has been greatly facilitated by the use of multiplex PCR (13, 44, 53). Several studies have shown that strains producing Shiga-like toxin 2 are more frequently found in cases of hemolytic-uremic syndrome than are those containing Shiga-like toxin 1 (30, 43, 46, 49).In the study reported here, we examined the distribution of phylogenetic groups and the prevalence of virulence genes in 659 genotypically unique E. coli strains isolated from humans and domestic animals in South Korea. In addition, we also tested 48 and 96 nonunique E. coli isolates from wild geese and the Yeongsan River, respectively, for phylogenetic distribution and virulence gene profiles. Here, we report that contrary to what has been previously reported in other parts of the world, no E. coli strains belonging to phylogenetic group B2 were found in domesticated animals and in humans from Jeonnam Province, South Korea. We also report that among the strains we examined, virulence genes were mainly found in phylogenetic group B1 strains isolated from beef cattle. Results of these studies may prove to be useful for the development of risk management strategies to maintain public health.     

Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers

The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log₁₀ copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log₁₀ copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log₁₀ copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution.Human and animal fecal pollution of coastal environments affects shellfish and recreational water quality and safety, in addition to causing economic losses from the closure of shellfish harvesting areas and from bathing restrictions (13, 19, 33). Human feces are known to contain human-specific enteric pathogens (3, 18, 28), but animals can also be reservoirs for numerous enteric human pathogens, such as Escherichia coli O157:H17, Salmonella spp., Mycobacterium spp., or Listeria spp., that may persist in the soil or surface waters (6, 8, 22, 24). Among animals, pigs are known to carry human pathogens that are excreted with fecal wastes. There are approximately 125 million pigs in the European Union (EU) and 114 million in North America (12, 36, 48), generating an estimated 100 and 91 million tons of pig slurry per year, respectively (4). France, the third largest pig producer in the EU, with about 23,000 farms, generates 8 to 10 million tons of pig slurry per year. Brittany accounts for 56.1% of the total national pig production on only 6% (27,200 km²) of the French territory, though it has 40% (2,700 km) of the coastline. This production could contaminate the environment when tanks on farms overflow, when slurry or compost is spread onto soils, or to a lesser extent, when lagoon surface waters are used for irrigation (38, 47, 52).Fecal contamination in shellfish harvesting and bathing areas is currently evaluated by the detection and enumeration of culturable facultative-anaerobic bacteria, such as E. coli, enterococci, or fecal coliforms (11), in shellfish and bathing waters (European Directives 2006/113/CE and 2006/7/CE). Pigs are among the potential sources of E. coli inputs to the environment; a pig produces approximately 1 × 10⁷ E. coli bacteria per gram of feces, which corresponds to an E. coli flow rate per day that is 28 times higher than that for one human (16, 34, 55).E. coli is not a good indicator of fecal sources of pollution in water because of its presence in both human and animal feces; therefore, alternative fecal indicators must be used. Microbial source tracking methods (44) are being developed to discriminate between human and nonhuman sources of fecal contamination and to distinguish contamination from different animal species (17, 46, 54). Many of these methods are library dependent, requiring a large number of isolates to be cultured and tested, which is time consuming and labor intensive. For these reasons, library-independent methods are preferred for the detection of host-specific markers.The detection of host-specific Bacteroidales markers is a promising library-independent method and has been used for identifying contamination from human and bovine origins (25, 29, 39, 40, 45). In this study, we selected Bacteroidales 16S rRNA gene markers and real-time PCR to focus on fecal contamination from pigs. To date, only one pig-specific Bacteroidales 16S rRNA gene marker has been developed and used on water samples for the identification of pig fecal contamination by real-time PCR assay (SYBR green) (37). When this pig-specific Bacteroidales marker was tested on a small number of fecal samples (n = 16), it showed some cross-reaction with human and cow feces.The present study investigated pig fecal contamination in a French catchment, the Daoulas estuary (Brittany), which has commercial and recreational shellfish harvesting areas and which is potentially subject to fecal contamination. The aims of the present study were (i) to design new primers for the detection and quantification of pig-specific Bacteroidales 16S rRNA genes by TaqMan analysis; (ii) to validate the sensitivity and specificity of the new primers and TaqMan assay using target (feces, slurry, compost, and lagoon water samples) and nontarget (human and other animal sources) DNA, respectively; and (iii) to evaluate the TaqMan assay for proper detection and quantitative estimation of pig-associated fecal pollution. The study represents the first application of pig-specific Bacteroidales markers using a TaqMan assay in Europe and included a monitoring study of marker levels throughout the various stages of slurry treatment.     

Enumeration and Characterization of Antimicrobial-Resistant Escherichia coli Bacteria in Effluent from Municipal,Hospital, and Secondary Treatment Facility Sources

We describe a modification of the most probable number (MPN) method for rapid enumeration of antimicrobial-resistant Escherichia coli bacteria in aqueous environmental samples. E. coli (total and antimicrobial-resistant) bacteria were enumerated in effluent samples from a hospital (n = 17) and municipal sewers upstream (n = 5) and downstream (n = 5) from the hospital, effluent samples from throughout the treatment process (n = 4), and treated effluent samples (n = 13). Effluent downstream from the hospital contained a higher proportion of antimicrobial-resistant E. coli than that upstream from the hospital. Wastewater treatment reduced the numbers of E. coli bacteria (total and antimicrobial resistant); however, antimicrobial-resistant E. coli was not eliminated, and E. coli resistant to cefotaxime (including extended-spectrum beta-lactamase [ESBL] producers), ciprofloxacin, and cefoxitin was present in treated effluent samples.The emergence and dissemination of antimicrobial resistance are well established as clinical problems that affect human and animal health. Escherichia coli is an important element of the flora of the human and animal intestine and a significant pathogen associated with gastrointestinal infection, urinary tract infections, and a variety of other extraintestinal infections (4). E. coli shed into the environment can survive for significant periods (7, 14, 23). Detection of E. coli in water and food is widely used as a microbiological indication of fecal contamination.Data on the significance of environmental contamination with antimicrobial-resistant E. coli for human health are limited. Previous reports have shown that antimicrobial-resistant strains of bacteria are present in various effluents, such as hospital effluent discharge (8, 10, 16, 21), inflow effluent to a wastewater treatment plant (WWTP) (15), and outflow-treated effluent from a wastewater treatment plant (2, 12, 13, 18, 27). A wastewater treatment plant treating effluent from hospitals may be associated with discharge of relatively high levels of antimicrobial-resistant E. coli compared with those of a plant treating municipal effluent that does not include hospital effluent discharge (22). There are few reports of quantitative data on antimicrobial-resistant E. coli bacteria in effluent, reflecting the lack of a convenient method for their enumeration (12, 15, 22). Previous methods available for the detection of antimicrobial-resistant E. coli in a water sample have generally involved the isolation of E. coli and the selection of some isolates for susceptibility testing. In such cases, the proportions of antimicrobial-resistant organisms are based only on those isolates selected and are therefore not representative of the entire population. By adding the antimicrobial agent of interest to the water sample before testing, we have adapted a commercial most probable number (MPN) method (the Colilert system) for enumerating the total number of E. coli isolates resistant to that agent in a sample.     

