Identification of protozoa in dairy lagoon wastewater that consume Escherichia coli O157:H7 preferentially

Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and is released in feces at numbers as high as 10 million cells/gram. EcO157 could survive in manure for as long as 21 months, but we observed a 90% decrease in cells of an outbreak strain of EcO157 within half a day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2×10(7) cells of EcO157/mL. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 10(4)/mL. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity.     

Incidence and tracking of Escherichia coli O157:H7 in a major produce production region in California

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.     

Role of Extracellular Structures of Escherichia coli O157:H7 in Initial Attachment to Biotic and Abiotic Surfaces

Infection by human pathogens through the consumption of fresh, minimally processed produce and solid plant-derived foods is a major concern of the U.S. and global food industries and of public health services. Enterohemorrhagic Escherichia coli O157:H7 is a frequent and potent foodborne pathogen that causes severe disease in humans. Biofilms formed by E. coli O157:H7 facilitate cross-contamination by sheltering pathogens and protecting them from cleaning and sanitation operations. The objective of this research was to determine the role that several surface structures of E. coli O157:H7 play in adherence to biotic and abiotic surfaces. A set of isogenic deletion mutants lacking major surface structures was generated. The mutant strains were inoculated onto fresh spinach and glass surfaces, and their capability to adhere was assessed by adherence assays and fluorescence microscopy methods. Our results showed that filament-deficient mutants bound to the spinach leaves and glass surfaces less strongly than the wild-type strain did. We mimicked the switch to the external environment—during which bacteria leave the host organism and adapt to lower ambient temperatures of cultivation or food processing—by decreasing the temperature from 37°C to 25°C and 4°C. We concluded that flagella and some other cell surface proteins are important factors in the process of initial attachment and in the establishment of biofilms. A better understanding of the specific roles of these structures in early stages of biofilm formation can help to prevent cross-contaminations and foodborne disease outbreaks.     

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

Background

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

Results

We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.

Conclusions

Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.     

Altered Protozoan and Bacterial Communities and Survival of Escherichia coli O157:H7 in Monensin-Treated Wastewater from a Dairy Lagoon

Surviving predation is a fitness trait of Escherichia coli O157:H7 (EcO157) that provides ample time for the pathogen to be transported from reservoirs (e.g. dairies and feedlots) to farm produce grown in proximity. Ionophore dietary supplements that inhibit rumen protozoa may provide such a selective advantage for EcO157 to proliferate in lagoons as the pathogen is released along with the undigested supplement as manure washings. This study evaluated the fate of an outbreak strain of EcO157, protozoan and bacterial communities in wastewater treated with monensin. Although total protozoa and native bacteria were unaffected by monensin, the time for 90% decrease in EcO157 increased from 0.8 to 5.1 days. 18S and 16S rRNA gene sequencing of wastewater samples revealed that monensin eliminated almost all colpodean and oligohymenophorean ciliates, probably facilitating the extended survival of EcO157. Total protozoan numbers remained high in treated wastewater as monensin enriched 94% of protozoan sequences undetected with untreated wastewater. Monensin stimulated 30-fold increases in Cyrtohymena citrina, a spirotrichean ciliate, and also biflagellate bicosoecids and cercozoans. Sequences of gram-negative Proteobacteria increased from 1% to 46% with monensin, but gram-positive Firmicutes decreased from 93% to 46%. It is noteworthy that EcO157 numbers increased significantly (P<0.01) in Sonneborn medium containing monensin, probably due to monensin-inhibited growth of Vorticella microstoma (P<0.05), a ciliate isolated from wastewater. We conclude that dietary monensin inhibits ciliate protozoa that feed on EcO157. Feed supplements or other methods that enrich these protozoa in cattle manure could be a novel strategy to control the environmental dissemination of EcO157 from dairies to produce production environments.     

Microbes in beach sands: integrating environment,ecology and public health

Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.     

