Nohelia Castro-del Campo

Genotypic Analyses of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.

Efficacy of chlorinated and ozonated water in reducing Salmonella typhimurium attached to tomato surfaces.

Abstract The purpose of this study was to compare chlorinated and ozonated water in reducing Salmonella typhimurium inoculated onto fresh ripe tomatoes. Surface-inoculated tomatoes were immersed/sprayed with chlorinated (200 mg l−1) and ozonated water (1 and 2 mg l−1) under 2 and 100 nefelometric turbidity units (NTU). Contact times were 120 and 30 s for immersing and spraying applications, respectively. Immersing in chlorinated water and low turbidity resulted in the most effective application with 3.61 log10 bacterial reduction, while 1 and 2 mg l−1 of ozone reduced 2.32 and 2.53 log10, respectively. High turbidity and chlorine reduced the bacterial counts by 3.39 log10, while 1 and 2 mg l−1 of ozonated water and low turbidity reduced the bacteria by 1.48 and 1.92 log10, respectively. Spraying chlorinated water reduced bacteria by 3 log10, and ozonated water at 1 and 2 mg l−1 reduced counts by 1.84 and 2.40 log10, respectively. No statistical differences were found between chlorine and ozone (2 mg l−1) during spraying applications (p < 0.05). The use of ozonated water both in immersing and spraying applications is suggested when water turbidity remains low.

Exposure and risk assessment of Salmonella in recycled residuals.

The potential health effects of Salmonella found in wastewater residuals is dependent on the exposure of individuals to the organism. This paper provides a risk assessment for human infection from Salmonella due to direct contact with Class B biosolids, and from contact with Class A biosolids following regrowth of Salmonella. In addition, a risk assessment is provided for infection via airborne transport of bioaerosols from Class B biosolids and biosolids in which regrowth had occurred, to off-site communities. Results of the risk characterization imply that the risk of human infection from direct contact with Class B land applied residuals and subsequent ingestion is low. In contrast, the risk from direct contact with Class A residuals following regrowth is greater. Risks from airborne transport of Salmonella via bioaerosols away from a Class B land application site are also low. However, once again the risk from aerosols resulting from biosolids in which regrowth had occurred was greater. Based on these analyses, we conclude that it is highly unlikely that Salmonella infections will occur from land applied Class A or B residuals. However, risks become significant if Class A biosolids are stored anaerobically i.e. saturated, prior to land application.

Produce Contamination Issues in México and Central America

Publisher Summary Mexico grows fruits and vegetables on about 4% of its agricultural land. The diversity of the natural environment in highland Central America has influenced the production of fruits and vegetables. Several large foodborne outbreaks have been linked to fresh produce from these regions, including crops such as cantaloupe, tomatoes, peppers, green onions, and berries. Although contributing factors have not been determined in all cases, quite a few notable causes have been proposed. In particular, cross-contamination with fecal matter of both domestic and wild animals has been suggested. In addition, contact with contaminated water also has been identified as a source of contamination. Moreover, the use of untreated manure or sewage as fertilizer, lack of field sanitary toilet facilities, poorly or unsanitized transportation vehicles, and contamination by handlers are also suggested as potential contributing factors. Private and government agencies need to identify mechanisms and the next steps for the way forward with appropriate partners in the development and implementation of a Good Agriculture Practice approach. Having Good Agricultural and Management practices in place ensures that the process is working correctly. It is important to understand where products are coming from and where they are going. This requires development, implementation, and verification of specifications. Collaborations are needed throughout the entire food chain from farm-to-fork.