In Vivo and Ex Vivo Evaluations of Bacteriophages e11/2 and e4/1c for Use in the Control of Escherichia coli O157:H7

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10¹⁰ CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10¹¹ PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.Escherichia coli O157:H7 has become a worldwide public health concern since it was first identified as a human pathogen in 1982 (31). This pathogen has a very low infectious dose (approximately 10 cells) in humans, and symptoms of infection range from watery diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and in some cases, death (22, 39). Ruminants are recognized as reservoirs for this pathogen and are the most common sources for food-borne outbreaks (8, 13, 25). It has been reported that the occurrence of E. coli O157:H7 in the feces and, in particular, the hide of cattle is a significant source of the pathogen on the carcass and in derived meat products (11, 12, 25). The control of this pathogen within the animal is difficult, because carriage in ruminants is asymptomatic and shedding can be intermittent and seasonal (12, 19).Research has highlighted the necessity for preharvest intervention strategies to control or reduce E. coli O157:H7 in the food chain (17, 18). Successful strategies to reduce the carriage of E. coli O157:H7 in ruminant animals could potentially reduce the risk of human exposure to this pathogen. There are currently no effective and reliable commercially available intervention strategies to control the carriage of E. coli O157:H7 in ruminants. However, research in this area is increasing, and numerous agents, such as vaccines, probiotics, and bacteriophages (phages), are being evaluated (15, 17, 18). The use of phages for the control of food-borne pathogens in the food chain is desirable, as they are natural, nontoxic viruses that target only specific bacteria (2) and are already being used in human and veterinary medicine, particularly prior to antibiotics (6, 14, 15, 30, 37). Many studies have investigated the use of different phages for the control of E. coli O157:H7 in various animals, including mice, calves, and sheep (4, 5, 35, 37, 41). Although the results between studies vary, some have reported the successful reduction of E. coli O157:H7 levels in animals (4), and one study has resulted in a U.S. patent (41). There are very few commercially available phage products to date, but research indicates promising outcomes for the use of phages for the control of E. coli O157:H7 within the food chain.The E. coli O157:H7-specific phages e11/2 and e4/1c were isolated from bovine slurry in a previous study (26) and have the potential to be used as biocontrol agents for E. coli O157:H7. Both phages have been found to be active against E. coli O157:H7 in a number of relevant test conditions involving different pHs, water activity, and temperatures (B. Coffey, L. Rivas, G. Duffy, A. Coffey, R. P. Ross, and O. McAuliffe, unpublished data). In addition, whole-genome sequencing revealed that neither phage encodes undesirable properties, such as virulence factors, that would hinder its use as a biocontrol agent for E. coli O157:H7 (B. Coffey, G. O''Flynn, A. Coffey, O. O''Sullivan, O. McAuliffe, and R. P. Ross, unpublished data). The objective of the present study was, first, to evaluate the effect of phages e4/1c and e11/2 against inoculated E. coli O157:H7 in an ex vivo model rumen system, and second, to assess the ability of a phage cocktail (e11/2 and e4/1c) to reduce the shedding of E. coli O157:H7 in experimentally inoculated cattle. Findings from ex vivo studies determined our phages to be effective against E. coli O157:H7 in a model rumen system; however, complete eradication of E. coli O157:H7 from cattle was not achieved.     

Phylogenetic Groups and Pathogenicity Island Markers in Fecal Escherichia coli Isolates from Asymptomatic Humans in China

The study of phylogenetic groups and pathogenicity island (PAI) markers in commensal Escherichia coli strains from asymptomatic Chinese people showed that group A strains are the most common and that nearly half of all fecal strains which were randomly selected harbor PAIs.Escherichia coli is a well-diversified commensal species in the intestine of healthy humans but also includes intestinal or extraintestinal pathogens. It has been reported that pathogenic E. coli may be derived from fecal strains by acquisition of virulence determinants (11). The relationship between the E. coli genetic background and the acquisition of virulence factors is now better understood (1, 5). Extraintestinal E. coli strains may harbor several virulence factors, such as adhesins, fimbriae, and hemolysin, which can contribute to bacterial pathogenesis. These traits are usually encoded on pathogenicity islands (PAIs), which have been studied in pathogenic E. coli previously (15). The E. coli population includes 4 major phylogroups (A, B₁, B₂, and D) (2). Pathogenic strains belong mainly to groups B₂ and D, while most fecal isolates belong to groups A and B₁. Strains of groups B₂ and D often carry virulence factors that are lacking in group A and B₁ strains (3, 9, 13).In this study, we examined the distribution of phylogroups and the prevalence of PAIs in commensal E. coli strains isolated from asymptomatic persons in one region of China.     

Role of Ceftiofur in Selection and Dissemination of blaCMY-2-Mediated Cephalosporin Resistance in Salmonella enterica and Commensal Escherichia coli Isolates from Cattle

Third-generation cephalosporin resistance of Salmonella and commensal Escherichia coli isolates from cattle in the United States is predominantly conferred by the cephamycinase CMY-2, which inactivates β-lactam antimicrobial drugs used to treat a wide variety of infections, including pediatric salmonellosis. The emergence and dissemination of bla_CMY-2--bearing plasmids followed and may in part be the result of selection pressure imposed by the widespread utilization of ceftiofur, a third-generation veterinary cephalosporin. This study assessed the potential effects of ceftiofur on bla_CMY-2 transfer and dissemination by (i) an in vivo experimental study in which calves were inoculated with competent bla_CMY-2-bearing plasmid donors and susceptible recipients and then subjected to ceftiofur selection and (ii) an observational study to determine whether ceftiofur use in dairy herds is associated with the occurrence and frequency of cephalosporin resistance in Salmonella and commensal E. coli. The first study revealed bla_CMY-2 plasmid transfer in both ceftiofur-treated and untreated calves but detected no enhancement of plasmid transfer associated with ceftiofur treatment. The second study detected no association (P = 0.22) between ceftiofur use and either the occurrence of ceftiofur-resistant salmonellosis or the frequency of cephalosporin resistance in commensal E. coli. However, herds with a history of salmonellosis (including both ceftiofur-resistant and ceftiofur-susceptible Salmonella isolates) used more ceftiofur than herds with no history of salmonellosis (P = 0.03) These findings fail to support a major role for ceftiofur use in the maintenance and dissemination of bla_CMY-2-bearing plasmid mediated cephalosporin resistance in commensal E. coli and in pathogenic Salmonella in these dairy cattle populations.The major mechanism of third-generation cephalosporin resistance among U.S. human and veterinary clinical isolates of Salmonella enterica subsp. enterica is the beta-lactamase CMY-2 (12, 17, 43, 44, 46). bla_CMY-2, which likely originated from the chromosomal AmpC locus of Citrobacter freundii, is disseminated among a group of similar plasmids harbored by diverse Enterobacteriaceae species (1, 2, 20, 26, 30, 31, 42, 45). In Salmonella, bla_CMY-2-bearing plasmids have been observed in more than 30 serovars, notably including serovar Newport, which has gained specific attention from public health officials as a rapidly emerging threat (2, 6, 31).Commensal Escherichia coli frequently harbors bla_CMY-2-bearing plasmids (15, 33, 44), and these plasmids may be transferable to pathogens, since bla_CMY-2 plasmids isolated from E. coli and S. enterica share extensive sequence similarity in addition to the bla_CMY-2 open reading frame (5, 12, 42, 44). This transfer may occur in the gastrointestinal tracts of cattle, where these bacterial species periodically coexist and where transconjugants may be subjected to specific antimicrobial selection pressure. In fact, in vivo transfer of bla_CMY-2 in the gastrointestinal tract has been reported between a Klebsiella pneumoniae bla_CMY-2 plasmid donor and a Salmonella enterica serovar Typhimurium isolate in cattle and goats (29).Ceftiofur is the only third-generation cephalosporin antimicrobial drug that is used in cattle production systems and is labeled for the treatment of pneumonia, postpartum metritis, necrotizing pododermatitis, and mastitis. Two ceftiofur preparations, ceftiofur sodium (Naxcel) and ceftiofur hydrochloride (Excenel) (Pfizer Animal Health, New York, NY), are unique in the veterinary pharmacopeia because they require no withholding and discard of milk collected from treated cows, making them frequent therapeutic choices in lactating animals (19, 35). Ceftiofur was licensed in 1988 (41) and its resistance in Salmonella spp. isolated from U.S. cattle, presumably conferred by bla_CMY-2, was first documented in 1998 (6).The effects of ceftiofur use on selection of bla_CMY-2-bearing commensal E. coli has been examined for cattle both epidemiologically and experimentally. Tragesser et al. studied 18 Ohio dairy herds and determined that the 11 herds that used ceftiofur in any capacity (labeled indications and/or extralabel use) were 25 times more likely to have E. coli with reduced susceptibility to ceftriaxone (an expected bla_CMY-2 phenotype) than the seven herds that reported no ceftiofur use (40). Interestingly, however, within eight herds that had detailed treatment records, no association was detected between the prevalence of E. coli with reduced susceptibility to ceftriaxone and use of ceftiofur on an individual-animal basis (40). In an experimental study by Jiang et al., ceftiofur administered to dairy calves was correlated with a 14% increase in ceftriaxone-resistant fecal E. coli compared to untreated controls (21). Together, these studies show a correlation between selection pressure within the gastrointestinal tracts at the individual-animal level and show that ceftiofur use may promote the dissemination of resistance in commensal E. coli at the whole-herd level.Whether or not ceftiofur treatment directly affects in vivo horizontal transfer of bla_CMY-2-bearing elements among E. coli and Salmonella has yet to be addressed. The diversity of bla_CMY-2 plasmid-bearing bacterial hosts is consistent with wide dissemination of this genetic element. One hypothesis that could explain this wide dissemination is that ceftiofur may itself promote the in vivo horizontal transfer of bla_CMY-2-bearing plasmids. Specifically, due to the relatively slow bactericidal activity of aminothiazolyl cephalosporins such as ceftiofur, it has been suggested that exposure to these compounds promotes filament formation in gram-negative bacteria prior to cell death that may increase the surface area and increase receptiveness of the cells for resistance plasmids (11).Because bla_CMY-2 may be disseminated by horizontal transfer of R plasmids and/or clonal expansion of individual strains, we examined the effect of ceftiofur use on these processes with two approaches; the first approach specifically considered the issue of horizontal transfer in an experimental in vivo calf model, while the second approach, a field study, assessed the overall relationship between ceftiofur use and bla_CMY-2 prevalence in the primary agricultural animal niche where it is used.     