Strain Differences in Fitness of Escherichia coli O157:H7 to Resist Protozoan Predation and Survival in Soil

Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C⁺) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C⁻ cells remained after 7 days. The C⁺ phenotype correlated negatively with D-values of EcO157 exposed to soil (r _s = −0.683; P = 0.036), Vorticella (r _s = −0.465; P = 0.05) or Colpoda (r _s = −0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C⁺ phenotype (Vorticella, r _s = 0.730, P = 0.0004; Colpoda, r _s = 0.625, P = 0.006) suggesting a preference for consumption of C⁺ cells, although they grew on C⁻ strains also. We speculate that the C⁻ phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments.     

Interactions among endophytic bacteria and fungi: Effects and potentials

Plants benefit extensively by harbouring endophytic microbes. They promote plant growth and confer enhanced resistance to various pathogens. However, the way the interactions among endophytes influence the plant productivity has not been explained. Present study experimentally showed that endophytes isolated from rice (Oryza sativa) used as the test plant produced two types of interactions; biofilms (bacteria attached to mycelia) and mixed cultures with no such attachments. Acidity, as measured by pH in cultures with biofilms was higher than that of fungi alone, bacteria alone or the mixed cultures. Production of indoleacetic acid like substances (IAAS) of biofilms was higher than that of mixed cultures, fungi or bacteria. Bacteria and fungi produced higher quantities of IAAS than mixed cultures. In mixed cultures, the potential of IAAS production of resident microbes was reduced considerably. There was a negative relationship between IAAS and pH of the biofilms, indicating that IAAS was the main contributor to the acidity. However, such a relationship was not observed in mixed cultures. Microbial acid production is important for suppressing plant pathogens. Thus the biofilm formation in endophytic environment seems to be very important for healthy and improved plant growth. However, it is unlikely that an interaction among endophytes takes place naturally in the endophytic environment, due to physical barriers of plant tissues. Further, critical cell density dependant quorum sensing that leads to biofilm formation may not occur in the endophytic environment as there is a limited space. As suchin vitro production and application of beneficial biofilmed inocula of endophytes are important for improved plant production in any agro-ecosystem. The conventional practice of plant inoculation with monocultures or mixed cultures of effective microbes may not give the highest microbial effect, which may only be achieved by biofilm formation.     

Application of antimicrobial ice for reduction of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Listeria monocytogenes) on the surface of fish

AIMS: The efficacy of antimicrobial ice was evaluated for the reduction of foodborne pathogens on the surface of fish. METHODS AND RESULTS: Antimicrobial ice containing chlorine dioxide (ClO2) was utilized to control foodborne pathogens in laboratory media and on fish skin. Escherichia coli O157:H7, Salmonella serotype Typhimurium and Listeria monocytogenes strains were treated with antimicrobial ice for 30 min on plates of selective agar and for 120 min on fish skin at room temperature, and then incubated for enumeration. After treatment with 100 ppm ClO2 for 30 min, 5.4, 4.4 and 3.2 log10 reduction was obtained with E. coli O157:H7, Salm. Typhimurium and L. monocytogenes on laboratory media, respectively. When antimicrobial ice (100 ppm ClO2) was applied to fish skin for 120 min, total reduction of E. coli O157:H7, Salm. Typhimurium and L. monocytogenes was 4.8, 2.6 and 3.3 log10, respectively. CONCLUSION: The initial load of foodborne pathogens was reduced by antimicrobial ice and the lowered microbial level was maintained during treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of antimicrobial ice is a simple and effective method for the safe preservation of fish.     

