Lucia Rivas

Isolation and characterisation of Arcobacter butzleri from meat

A survey was conducted to determine the prevalence of Arcobacter in ground chicken, pork, beef and lamb meats. Meat samples were enriched in Arcobacter broth (AB) containing cefoperazone, amphotericin and teicoplanin (CAT) supplement. Samples were screened for the presence of Arcobacter spp. using a multiplex polymerase chain reaction (PCR) followed by isolation on blood and selective agar. Arcobacter butzleri was the only species of Arcobacter isolated from 35% of 88 samples of ground meats. A. butzleri was more frequently isolated from poultry (73%) than pork (29%), beef (22%) or lamb (15%) samples. No significant differences were found in the isolation rates and from the different regions sampled. Isolates were characterised by pulsed-field gel electrophoresis (PFGE) using SacII, EagI and SmaI restriction endonucleases. A number of isolates with indistinguishable PFGE fingerprints were found to be epidemiologically related, which may indicate cross-contamination of common types of Arcobacter from different meat species or between meat species. The public health significance of Arcobacter in ground meat needs to be determined.

Inhibition of verocytotoxigenic Escherichia coli in model broth and rumen systems by carvacrol and thymol

The antimicrobial activities of thymol and carvacrol were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11) and other bacterial species and spoilage bacteria (n=7) using a model broth system. The effects of pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competing microflora on the activities of thymol and carvacrol against E. coli O157:H7 strain 380-94 were also determined. The minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) and numbers of surviving E. coli O157:H7 were determined following incubation. The mean numbers of VTEC surviving exposure to thymol or carvacrol at concentrations of >/=500mug/ml were between 2.0 and 7.8log cfu/ml less than the numbers in the corresponding controls. The susceptibility of E. coli O157:H7 to carvacrol or thymol was found to increase with decreasing storage temperature, water activity, pH and E. coli O157:H7 inoculum size. Sodium chloride (0.5-2.5%) and the presence of a microflora cocktail did not significantly (p>0.05) affect the antimicrobial activities of thymol or carvacrol against E. coli O157:H7. The antimicrobial activity of carvacrol against E. coli O157:H7 was also tested in a model rumen system. A MIC of 500mug/ml carvacrol reduced E. coli O157:H7 inoculated at levels of 10(3) and 10(6)cfu/ml to undetectable levels in the system after 24h incubation. This concentration of carvacrol significantly (p<0.05) decreased the total gas production and volatile fatty acid concentrations in the model rumen assay.

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

ABSTRACT This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichiacoli 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.

Assessment of Escherichia coli O157:H7-specific bacteriophages e11/2 and e4/1c in model broth and hide environments.

The efficacy of bacteriophages e11/2 and e4/1c as potential biocontrol agents for Escherichia coli O157:H7 in food applications was assessed under conditions relevant to the food chain environment. The stability of each phage was determined following exposure to varying environmental conditions (pH, temperature, water activity, and sodium chloride) and the ability of each phage to infect and reduce E. coli O157:H7 numbers under selected conditions was also examined. Both e11/2 and e4/1c significantly (p<0.05) reduced numbers of E. coli O157:H7 when exposed to pH values ranging from pH>4 to pH 9, temperatures from 4 °C to 37 °C, water activity values of 0.87 or 0.91 to 1.00 and NaCl concentrations of 1% to 2.5%. Subsequently, a cocktail of both phages was used (e11/2 and e4/1c) to assess reduction of E. coli O157:H7 on cattle hide pieces. This involved inoculating pieces of hide (20×20 cm) with E. coli O157:H7 (approximately 10⁶cfu/cm²) which were subsequently treated with either a suspension of a phage cocktail, consisting of e11/2 and e4/1c (multiplicity of infection of 1000 and 10,000, respectively) or water or not treated. Two different investigations were carried out; immediately or 1h after treatment application was performed in different experiments. Swab samples taken immediately after phage treatment showed no significant (p>0.05) reduction of E. coli O157:H7 numbers compared to the water treated or untreated samples. However, an extended exposure time of 1h following phage application revealed a significant reduction (p<0.05) (1.5 log₁₀ cfu/cm² reduction) in E. coli O157:H7 numbers compared to the numbers recovered on samples treated with water only. These findings demonstrate the potential use of e11/2 and e4/1c phages as a biocontrol agent for E. coli O157:H7 within various stages of the food chain, including on cattle hide.

Physicochemical properties of Shiga toxigenic Escherichia coli

Aims:  To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture.

Expression and putative roles in attachment of outer membrane proteins of Escherichia coli O157 from planktonic and sessile culture.