Characterization of Environmentally Persistent Escherichia coli Isolates Leached from an Irish Soil

Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.Escherichia coli is a well-established indicator of fecal contamination in the environment. The organism''s validity as an indicator of water pollution is dependent, among other factors, on its fecal specificity and its inability to multiply outside the primary host, the gastrointestinal tracts of humans and warm-blooded animals (9). While many pathogens and indicator organisms are considered to be poorly adapted for long-term survival, or proliferation, outside their primary hosts (24), there is increasing evidence that this view needs to be reconsidered with respect to E. coli (17, 38). In particular, questions remain about its fate and survival capacity in environmental matrices, such as soil. While the habitat within the primary host is characterized by constant warm temperature conditions and a ready availability of nutrients and carbon, that of soil is often characterized by oligotrophic and highly dynamic conditions, temperature and pH variation, predatory populations, and competition with environmentally adapted indigenous microflora (39). Soils are thus typically considered to be suboptimal environments for enteric organisms, and growth is thought to be negligible, with die-off of organisms at rates reported to be a function of the interaction of numerous factors, including the type and physiological state of the microorganism, the physical, chemical, and biological properties of the soil, atmospheric conditions (including sunlight, moisture, and temperature), and organism application method (10).In recent years, the growth of E. coli in soils, sediments, and water in tropical and subtropical regions has been widely documented, and the organism is considered to be an established part of the soil biota within these regions (4, 5, 7, 12, 14, 19, 25, 32). The integration of E. coli as a component of the indigenous microflora in soils of tropical and subtropical regions may be attributable to the nutrient-rich nature and warm temperatures of these habitats (21, 39), combined with the metabolic versatility of the organism and its simple nutritional requirements (21). In addition to tropical and subtropical regions, the presence of autochthonous E. coli populations in the cooler soils of temperate and northern temperate regions has also been reported (6, 20, 22, 37), with one report on an alpine soil (34) and, most recently, a report on a maritime temperate grassland soil (3). The growth of E. coli within soils can act as a reservoir for the further contamination of bodies of water (20, 31, 32), compromising the indicator status of E. coli within these regions. As such, an understanding of the ecological characteristics of E. coli in soil is critical to its validation as an indicator organism. With respect to the input of pathogenic E. coli into the environment, this knowledge becomes essential for assessing the potential health risk to human and animal hosts from agricultural activities such as landspreading of manures and slurries (24).It has been suggested that E. coli can sustain autochthonous populations within soils in temperate regions, wherever favorable conditions exist (21). The phenotypic traits of the organism (including its metabolic diversity and its ability to grow both aerobically and anaerobically in a broad temperature range) may assist the persistence, colonization, and growth of E. coli when conditions permit. The challenging nature of the soil environment and the disparity of conditions between the primary host and the secondary habitat raises the question of how these E. coli populations survive and compete for niche space among the highly competitive and diverse coexisting populations of the indigenous microflora (15, 21). There is some evidence that naturalized E. coli may form genetically distinct populations in the environment (17, 20, 34, 36). This suggests that autochthonous E. coli populations in soil may have increased environmental fitness, facilitating their residence in soil (20, 34, 38). Little is known, however, of the physiology of these organisms, and their capacity for survival in soil remains poorly understood (21).Previous work (3) recorded continuous low-level leaching of viable E. coli from lysimeters of a poorly drained Luvic Stagnosol soil type, more than 9 years after the last application of fecal material. This finding was indicative of the growth of E. coli within the soil and suggested the presence of autochthonous E. coli populations within the soil that could be leached subsequently. To our knowledge, prior to this report, naturalized autochthonous E. coli populations persisting under the relatively oligotrophic, low-temperature conditions of maritime temperate soil environments had not been described previously. Growth within this soil was attributed chiefly to favorable characteristics of the soil, which include high clay and moisture contents, nutrient retention, and the presence of anaerobic zones. The objective of this work was to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached. In particular, we were interested in determining if the isolates possessed phenotypic characteristics that may enhance their capacity to survive and occupy niche space within the soil. This study tested the hypothesis that E. coli clones persisting in lysimeters of this soil form a genetically distinct grouping and possess a physiology tailored to the soil environment.     

Lyophilization Prior to Direct DNA Extraction from Bovine Feces Improves the Quantification of Escherichia coli O157:H7 and Campylobacter jejuni