Development of membrane filter holder (MFH) method for recovery of heat-injured Escherichia coli O157:H7 and Salmonella typhimurium

AIMS: A method of recovering sublethally heat-injured bacteria was developed with specific apparatus (membrane filter holder; MFH) which was originally used for Iso-Grid Hydrophobic membrane. filter holder. METHODS AND RESULTS: The procedure used a non-selective agar underlayed with a selective medium with a MFH. A non-selective agar was poured on upper part (compartment A) of MFH, and then injured foodborne pathogens were inoculated on the non-selective medium. After 3-h repair incubation period, selective agar was added to the bottom of the chamber (compartment B) of the MFH and further incubated. By diffusing through the non-selective top agar, selective agents from the underlay medium impart selectivity to the system. CONCLUSIONS: Using the MFH method, recovery of heat-injured foodborne pathogens (Escherichia coli O157:H7 and Salmonella typhimurium) were not different (P > 0.05) from recoveries with non-selective media (TSA). However, the recoveries of foodborne pathogens on MFH were significantly higher (P < 0.05) than those of direct plating on selective medium such as SMAC (MacConkey Sorbitol Agar) or XLD (Xylose Lysine Desoxycholate). SIGNIFICANCE AND IMPACT OF THE STUDY: In conclusion, the MFH method is a simple and convenient method for recovery of heat-injured foodborne pathogens.     

Influence of Cr3+ on microbial cluster formation in biofilm and on steel corrosion

Pit corrosion of mild steel in seawater increased with Cr³⁺ concentration. SEM observations showed that increasing Cr³⁺ concentration caused microbes in biofilm on the steel surface to aggregate forming clusters. AFM images suggested that pit corrosion occurred largely on the mild steel surface between clusters, and only little corrosion on the surface covered by microbes.     

A comparative heat inactivation study of indigenous microflora in beef with that of Listeria monocytogenes,Salmonella serotypes and Escherichia coli O157:H7

AIMS: Thermal inactivation of a mixture of five strains of Listeria monocytogenes, four strains of Escherichia coli O157:H7 and eight serotypes of Salmonella were compared with that of indigenous microflora in 75% lean ground beef. METHODS AND RESULTS: Inoculated meat was packaged in bags that were completely immersed in a circulating water bath and held at 55, 57.5 and 60 degrees C for predetermined lengths of time. The surviving cell population was enumerated by spiral plating heat-treated samples onto tryptic soya agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. D-values, determined by linear regression, in beef were 77.49, 21.9, and 10.66 min at 55, 57.5, and 60 degrees C, respectively, for indigenous microflora (z = 5.81 degrees C). When either of the three pathogens were heated in beef, their D-values calculated were significantly lower (P < 0.05) than those of indigenous microflora at all temperatures. The slope of the thermal death time curve for L. monocytogenes, E. coli O157:H7 and indigenous microflora were similar. Using a survival model for nonlinear survival curves, the D1-values at all temperatures for L. monocytogenes were significantly higher (P < 0.05) compared with those for Salmonella serotypes, E. coli O157:H7 or indigenous microflora. However, higher recovery of a subpopulation of the indigenous microflora in beef exposed to heating at 55, 57.5 or 60 degrees C resulted in significantly higher (P < 0.05) D2-values at all three temperatures, compared with those of the three pathogens at the same test temperatures. CONCLUSIONS: If the thermal process is designed to ensure destruction of indigenous microbial flora, it should also provide an adequate degree of protection against L. monocytogenes, Salmonella serotypes or E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study will assist the retail food industry in designing acceptance limits on critical control points that ensure safety, without introducing pathogens in a retail food environment, against L. monocytogenes, E. coli O157:H7 and Salmonella in cooked ground beef.     

Regional Variation in the Prevalence of E. coli O157 in Cattle: A Meta-Analysis and Meta-Regression

Background

Escherichia coli O157 (EcO157) infection has been recognized as an important global public health concern. But information on the prevalence of EcO157 in cattle at the global and at the wider geographical levels is limited, if not absent. This is the first meta-analysis to investigate the point prevalence of EcO157 in cattle at the global level and to explore the factors contributing to variation in prevalence estimates.

Methods

Seven electronic databases- CAB Abstracts, PubMed, Biosis Citation Index, Medline, Web of Knowledge, Scirus and Scopus were searched for relevant publications from 1980 to 2012. A random effect meta-analysis model was used to produce the pooled estimates. The potential sources of between study heterogeneity were identified using meta-regression.