Many strains of Shiga toxigenic Escherichia coli (STEC), particularly the serotype O157:H7, are foodborne pathogens causing disease in many countries throughout the world. E. coli O157:H7 is able to attach and survive on various surfaces such as stainless steel (SS) found within the food processing environment. We examined the outer membrane protein (OMP) profiles of four E. coli O157 (three toxigenic O157:H7 and one nontoxigenic O157:HR) and one non-STEC strain (O1:H7), previously reported to have different abilities to attach to SS following growth in planktonic (nutrient broth) and sessile (nutrient agar) culture. The OMPs of the five E. coli strains grown in planktonic and sessile culture were extracted using N-lauroyl sarcosine and the OMP profiles were separated using two-dimensional (2D) gel electrophoresis. Qualitative and quantitative variations in the total number of OMPs expressed between planktonic and sessile cultures were found for all E. coli isolates tested. A number of differentially expressed protein spots were selected from 2D gels and were identified. FlgE was found to be expressed in planktonic culture but not sessile culture. MipA and OmpX had higher expression in sessile culture than planktonic culture, while expression of OmpA did not differ between E. coli strains or between the two modes of growth. Although differential expression of OMPs was found between isolates grown in planktonic and sessile culture, further investigations are required to determine a role of some of these identified proteins during growth of E. coli in planktonic and sessile culture and their influence during the attachment process.

Attachment of shiga toxigenic Escherichia coli to beef muscle and adipose tissue.

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (Sr) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. Sr values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and Sr values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

Inhibition of verocytotoxigenic Escherichia coli by antimicrobial peptides caseicin A and B and the factors affecting their antimicrobial activities

The antimic robial activities of caseicin A and B antimicrobial peptides (AMPs) were assessed against a selection of verocytotoxigenic Escherichia coli (VTEC) strains (n=11), other bacterial pathogenic and spoilage bacteria (n=7), using a model broth system. The ability of the AMPs to retain their antimicrobial activities against a strain of E. coli O157:H7 380-94 under various test conditions (pH, temperature, water activity, sodium chloride concentrations, inoculum size and the presence of competitive microflora) was assessed and the minimum inhibitory concentrations (MIC) and number of surviving E. coli O157:H7 calculated. The mean number of VTEC surviving after exposure to 2 mg/ml caseicin A and B was reduced by 4.96 and 4.19 log(10) cfu/ml compared to the respective controls. The susceptibility of E. coli O157:H7 to the caseicin AMPs decreased as temperature, pH, water activity and inoculum size were reduced. The presence of sodium chloride (0.5-2.5%) did not affect the activity of caseicin A (p>0.05), however it did inhibit the activity of caseicin B. The presence of a competitive microflora cocktail did not significantly (p>0.05) affect the activities of the AMPs for the majority of the concentrations tested. Using a quantitative PCR assay, the levels of verotoxins (vt1 and vt2) expressed by E. coli O157:H7 following exposure to a sub-inhibitory concentration (0.5 mg/ml) of caseicin A showed that the verotoxin levels did not differ from the levels produced by the control cultures. The antimicrobial activity of caseicin A against E. coli O157:H7 was also tested in a model rumen system, however concentrations of ≥2 mg/ml did not significantly (p>0.05) reduce E. coli O157:H7 numbers in the model system over a 24 h period. The application of caseicin AMPs in food and/or animal production may be valuable in combination with other antimicrobials although further research is required.

Isolation and Detection of Pathogenic Escherichia coli in Foods

There is currently no single method that can be used to enrich, isolate, or select for the various E. coli pathotypes that exist. Isolation and detection methods for foodborne E. coli has focused on the Shiga-toxin producing Escherichia coli (STEC) group, particularly for the serogroup O157. However, this is expanding to include non-O157 serogroups as their role in foodborne disease is further elucidated. Traditional methods of detecting pathogenic E. coli are often time-consuming and labor-intensive as they require the growth of the pathogen in culture media, followed by isolation and biochemical and/or serological identification or even further confirmation steps. Various methods have been developed to rapidly identify pathogens including phenotypic assays, such as antibody and modifications of conventional tests, and molecular detection assays. Molecular detection methods can be used to rapidly screen food samples for E. coli pathotypes. However, popular methods such as Polymerase Chain Reaction (PCR) have limitations, including the inability to differentiate live cells from dead and the inhibition of PCR by complex media. One major limitation of all detection methods is the lack of sensitivity to directly detect the small number of pathogenic cells in a food sample. However, further developments and the use of a combination of methods may facilitate the accurate detection and possible enumeration of these pathogens in the future.

Introduction to Pathogenic Escherichia coli

Escherichia coli is one of the predominant facultative anaerobes in the human gastrointestinal tract. However, several pathogenic E. coli strains have emerged that cause disease in humans. Pathogenic E. coli can be divided into intestinal pathogens causing diarrhea, and extra intestinal E. coli causing a variety of infections in both humans and animals. This review will concentrate on the E. coli pathotypes; Enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC), which includes a subgroup known as Enterohemorrahagic E. coli (EHEC), Enteropathogenic E. coli (EPEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC), and Diffusely Adherent E. coli (DAEC). The pathotypes of interest in this review are those that are associated with intestinal infections, with a particular focus on strains important for food safety and/or cause diarrheal disease. Foodborne diseases, particularly diarrheal diseases, are an important cause of morbidity and mortality and are a public health concern worldwide with STEC/EHEC group emerging as a significant cause of foodborne disease. An estimate of the global burden of foodborne disease, including E. coli, is unknown. Surveillance and outbreak investigations are vital in gaining valuable information on the epidemiology of these pathogens and identifying other “emerging” strains, which, in turn, will aid in formulating control and intervention measures with the food industry.