Lyophilization was used to concentrate bovine feces prior to DNA extraction and analysis using real-time PCR. Lyophilization significantly improved the sensitivity of detection compared to that in fresh feces and was associated with reliable quantification of both Escherichia coli O157:H7 and Campylobacter jejuni bacteria present in feces at concentrations ranging between 2 log₁₀ and 6 log₁₀ CFU g⁻¹.Bovines are a reservoir for verotoxigenic Escherichia coli O157:H7 and Campylobacter jejuni, pathogenic microorganisms responsible for severe human gastrointestinal disease (5, 12). Qualitative and quantitative detection of these organisms in bovine feces is essential for evaluating risk to human health. Real-time PCR (quantitative PCR [qPCR]) assays have been developed to detect and quantify both E. coli O157:H7 and C. jejuni bacteria by using DNA directly extracted from animal feces (20, 22). Analysis of DNA extracted from bovine feces can generate a high level of correlation between the actual target cell density and the PCR signal (7, 8). However, the detection of E. coli O157:H7 and C. jejuni by direct DNA extraction is less sensitive and more variable than detection by procedures based on a preliminary enrichment step (e.g., laboratory culture) (7, 9, 16, 20). We explored the potential of lyophilization for improving overall detection by qPCR through increasing the amount of bovine fecal material available for DNA extraction.Four sets of five fresh bovine fecal samples were collected, and each sample was divided into four equal portions. Samples were seeded with either (i) E. coli O157:H7 (strain NZRM 3614) grown for 18 h at 37°C in tryptic soy broth (BD, Sparks, MD) or (ii) C. jejuni (strain NZRM 1958) grown for 48 h at 42°C in Exeter broth (11) to obtain the following concentrations: set 1, 0 CFU g⁻¹ (unseeded) and 3.5 log₁₀, 4.5 log₁₀, and 5.5 log₁₀ CFU of E. coli O157:H7 g⁻¹, and set 2, 0 CFU g⁻¹ (unseeded) to 5.2 log₁₀ CFU of E. coli O157:H7 g⁻¹. Set 3 and 4 concentrations varied from 0 CFU g⁻¹ (unseeded) to 6.4 log₁₀ C. jejuni CFU g⁻¹. DNA was either extracted directly from fresh samples or extracted from samples after lyophilization. Lyophilization involved mixing of prepared fecal samples in phosphate-buffered saline (145 mM NaCl, 59 mM Na₂HPO₄, 8 mM KH₂PO₄, pH 7.5) at a ratio of 1:10 (wt/vol), homogenization with a lab blender model 400 (Seward Medical, London, United Kingdom), cooling to −35°C, and concentration using a 1015GP lyophilizer (Cuddon Ltd., Blenheim, New Zealand). Total DNA was extracted from 0.2 g of a fresh or lyophilized fecal sample by using a QIAamp DNA stool minikit (Qiagen Inc., Mississauga, Canada). DNA was amplified using either a TaqMan E. coli O157:H7 detection kit (Applied Biosystems, Foster City, CA) or mapA primers and a corresponding probe (1). Amplification and fluorescence data were collected with optical-grade 96-well plates by using a TaqMan 7300 PCR system (Applied Biosystems). For each DNA sample, a mean threshold cycle (C_T) value for triplicate qPCR runs was calculated. When no C_T value was obtained, an arbitrary C_T value of 40 was assigned. All data were reported as equivalent concentrations in fresh feces. Significance levels were determined by one-way analysis of variance. The relationship between the log₁₀ numbers of CFU g⁻¹ fresh feces (viable-cell counts) and C_T values was analyzed using GenStat software (version 10.2.0.175; VSN International, Oxford, United Kingdom). Confidence intervals were obtained using the software program Flexi (21).Lyophilized samples were associated with significantly improved sensitivity (P < 0.001) at seeding levels of 4.5 and 5.5 log₁₀ E. coli O157:H7 CFU g⁻¹ (Table ​(Table1).1). At 3.5 log₁₀ CFU g⁻¹, the rate of E. coli O157:H7 detection was also higher, with all lyophilized samples producing a C_T value of <40 (Table ​(Table1).1). Individual C_T values for the three qPCR amplification runs were sufficiently similar to allow averaging (P > 0.05). Regression analysis of the averaged set 2 and 3 data (Fig. ​(Fig.1)1) demonstrated that the detection of both E. coli O157:H7 and C. jejuni was linear for seeding levels ranging from ca. 2 log₁₀ to 6 log₁₀ CFU g⁻¹ fresh feces. The range of concentrations used reflects the reported range of concentrations of these bacteria in feces (i.e., 0 to 6 log₁₀ CFU g⁻¹) as determined by conventional culture (3, 4, 18, 19). The high coefficients of correlation for the relationships between the log₁₀ numbers of CFU g⁻¹ feces and the C_T values indicated the specific amplification of the target DNA. The reproducibility of detection of E. coli O157:H7 was reduced at the lowest seeding concentration (i.e., 2.2 log₁₀ CFU g⁻¹ feces), with 75% of the samples giving a C_T value of <40. The limit for 100% successful detection after lyophilization was 2.9 log₁₀ E. coli O157:H7 CFU g⁻¹. The detection of C. jejuni by qPCR varied between sets. For set 3, 100% reproducibility occurred at 2.2 log₁₀ C. jejuni CFU g⁻¹. For set 4, satisfactory detection was obtained only after dilution of the DNA extract prior to qPCR. Despite this requirement for dilution, C. jejuni was still detected in 80% of the samples of set 4 seeded at a density of 2.2 log₁₀ C. jejuni CFU g⁻¹.Open in a separate windowFIG. 1.Ranges of quantification of E. coli O157:H7 (A) and C. jejuni (B) bacteria obtained from lyophilized fecal samples by real-time PCR. Each point represents the average C_T value for triplicate runs of one fecal sample at one seeding concentration. The hatched areas represent the 95% confidence intervals.

TABLE 1.

Difference in C_T values obtained for real-time PCR detection of E. coli O157:H7 in seeded fecal samples (n = 5) with and without lyophilization

Longus,a Type IV Pilus of Enterotoxigenic Escherichia coli,Is Involved in Adherence to Intestinal Epithelial Cells

Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of traveler''s diarrhea. The main hallmarks of this type of bacteria are the expression of one or more enterotoxins and fimbriae used for attachment to host intestinal cells. Longus is a pilus produced by ETEC. These bacteria grown in pleuropneumonia-like organism (PPLO) broth at 37°C and in 5% CO₂ produced longus, showing that the assembly and expression of the pili depend on growth conditions and composition of the medium. To explore the role of longus in the adherence to epithelial cells, quantitative and qualitative analyses were done, and similar levels of adherence were observed, with values of 111.44 × 10⁴ CFU/ml in HT-29, 101.33 × 10⁴ CFU/ml in Caco-2, and 107.11 × 10⁴ CFU/ml in T84 cells. In addition, the E9034AΔlngA strain showed a significant reduction in longus adherence of 32% in HT-29, 22.28% in Caco-2, and 21.68% in T84 cells compared to the wild-type strain. In experiments performed with nonintestinal cells (HeLa and HEp-2 cells), significant differences were not observed in adherence between E9034A and derivative strains. Interestingly, the E9034A and E9034AΔlngA(pLngA) strains were 30 to 35% more adherent in intestinal cells than in nonintestinal cells. Twitching motility experiments were performed, showing that ETEC strains E9034A and E9034AΔlngA(pLngA) had the capacity to form spreading zones while ETEC E9034AΔlngA does not. In addition, our data suggest that longus from ETEC participates in the colonization of human colonic cells.Enterotoxigenic Escherichia coli (ETEC) is an important cause of infant diarrhea in developing countries, a leading cause of traveler''s diarrhea, and a reemergent diarrheal pathogen in the United States (1, 25, 29, 33, 38, 40, 41, 44, 51, 52, 55). ETEC strains were first recognized as a cause of diarrheal disease in animals, especially in piglets and calves, where the disease continues to cause lethal infection in newborn animals (3, 37). Studies of ETEC in piglets first elucidated the mechanisms of disease, including the presence of two plasmid-encoded enterotoxins. In humans, the clinical appearance of ETEC infection is identical to that of cholera, with severe dehydrating illness not commonly seen in adults (38, 46). DuPont et al. (12) subsequently showed that ETEC strains were able to cause diarrhea in adult volunteers. ETEC strains cause watery diarrhea similar to that caused by Vibrio cholerae through the action of two enterotoxins, the cholera-like heat-labile and heat-stable enterotoxins (LT and ST, respectively) (38). These strains may express an LT only, an ST only, or both LT and ST. To cause diarrhea, ETEC strains must first adhere to small bowel enterocytes, an event mediated by a variety of surface fimbrial appendages called colonization factor antigens (CFAs), coli surface antigens (CSs), and putative colonization factors (PCF) (22, 33, 38). Transmission electron microscopy (TEM) of ETEC strains typically reveals many peritrichously arranged fimbriae around the bacterium; often, multiple fimbrial morphologies can be visualized on the same bacterium (6, 19, 31, 38). ETEC strains also express the K99 fimbriae, which are pathogenic for calves, lambs, and pigs, whereas K88-expressing organisms are able to cause disease only in pigs (8). Human ETEC strains possess their own array of colonization fimbriae, the CFAs usually encoded in plasmids (10). Currently, more than 20 CFAs known in human ETEC infections have been described (17). The CFAs can be subdivided based on their morphological characteristics. Three major morphological varieties exist: rigid rods (CFA I), bundle-forming flexible rods (CFA III), and thin, flexible, wiry structures (CFA II and CFA IV) (7, 8, 26, 30, 49, 53, 54).A high proportion of human ETEC strains contain a plasmid-encoded type IV pilus (T4P) antigen (CS20) also called longus for its length (19, 21). Longus is a T4P composed of a repeating structural subunit called LngA of 22 kDa, and its N-terminal amino acid sequences shares similarities with the class B type IV pili. These pili include the CFA III pilin subunit CofA of ETEC, the toxin-coregulated pilin (TCP) of V. cholerae, and the bundle-forming pilin (BFP) found in enteropathogenic E. coli (EPEC) and in a small percentage in other Gram-negative pathogens (21, 23). The lngA gene, which encodes the longus pilus in ETEC strains, is widely distributed in different geographic regions such Bangladesh, Chile, Brazil, Egypt, and Mexico (23). Interestingly, the lngA gene has been observed in association with ETEC strain producers of LT and ST (23). Sequence analysis of the fimbrial genes provided insight into the evolutionary history of longus. It appears that the highly conserved nonstructural lngA genes evolved in a similar manner to that of housekeeping genes.Recently, another important adherence factor called E. coli common pilus (ECP) has been identified; it is composed of a 21-kDa pilin subunit whose amino acid sequence corresponds to the product of the yagZ (renamed ecpA) gene present in all E. coli genomes sequenced to date (47). ECP production was demonstrated in strains representing intestinal (enterohemorrhagic E. coli [EHEC], EPEC, and ETEC) and extraintestinal pathogenic E. coli as well as normal-flora E. coli.In this study we report that longus plays an important role in the adherence to colonic epithelial cells. In addition to mediating cell adherence, longus is also associated with other pathogenicity attributes exhibited by other Gram-negative pathogenic bacteria producing T4P, which can contribute in part to the virulence of ETEC.     