Principal findings

A total of 140 studies consisting 220,427 cattle were included in the meta-analysis. The prevalence estimate of EcO157 in cattle at the global level was 5.68% (95% CI, 5.16–6.20). The random effects pooled prevalence estimates in Africa, Northern America, Oceania, Europe, Asia and Latin America-Caribbean were 31.20% (95% CI, 12.35–50.04), 7.35% (95% CI, 6.44–8.26), 6.85% (95% CI, 2.41–11.29), 5.15% (95% CI, 4.21–6.09), 4.69% (95% CI, 3.05–6.33) and 1.65% (95% CI, 0.77–2.53), respectively. Between studies heterogeneity was evidenced in most regions. World region (p<0.001), type of cattle (p<0.001) and to some extent, specimens (p = 0.074) as well as method of pre-enrichment (p = 0.110), were identified as factors for variation in the prevalence estimates of EcO157 in cattle.

Conclusion

The prevalence of the organism seems to be higher in the African and Northern American regions. The important factors that might have influence in the estimates of EcO157 are type of cattle and kind of screening specimen. Their roles need to be determined and they should be properly handled in any survey to estimate the true prevalence of EcO157.     

Effects of organic matter on acidic electrolysed water for reduction of foodborne pathogens on lettuce and spinach

Aims: To evaluate the efficacy of acidic electrolysed water (EW) in the presence of organic matter (bovine serum) on the inoculated surfaces of lettuce and spinach. Materials and Results: Lettuce and spinach leaves were inoculated with a cocktail of three strains each of Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes and treated with deionized water, acidic EW and acidic EW containing bovine serum (5, 10, 15 and 20 ml l^?1) for 15 s, 30 s, 1 min, 3 min and 5 min at room temperature (22 ± 2°C). In the absence of bovine serum, acidic EW treatment reduced levels of cells below the detection limit (0·7 log) in 5 min. In the presence of bovine serum, bactericidal activity of acidic EW decreased with increasing serum concentration. Conclusions: Organic matter reduces the effectiveness of acidic EW for reducing pathogens on the surfaces of lettuce and spinach. Significance and Impact of the Study: From a practical standpoint, organic matter reduces the efficacy of acidic EW. This study was conducted to confirm the effect of organic matter on the properties of acidic EW in the inactivation of foodborne pathogens on the surface of vegetables.     

A comparative study of biofilm formation by Shiga toxigenic Escherichia coli using epifluorescence microscopy on stainless steel and a microtitre plate method

Attachment of Shiga toxigenic Escherichia coli (STEC) to surfaces and the formation of biofilms may enhance persistence in a food processing environment and present a risk of contaminating products. Seven strains of STEC and three non-STEC strains were selected to compare two biofilm quantification methods; epifluorescence microscopy on stainless steel (SS) and a microtitre plate assay. The influence of prior growth in planktonic (nutrient broth) and sessile (nutrient agar) culture on biofilm production, as well as expression of surface structures and the possession of antigen 43 (encoded by agn43) on biofilm formation were also investigated. Biofilms were produced in diluted nutrient broth at 25 degrees C for 24 and 48 h. Curli expression was determined using congo red indicator agar, while the presence of agn43 was determined using polymerase chain reaction. No correlation was found between counts for epifluorescence microscopy on SS and the absorbance values obtained with the microtitre plate method for planktonic and sessile grown cultures. Different abilities of individual STEC strains to attach to SS and microtitre plates were found with some strains attaching better to each surface following growth in either planktonic or sessile culture. All O157 STEC strains had low biofilm counts on SS for planktonic and sessile grown cultures; however, one STEC O157:H- strain (EC516) had significantly greater (p<0.05) biofilm production on microtitre plates compared to the other O157 STEC strains. EC516 and other STEC (O174:H21 and O91:H21) strains expressing curli fimbriae were found to produce significantly greater (p<0.05) biofilms on microtitre plates compared to the non-curli expressing strains. No relationship was found between the production of type-I fimbriae, motility, agn43 and bacterial physicochemical properties (previously determined) and biofilm formation on SS or microtitre plates. Variations between the two biofilm determination methods may suggest that the biofilm production on microtitre plates may not be appropriate to represent other surfaces such as SS and that caution should be taken when selecting a method to quantify biofilm production on a surface.     