Transmission and infectious dose of Escherichia coli O157:H7 in swine

Escherichia coli O157:H7 is only occasionally isolated from healthy swine, but some experimentally infected animals will shed the organism in their feces for at least 2 months. Potential explanations for the paucity of naturally occurring infections in swine, as compared to cattle, include a lack of animal-to-animal transmission so that the organism cannot be maintained within a herd, a high infectious dose, or herd management practices that prevent the maintenance of the organism in the gastrointestinal tract. We hypothesized that donor pigs infected with E. coli O157:H7 would transmit the organism to naïve pigs. We also determined the infectious dose and whether housing pigs individually on grated floors would decrease the magnitude or duration of fecal shedding. Infected donor pigs shedding <10⁴ CFU of E. coli O157:H7 per g transmitted the organism to 6 of 12 naïve pigs exposed to them. The infectious dose of E. coli O157:H7 for 3-month-old pigs was approximately 6 × 10³ CFU. There was no difference in the magnitude and duration of fecal shedding by pigs housed individually on grates compared to those housed two per pen on cement floors. These results suggest that swine do not have an innate resistance to colonization by E. coli O157:H7 and that they could serve as a reservoir host under suitable conditions.Escherichia coli O157:H7 and other serotypes of Shiga toxigenic E. coli (STEC) cause an estimated 110,000 cases of human illness yearly in the United States (26). Most cases are thought to occur as a result of the ingestion of contaminated food or water, although direct contacts with animals and person-to-person transmission have also been documented (4). Cattle are considered to be the major reservoir of STEC, and the prevalence of E. coli O157:H7 in the U.S. herd ranges from 2 to 28%, depending on the culture techniques used, the age of the animals, and the season in which samples are collected (10, 12, 15, 17, 29, 33). E. coli O157:H7 has also been recovered from other ruminants such as deer (22, 30) and sheep (24). E. coli O157:H7 has occasionally been isolated from nonruminant animals such as wild birds (32) and raccoons (18), but the bulk of the data suggests that the prevalence of STEC is greater in ruminants than it is in other animals.In the last several years, there have been reports that E. coli O157:H7 has been isolated from healthy swine in Japan, The Netherlands, Sweden, Canada, Norway, and the United States (11, 13, 19, 20, 27; C. L. Gyles, R. Friendship, K. Ziebell, S. Johnson, I. Yong, and R. Amezcua, Proc. 2002 Congr. Int. Pig Vet. Soc., abstr. 191). The prevalence of the organism in these studies is generally low (0.1 to 6%), and no human outbreaks have been specifically traced back to pork, although sausage containing both beef and pork was implicated as the source of human infection in at least one outbreak (28). In Chile, the prevalence of E. coli O157:H7 reported from pigs (10.8%) was greater than that reported from cattle (2.9%), suggesting that swine may be an important source of this organism in some countries (3). Previously, we have shown that some market-weight pigs experimentally infected with E. coli O157:H7 will shed the organism for at least 2 months (2). These animals do not become clinically ill, and the magnitude and duration of fecal shedding of E. coli O157:H7 are reminiscent of those seen in experimentally infected ruminants (6, 7). This suggests that swine have the biological potential to emerge as a reservoir for E. coli O157:H7 and other STEC strains pathogenic for humans. In order for swine to serve as a reservoir host, not only must the organism colonize the gastrointestinal tract of individual animals, it must also be transmitted from colonized animals to naïve animals to be maintained within the herd. In addition, the infectious dose must be of such a magnitude that a natural infection could be perpetuated within the herd. We hypothesized that E. coli O157:H7 would be transmitted from infected donor pigs to naïve pigs at levels that could be sustained in a natural infection. In addition, we determined the infectious dose of in vitro-grown E. coli O157:H7 for 3-month-old pigs and determined whether housing pigs individually on raised decks or in groups on cement floors affected the magnitude and duration of fecal shedding in infected animals.(A preliminary report of this work was presented at the International Symposium on Shiga Toxin-Producing E. coli, Kyoto, Japan, 2000, and Edinburgh, Scotland, 2003.)     

Longitudinal Study of Escherichia coli O157:H7 in a Beef Cattle Feedlot and Role of High-Level Shedders in Hide Contamination

The objectives of the study described here were (i) to investigate the dynamics of Escherichia coli O157:H7 fecal and hide prevalence over a 9-month period in a feedlot setting and (ii) to determine how animals shedding E. coli O157:H7 at high levels affect the prevalence and levels of E. coli O157:H7 on the hides of other animals in the same pen. Cattle (n = 319) were distributed in 10 adjacent pens, and fecal and hide levels of E. coli O157:H7 were monitored. When the fecal pen prevalence exceeded 20%, the hide pen prevalence was usually (25 of 27 pens) greater than 80%. Sixteen of 19 (84.2%) supershedder (>10⁴ CFU/g) pens had a fecal prevalence greater than 20%. Significant associations with hide and high-level hide (≥40 CFU/100 cm²) contamination were identified for (i) a fecal prevalence greater than 20%, (ii) the presence of one or more high-density shedders (≥200 CFU/g) in a pen, and (iii) the presence of one or more supershedders in a pen. The results presented here suggest that the E. coli O157:H7 fecal prevalence should be reduced below 20% and the levels of shedding should be kept below 200 CFU/g to minimize the contamination of cattle hides. Also, large and unpredictable fluctuations within and between pens in both fecal and hide prevalence of E. coli O157:H7 were detected and should be used as a guide when preharvest studies, particularly preharvest intervention studies, are designed.It is now well established that at the time of harvest, hides are the major source of Escherichia coli O157:H7 contamination on beef carcasses (1, 4, 22). Thus, reducing the levels of food-borne pathogens on cattle hides has been the focus of many pre- and postharvest research efforts. For postharvest applications, hide interventions (i.e., washing of hide-on carcasses with various antimicrobial agents) are direct approaches and have been shown to be efficacious for reducing hide and carcass contamination rates (2, 4, 5, 22).In the area of preharvest research, several approaches have been taken to reduce the prevalence of E. coli O157:H7 in feces of cattle presented for slaughter. These approaches include, among others, feeding cattle probiotics (dietary administration of beneficial bacteria to compete with E. coli O157:H7), vaccination, and bacteriophage treatment (8, 24, 30). These intervention approaches are indirect. By reducing the fecal pathogen load, the pathogen prevalence and the level on hides are reduced through lower cross-contamination at the feedlot, and subsequently, carcass contamination rates decrease. While the effectiveness of preharvest interventions varies, no preharvest intervention is 100% effective in reducing the fecal prevalence of E. coli O157:H7. It is not known what level of pathogen reduction in feces would be necessary to significantly reduce hide and carcass contamination during processing. Key pieces of information needed to address this question are the number of shedding cattle in a pen needed to contaminate the hides of most of the cattle in the same pen and at what level the shedding cattle are contaminated.Aside from the number of cattle shedding a pathogen, the concentration of the pathogen in feces plays a pivotal role in spreading the pathogen between animals. Recently, cattle shedding E. coli O157:H7 at levels of >10⁴ CFU/g (“supershedders”) have been associated with high rates of transmission of the pathogen between cohort animals (18, 23). Matthews et al. reported that 20% of the E. coli O157:H7 infections in cattle on Scottish farms were responsible for 80% of the transmission of the organism between animals (18). Another study reported similar findings; 9% of the animals shedding E. coli O157:H7 produced over 96% of the total E. coli O157:H7 fecal load for the group (23). While a number of studies have indicated the importance of supershedders in fecal transmission dynamics, there is a general lack of information concerning the effects of high shedding rates on hide prevalence and load. Accordingly, the objectives of this study were (i) to investigate the dynamics of E. coli O157:H7 prevalence and levels in feces and on hides of feedlot cattle over time and (ii) to determine how pathogen prevalence and levels on hides in a pen are affected by individuals shedding E. coli O157:H7 at high levels.In the analysis presented here, fecal shedding was analyzed using the following three categories based on the level of E. coli O157:H7 being shed: shedding positive (presumed concentration, ≥1 CFU/g), high-density shedder (≥200 CFU/g), and supershedder (≥10⁴ CFU/g). Several definitions of E. coli O157:H7 supershedders have been offered previously. One-time shedding levels of >10³ or >10⁴ CFU/g have been used in multiple studies (17, 23, 24), while other groups have required persistent colonization of the rectoanal junction, as well as high cell counts, for an animal to qualify as a supershedder (10). Recently, Chase-Topping et al. (9) reviewed the requirements for supershedder status and provided a working definition: an animal that excretes >10⁴ CFU/g. In doing this, Chase-Topping et al. noted the high stringency of this definition and acknowledged that with such a definition some supershedders will be missed if they are sampled at times other than peak shedding times (9). In the current study, this was a concern. In an attempt to investigate the link between high-shedding-level animals and hide contamination, greater leeway was needed in the classification. When it is sampled on a monthly basis, an animal shedding at high levels can have a large impact on the hide status of pen cohorts between sampling intervals but not be shedding at peak levels on the day of sample collection. Hence, the categories described above were selected to analyze the relationship between fecal shedding and hide contamination.     

Rapid Microarray-Based Genotyping of Enterohemorrhagic Escherichia coli Serotype O156:H25/H?/Hnt Isolates from Cattle and Clonal Relationship Analysis

Since enterohemorrhagic Escherichia coli (EHEC) isolates of serogroup O156 have been obtained from human diarrhea patients and asymptomatic carriers, we studied cattle as a potential reservoir for these bacteria. E. coli isolates serotyped by agglutination as O156:H25/H−/Hnt strains (n = 32) were isolated from three cattle farms during a period of 21 months and characterized by rapid microarray-based genotyping. The serotyping by agglutination of the O156 isolates was not confirmed in some cases by the results of DNA-based serotyping as only 25 of the 32 isolates were conclusively identified as O156:H25. In the multilocus sequence typing (MLST) analysis, all EHEC O156:H25 isolates were characterized as sequence type 300 (ST300) and ST688, which differ by a single-nucleotide exchange in the purA gene. Oligonucleotide microarrays allow simultaneous detection of a wider range of EHEC-associated and other E. coli virulence markers than other methods. All O156:H25 isolates showed a wide spectrum of virulence factors typical for EHEC. The stx₁ genes combined with the EHEC hlyA (hlyA_EHEC) gene, the eae gene of the ζ subtype, as well as numerous other virulence markers were present in all EHEC O156:H25 strains. The behavior of eight different cluster groups, including four that were EHEC O156:H25, was monitored in space and time. Variations in the O156 cluster groups were detected. The results of the cluster analysis suggest that some O156:H25 strains had the genetic potential for a long persistence in the host and on the farm, while other strains did not. As judged by their pattern of virulence markers, E. coli O156:H25 isolates of bovine origin may represent a considerable risk for human infection. Our results showed that the miniaturized E. coli oligonucleotide arrays are an excellent tool for the rapid detection of a large number of virulence markers.Shiga toxin-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (45). In humans, infections with some STEC serotypes may result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by the hemolytic uremic syndrome (HUS) (32). These STEC strains are also designated enterohemorrhagic Escherichia coli (EHEC). Consequently, EHEC strains represent a subgroup of STEC with high pathogenic potential for humans. Although E. coli O157:H7 is the most frequent EHEC serotype implicated in HUS, other serotypes can also cause this complication. Non-O157:H7 EHEC strains including serotypes O26:H11/H−, O103:H2/H−, O111:H8/H10/H−, and O145:H28/H25/H− and sorbitol-fermenting E. coli O157:H− isolates are present in about 50% of stool cultures from German HUS patients (10, 42). However, STEC strains that cause human infection belong to a large number of E. coli serotypes, although a small number of STEC isolates of serogroup O156 were associated with human disease (7). Strains of the serotypes O156:H1/H8/H21/H25 were found in human cases of diarrhea or asymptomatic infections (9, 22, 25, 26). The detection of STEC of serogroup O156 from healthy and diseased ruminants such as cattle, sheep, and goats was reported by several authors (1, 11-13, 21, 39, 46, 50, 52). Additional EHEC-associated virulence genes such as stx, eae, hlyA_EHEC, or nlaA were found preferentially in the serotypes O156:H25 and O156:H− (11-13, 21, 22, 50, 52).Numerous methods exist for the detection of pathogenic E. coli, including genotypic and phenotypic marker assays for the detection of virulence genes and their products (19, 47, 55, 57). All of these methods have the common drawback of screening a relatively small number of determinants simultaneously. A diagnostic DNA microarray based on the ArrayTube format of CLONDIAG GmbH was developed as a viable alternative due to its ability to screen multiple virulence markers simultaneously (2). Further microarray layouts working with the same principle but different gene targets were developed for the rapid identification of antimicrobial resistance genes in Gram-negative bacteria (5) and for the rapid DNA-based serotyping of E. coli (4). In addition, a protein microarray for E. coli O serotyping based on the ArrayTube format was described by Anjum et al. (3).The aim of our study was the molecular genotyping of bovine E. coli field isolates of serogroup O156 based on miniaturized E. coli oligonucleotide arrays in the ArrayStrip format and to combine the screening of E. coli virulence markers, antimicrobial resistance genes, and DNA serotyping targets, some of which were partially described previously for separate arrays (2, 4, 5). The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O156 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.     

Prevalence and Persistence of Escherichia coli Strains with Uropathogenic Virulence Characteristics in Sewage Treatment Plants

We investigated the prevalence and persistence of Escherichia coli strains in four sewage treatment plants (STPs) in a subtropical region of Queensland, Australia. In all, 264 E. coli strains were typed using a high-resolution biochemical fingerprinting method and grouped into either a single or a common biochemical phenotype (S-BPT and C-BPT, respectively). These strains were also tested for their phylogenetic groups and 12 virulence genes associated with intestinal and extraintestinal E. coli strains. Comparison of BPTs at various treatment stages indicated that certain BPTs were found in two or all treatment stages. These BPTs constituted the highest proportion of E. coli strains in each STP and belonged mainly to phylogenetic group B2 and, to a lesser extent, group D. No virulence genes associated with intestinal E. coli were found among the strains, but 157 (59.5%) strains belonging to 14 C-BPTs carried one or more virulence genes associated with uropathogenic strains. Of these, 120 (76.4%) strains belonged to seven persistent C-BPTs and were found in all four STPs. Our results indicate that certain clonal groups of E. coli with virulence characteristics of uropathogenic strains can survive the treatment processes of STPs. These strains were common to all STPs and constituted the highest proportion of the strains in different treatment tanks of each STP.Community sewage treatment plants (STPs) receive waste from diverse sources, including residential, industrial, and recreational facilities (31). Waste generated from these facilities contains the liquid and fecal discharges of humans and animals, household wastes, industry-specific materials, and storm water runoff (31). These materials are treated through primary, secondary, and tertiary sedimentation processes (18). Following these processes, effluent is normally clear and thus often recycled for nonpotable use (20), with excess water released into receiving waterways. However, due to possible malfunctions or poor management of wastewater systems (1), effluent containing pathogenic bacteria can be discharged into receiving waterways (11, 34). It has been speculated that waters contaminated with feces are a great risk to human health, as they are likely to contain human-specific enteric pathogens, including Salmonella spp. (30), Shigella spp. (10), enteroviruses (12), hepatitis A virus (13), and pathogenic Escherichia coli (30).E. coli, while widely used as an indicator bacterium (30, 35), can actually be pathogenic and be responsible for both intestinal and extraintestinal diseases (16). Intestinal pathogenic strains of E. coli are rarely encountered in the fecal flora of healthy hosts. Extraintestinal pathogenic E. coli (ExPEC) strains commonly cause infections of any organ or anatomical site (28). The ability of these pathogenic bacteria to cause disease is due to their acquisition of specialized virulence factors, which commensal E. coli strains typically lack. These specialized virulence factors allow them to cause a broad spectrum of diseases (17, 28), such as gastroenteritis (34), diarrhea (16), urinary tract infections and meningitis (29), and soft tissue infections and bacteremia (28). E. coli strains belong to four main phylogenetic groups (A, B1, B2, and D) (2), with pathogenic strains belonging mostly to phylogenetic group B2 and, to a lesser extent, group D. Another phylogenetic group (group E) has also been identified; however, it is uncommon and is not widely used (5).Presently, chlorination is an extremely widespread practice aimed at reducing the pathogen load in the final effluent to levels low enough to ensure that the organisms will not cause disease when the wastewater is discharged (31). Despite this, some pathogenic strains of E. coli may survive to become a significant public health risk (14, 35). The aim of this study was to investigate the presence and survival of these pathogenic E. coli strains during the treatment processes of four community STPs with different capacities in South East Queensland, Australia.     