Biofilm formation by enteric pathogens and its role in plant colonization and persistence

The significant increase in foodborne outbreaks caused by contaminated fresh produce, such as alfalfa sprouts, lettuce, melons, tomatoes and spinach, during the last 30 years stimulated investigation of the mechanisms of persistence of human pathogens on plants. Emerging evidence suggests that Salmonella enterica and Escherichia coli, which cause the vast majority of fresh produce outbreaks, are able to adhere to and to form biofilms on plants leading to persistence and resistance to disinfection treatments, which subsequently can cause human infections and major outbreaks. In this review, we present the current knowledge about host, bacterial and environmental factors that affect the attachment to plant tissue and the process of biofilm formation by S. enterica and E. coli, and discuss how biofilm formation assists in persistence of pathogens on the plants. Mechanisms used by S. enterica and E. coli to adhere and persist on abiotic surfaces and mammalian cells are partially similar and also used by plant pathogens and symbionts. For example, amyloid curli fimbriae, part of the extracellular matrix of biofilms, frequently contribute to adherence and are upregulated upon adherence and colonization of plant material. Also the major exopolysaccharide of the biofilm matrix, cellulose, is an adherence factor not only of S. enterica and E. coli, but also of plant symbionts and pathogens. Plants, on the other hand, respond to colonization by enteric pathogens with a variety of defence mechanisms, some of which can effectively inhibit biofilm formation. Consequently, plant compounds might be investigated for promising novel antibiofilm strategies.     

Effects of Ractopamine HCl on Escherichia coli O157:H7 and Salmonella In Vitro and on Intestinal Populations and Fecal Shedding in Experimentally Infected Sheep and Pigs

The effects of the β-agonist ractopamine, approved for use in finishing swine and cattle to improve carcass quality and performance, were examined on two important foodborne pathogens, Escherichia coli O157:H7 and Salmonella. Ractopamine, administered to sheep before and after oral inoculation with E. coli O157:H7, increased (P < 0.01) fecal shedding and tended to increase (P = 0.08) cecal populations of the challenge strain. Pigs receiving ractopamine in the diet and then experimentally infected with Salmonella Typhimurium, had decreased (P < 0.05) fecal shedding and fewer (P = 0.05) liver samples positive for the challenge strain of Salmonella. Pure cultures of E. coli O157:H7 (used in the present sheep study), E. coli O157:H19 (isolated from pigs with postweaning diarrhea), Salmonella Typhimurium (used in the present pig study), and Salmonella Choleraesuis were incubated with varying concentrations of ractopamine to determine if ractopamine has a direct effect on bacterial growth. No differences in growth rate were observed for either strain of E. coli or for Salmonella Typhimurium when incubated with increasing concentrations of ractopamine. The growth rate for Salmonella Choleraesuis was increased with the addition of 2.0 μg ractopamine/ml compared with the other concentrations examined. Collectively, these results indicate that ractopamine may influence gut populations and fecal shedding of E. coli O157:H7 and Salmonella. Because ractopamine is currently approved to be fed to finishing cattle and swine immediately before slaughter, any potential for decreasing foodborne pathogens has exciting food safety implications. Mention of trade names, proprietary products, or specific equipment does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Colonization of Arabidopsis thaliana with Salmonella enterica and enterohemorrhagic Escherichia coli O157:H7 and competition by Enterobacter asburiae

Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 10(9) CFU g(-1) on A. thaliana roots and to 2 x 10(7) CFU g(-1) on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.