Escherichia coli O157:H7 Strain Origin,Lineage, and Shiga Toxin 2 Expression Affect Colonization of Cattle

Enterohemorrhagic Escherichia coli O157:H7 has evolved into an important human pathogen with cattle as the main reservoir. The recent discovery of E. coli O157:H7-induced pathologies in challenged cattle has suggested that previously discounted bacterial virulence factors may contribute to the colonization of cattle. The objective of the present study was to examine the impact of lineage type, cytotoxin activity, and cytotoxin expression on the amount of E. coli O157:H7 colonization of cattle tissue and cells in vitro. Using selected bovine- and human-origin strains, we determined that lineage type predicted the amount of E. coli O157:H7 strain colonization: lineage I > intermediate lineages > lineage II. All E. coli O157:H7 strain colonization was dose dependent, with threshold colonization at 10³ to 10⁵ CFU and maximum colonization at 10⁷ CFU. We also determined that an as-yet-unknown factor of strain origin was the most dominant predictor of the amount of strain colonization in vitro. The amount of E. coli O157:H7 colonization was also influenced by strain cytotoxin activity and the inclusion of cytotoxins from lineage I or intermediate lineage strains increased colonization of a lineage II strain. There was a higher level of expression of the Shiga toxin 1 gene (stx₁) in human-origin strains than in bovine-origin strains. In addition, lineage I strains expressed higher levels of the Shiga toxin 2 gene (stx₂). The present study supports a role for strain origin, lineage type, cytotoxin activity, and stx₂ expression in modulating the amount of E. coli O157:H7 colonization of cattle.Enterohemorrhagic Escherichia coli O157:H7 is a bacterium that causes serious human disease outbreaks through the consumption of contaminated food or water (39). Mature cattle are considered the primary reservoir for E. coli O157:H7 and historically were reported to have no symptoms or pathologies (17, 23, 38); this was attributed both to a lack of receptors for a critical E. coli O157:H7 virulence factor, Shiga toxin 1 (Stx1 [29]), and to a differential expression of type III protein secretion system effector molecules such as EspA, EspD, and Iha (25, 30) in cattle compared to humans. In 2008, it was established for the first time that E. coli O157:H7 causes mild to severe intestinal pathology in persistent shedding cattle (5, 26) and that the secreted cytotoxins enhanced E. coli O157:H7 colonization of intestinal tissues of cattle (6). This suggested that cattle were susceptible to E. coli O157:H7 infection and that previously discounted virulence factors could influence the amount of colonization in cattle.Three distinct E. coli O157:H7 lineages have been identified based on the lineage specific polymorphism assay (LSPA-6) that suggests both the evolutionary history of the strain and their propensity to be present among animals, the environment, and clinical human isolates (21, 22, 24, 33, 40, 42). Typically, two predominant lineages have been described, lineages I and II (22, 40) and, more recently, intermediate lineages that have characteristics of lineage I and/or II have been reported at higher frequency among cattle (34). Although all E. coli O157:H7 lineages have been isolated from feedlot cattle, the predominant recovery of lineage I from clinical human illnesses suggests that this particular lineage type has unique expression patterns that may contribute to its preferential colonization of humans. There is some evidence to suggest that lineage I strains do not express certain virulence factors in bovine hosts, whereas other factors such as cytotoxins are expressed equally irrespective of host (30). One virulence factor associated with all lineages is the bacterium''s ability to form intimate attaching-and-effacing lesions or colonization sites in the ilea of susceptible animals (28). The amount of colonization is enhanced by the expression of Shiga toxin 2 (Stx2) through both an increase in the expression of alternative non-TIR (translocated intimin receptor) colonization sites (31) and toxicity to the absorptive epithelial cells (32). In cattle, attaching-and-effacing lesions are also formed (5), and Stx2 increases colonization but is not cytotoxic to epithelial cells from the jejuna and descending colons of cattle (4). Differential expression of stx₂ among E. coli O157:H7 lineages is also linked to the increased pathogenicity of lineage I strains in humans (25), and this may affect cattle similarly. Together, this information suggests that at least some similar virulence factors affecting E. coli O157:H7 colonization in humans also function in cattle.In order to gain a better understanding of the factors modulating E. coli O157:H7 colonization in cattle, we compared the ability of lineage I, lineage II, and intermediate lineages isolated from human sources to colonize the jejunum tissue and a colonic cell line from cattle. We hypothesized that the bovine colonic cell line could be used as a model system to reflect E. coli O157:H7 colonization of tissue. To confirm the value of this model, the role of strain origin in colonization of cattle was examined. In order to understand the differences in colonization associated with lineage and strain origins, we assessed cytotoxin expression, secreted cytotoxin activity, and cytotoxin-induced changes in E. coli O157:H7 colonization. Given the known lack of Stx1 activity in cattle, we examined the effects of LSPA-6 genotype, strain origin (human versus bovine), and cytotoxin activity on E. coli O157:H7 colonization of cattle.     

The Pentapeptide Repeat Proteins MfpAMt and QnrB4 Exhibit Opposite Effects on DNA Gyrase Catalytic Reactions and on the Ternary Gyrase-DNA-Quinolone Complex

MfpA_Mt and QnrB4 are two newly characterized pentapeptide repeat proteins (PRPs) that interact with DNA gyrase. The mfpA_Mt gene is chromosome borne in Mycobacterium tuberculosis, while qnrB4 is plasmid borne in enterobacteria. We expressed and purified the two PRPs and compared their effects on DNA gyrase, taking into account host specificity, i.e., the effect of MfpA_Mt on M. tuberculosis gyrase and the effect of QnrB4 on Escherichia coli gyrase. Whereas QnrB4 inhibited E. coli gyrase activity only at concentrations higher than 30 μM, MfpA_Mt inhibited all catalytic reactions of the M. tuberculosis gyrase described for this enzyme (supercoiling, cleavage, relaxation, and decatenation) with a 50% inhibitory concentration of 2 μM. We showed that the D87 residue in GyrA has a major role in the MfpA_Mt-gyrase interaction, as D87H and D87G substitutions abolished MfpA_Mt inhibition of M. tuberculosis gyrase catalytic reactions, while A83S modification did not. Since MfpA_Mt and QnrB4 have been involved in resistance to fluoroquinolones, we measured the inhibition of the quinolone effect in the presence of each PRP. QnrB4 reversed quinolone inhibition of E. coli gyrase at 0.1 μM as described for other Qnr proteins, but MfpA_Mt did not modify M. tuberculosis gyrase inhibition by fluoroquinolones. Crossover experiments showed that MfpA_Mt also inhibited E. coli gyrase function, while QnrB4 did not reverse quinolone inhibition of M. tuberculosis gyrase. In conclusion, our in vitro experiments showed that MfpA_Mt and QnrB4 exhibit opposite effects on DNA gyrase and that these effects are protein and species specific.The pentapeptide repeat protein (PRP) family includes more than 500 proteins in the prokaryotic and eukaryotic kingdoms (45). PRPs are characterized by the repetition of the pentapeptide repeat motif [S,T,A,V][D,N][L,F][S,T,R][G] (6), which results in a right-handed β-helical structure (8, 17). The functions of the majority of the members of this large and heterogeneous family remain unknown, but three PRPs, McbG (from Escherichia coli), MfpA_Mt (from Mycobacterium tuberculosis), and Qnr (from Klebsiella pneumoniae and other enterobacteria) were reported to interact with DNA gyrase, at least with the E. coli enzyme (17, 33, 35, 44). McbG was shown to protect E. coli DNA gyrase from the toxic action of microcin B17 (33). Qnr and MfpA_Mt were involved in resistance to fluoroquinolones, which are synthetic antibacterial agents prescribed worldwide for the treatment of various infectious diseases, including tuberculosis (7).DNA gyrase is an essential ATP-dependent enzyme that transiently cleaves a segment of double-stranded DNA, passes another piece of DNA through the break, and reseals it (12). DNA gyrase is unique in catalyzing the negative supercoiling of DNA in order to facilitate the progression of RNA polymerase. Most eubacteria, such as E. coli, have two type II DNA topoisomerases, i.e., DNA gyrase and topoisomerase IV, but a few, such as M. tuberculosis, harbor only DNA gyrase (11).Quinolones target type II topoisomerases, and their activity is measured by the inhibition of supercoiling by gyrase or decatenation by topoisomerase IV and stabilization of complexes composed of topoisomerase covalently linked to cleaved DNA (16). The DNA gyrase active enzyme is a GyrA₂GyrB₂ heterotetramer. The quinolone-gyrase interaction site in gyrase is thought to be located at the so-called quinolone resistance-determining regions (QRDR) in the A subunit (amino acids 57 to 196 in GyrA) and the B subunit (amino acids 426 to 466 in GyrB), which contain the majority of mutations conferring quinolone resistance (19). The GyrB QRDR is thought to interact with the GyrA QRDR to form a drug-binding pocket (18). Resistance to quinolones is usually due to chromosomal mutations either in the structural genes encoding type II topoisomerases (QRDR) (19, 22) or in regulatory genes producing decreased cell wall permeability or enhancement of efflux pumps (36). The recent emergence of plasmid-borne resistance genes, such as qnr (9, 13, 31, 38, 46), aac(6′)-Ib-cr (32, 39) and qepA (34, 47), renewed interest in quinolone resistance, and especially interest in the new Qnr-based mechanism. Three qnr determinants have been identified so far: qnrA (variants A1 to A6), qnrB (variants B1 to B19), and qnrS (variants S1 and S2) (15, 21, 23, 27). Qnr confers a new mechanism of quinolone resistance by mediating DNA gyrase protection (42): in vitro, QnrA1 and QnrB1 protect E. coli DNA gyrase and topoisomerase IV from the inhibitory effect of fluoroquinolones in a concentration-dependent manner (23, 42-44). Although Qnr was shown to bind GyrA and GyrB and compete with DNA binding, the consequences of Qnr binding for enzyme performance are not yet clear.mfpA, a chromosomal gene that encodes a 192-amino-acid PRP, is an intrinsic quinolone resistance determinant of Mycobacterium smegmatis (29). A similar gene, mfpA_Mt, was found in the M. tuberculosis genome, and MfpA_Mt shows 67% identity with MfpA. Recent crystallography analysis of MfpA_Mt showed that its atomic structure displays size, shape, and electrostatic similarity to B-form DNA, and MfpA_Mt has been suggested to interact with DNA gyrase via DNA mimicry (17). The effect of MfpA_Mt was studied by testing E. coli DNA gyrase, and MfpA_Mt showed catalytic inhibition (17, 37), but whether it protects gyrase from quinolones was not assessed. Because the structure and functions of the M. tuberculosis gyrase, as well as its interaction with quinolones, differ from those of the E. coli gyrase (2, 3, 20, 26, 28), we suspected that the PRP-topoisomerase interaction exhibits species specificity, i.e., depends on the proteins issued from the same host.Our objective was to compare the effects of MfpA_Mt and Qnr on their respective targets, i.e., the effect of MfpA_Mt on the M. tuberculosis gyrase and the effect of Qnr on the E. coli gyrase, by assessing (i) the catalytic reactions of the enzyme and (ii) the interaction with the DNA gyrase-DNA-fluoroquinolone ternary complex. Among the Qnr proteins, we selected the QnrB4 protein, which is a frequent variant of QnrB and has not yet been purified and studied. We cloned, expressed, and purified the two PRPs, MfpA_Mt and QnrB4, as recombinant His tag fusion proteins and assessed their functions under the same experimental conditions.     

Genomic Regions Conserved in Lineage II Escherichia coli O157:H7 Strains

Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (<6%) of the 17 lineage I strains. Several of these linage II-related fragments contain insertions/deletions that may play an important role in virulence. These lineage II-related loci were also shown to be useful markers for genotyping of E. coli O157:H7 strains isolated from human and animal sources.Enterohemorrhagic Escherichia coli is associated with diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans (31). E. coli serotype O157:H7 predominates in epidemics and sporadic cases of enterohemorrhagic E. coli-related infections in the United States, Canada, Japan, and the United Kingdom (12). Cattle are considered the most important reservoir of E. coli O157:H7 (10, 24, 37, 41), and foods contaminated with bovine feces are thought to be the most common source of human infection with this pathogen (27, 33). The two most important virulence factors of the organism are the production of one or more Shiga toxins (Stx) (6, 20, 32) and the ability to attach to and efface microvilli of host intestinal cells (AE). Stx genes are encoded by temperate bacteriophage inserted in the bacterial chromosome, and genes responsible for the AE phenotype are located on the locus of enterocyte effacement (LEE) as well as other pathogenicity islands (4, 17). All E. coli O157:H7 strains also possess a large plasmid which is thought to play a role in virulence (10, 40, 42).Octamer-based genome scanning (OBGS) was first used to show that E. coli O157 strains from the United States and Australia could be subdivided into two genetically distinct lineages (21, 22, 46). While both E. coli O157:H7 lineages are associated with human disease and are isolated from cattle, there is a bias in the host distribution between the two lineages, with a significantly higher proportion of lineage I strains isolated from humans than lineage II strains. Several recent studies have shown that there are inherent differences in gene content and expression between populations of lineage I and lineage II E. coli O157:H7 strains. Lejeune et al. (26) reported that the antiterminator Q gene of the stx₂-converting bacteriophage 933W was found in all nine OBGS lineage I strains examined but in only two of seven lineage II strains, suggesting that there may be lineage-specific differences in toxin production. Dowd and Ishizaki (9) used DNA microarray analysis to examine expression of 610 E. coli O157:H7 genes and showed that lineage I and lineage II E. coli O157:H7 strains have evolved distinct patterns of gene expression which may alter their virulence and their ability to survive in different microenvironments and colonize the intestines of different hosts (9, 28, 38).The observations of lineage host bias have been supported and extended by studies using a six-locus-based multiplex PCR termed the lineage-specific polymorphism assay (LSPA-6) (46). However, Ziebell et al. (48) have recently shown that not all LSPA-6 types within lineage II are host biased; e.g., LSPA-6 type 211111 isolation rates from humans and cattle were significantly different from those of other lineage II LSPA-6 types. Therefore, a clearer definition is required of not only the differences between lineages but also the differences among clonal groups within lineages.The genome sequences of two E. coli O157:H7 strains, Sakai and EDL933 (14, 36), have been determined; however, both of these strains are of lineage I, and there are presently no completed and fully annotated genome sequences available for lineage II strains. In our laboratory, comparative studies utilizing suppression subtractive hybridization (SSH) and comparative genomic hybridization revealed numerous potential virulence factors that are conserved in lineage I strains and that are rare or absent in lineage II strains (42, 47). In this study, we have used SSH to identify genomic regions present in E. coli O157:H7 lineage II strains that are absent from lineage I strains. We wished to examine the distribution of these novel gene segments in E. coli O157:H7 strains and gain insight into their origins and functions. We also attempted to identify molecular markers specific to lineage II strains as well as other markers that would be useful in the genetic subtyping or molecular fingerprinting of E. coli O157:H7 strains in population and epidemiological studies (25). This information may be helpful in the identification of genotypes of the organism associated with specific phenotypes of both lesser and greater virulence (29).