Point-of-use Unit Based on Gravity Ultrafiltration Removes Waterborne Gastrointestinal Pathogens from Untreated Water Sources in Rural Communities

OBJECTIVE In developing countries, rural communities often face the lack of potable water infrastructure and must rely on untreated sources for drinking, which are often contaminated with waterborne pathogens. The use of home water treatment devices is seen as one means of reducing the risk of exposure to waterborne pathogens. The aim of this study was to evaluate the microbiological and physicochemical performance of a simple in-home point-of-use device based on gravity ultrafiltration through an ultrafilter membrane. METHODS Twenty-five randomly selected households from 2 rural communities in Culiacán, Mexico, were enrolled. Water samples were collected before and after treatment and during storage for a period of 8 weeks. Heterotrophic bacteria, total coliforms, fecal coliforms, Escherichia coli, and Giardia spp were quantified, as well as various physicochemical parameters. RESULTS All of the untreated water samples contained high levels of indicator bacteria, but none were detected in the treated water fulfilling the requirements set by the Mexican Norm (NOM-127-SSA1-1994) and the World Health Organization guidelines for drinking water. However, indicator bacteria (fecal coliforms and E coli) were detected in every sample from water stored 24 hours after treatment. CONCLUSION This study demonstrated that point-of-use filters using gravity-fed ultrafilters are a low-cost, effective water treatment technology for water of poor microbial quality. However, further identification of the sources and mechanisms by which water is contaminated when stored after treatment will help with designing and implementing better strategies for keeping water safe for domestic use.

Bacteriophage cocktail for biocontrol of Escherichia coli O157:H7: Stability and potential allergenicity study

Escherichia coli O157:H7 has become a global public health and a food safety problem. Despite the implementation of control strategies that guarantee the safety in various products, outbreaks persist and new alternatives are necessary to reduce this pathogen along the food chain. Recently, our group isolated and characterised lytic bacteriophages against E. coli O157:H7 with potential to be used as biocontrol agents in food. To this end, phages need certain requirements to allow their manufacture and application. The aim of this study was to determine the physical stability and allergenic potential of free and microencapsulated (ME) bacteriophage cocktails against E. coli O157:H7. In vitro and in vivo studies were performed to determine phage survival under different pH, gastrointestinal conditions, temperature and UV light intensities. Results showed that the stability of ME phages was significantly (P<0.05) higher than free phages after ultraviolet irradiation, pH conditions between 3 to 7, and exposure to temperatures between at -80°C and 70°C. Both formulations were highly sensitive to very low pH in simulated gastric fluid, but stable in bile salts. In vivo studies in mice confirmed these phages passed through the gastrointestinal tract and were excreted in faeces. In silico, full-length alignment analysis showed that all phage proteins were negative for allergenic potential, but different predicting criteria classified seven phage proteins with a very low probability to be an allergen. In conclusion, these data demonstrated that microencapsulation provided a greater stability to phage formulation under stress conditions and assure a more suitable commercial formulation for the biological control of E. coli O157:H7.

Differences in carbon source utilization of Salmonella Oranienburg and Saintpaul isolated from river water

Abstract Long-term exposure to river water by non-indigenous micro-organisms such as Salmonella may affect metabolic adaptation to carbon sources. This study was conducted to determine differences in carbon source utilization of Salmonella Oranienburg and Salmonella Saintpaul (isolated from tropical river water) as well as the control strain Salmonella Typhimurium exposed to laboratory, river water, and host cells (Hep-2 cell line) growth conditions. Results showed that Salmonella Oranienburg and Salmonella Saintpaul showed better ability for carbon source utilization under the three growth conditions evaluated; however, S. Oranienburg showed the fastest and highest utilization on different carbon sources, including D-Glucosaminic acid, N-acetyl-D-Glucosamine, Glucose-1-phosphate, and D-Galactonic acid, while Salmonella Saintpaul and S. Typhimurium showed a limited utilization of carbon sources. In conclusion, this study suggests that environmental Salmonella strains show better survival and preconditioning abilities to external environments than the control strain based on their plasticity on diverse carbon sources use.

Genomic Analysis of Broad-Host-Range Enterobacteriophage Av-05

ABSTRACT Lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phage relies on appropriate genomic characterization. In this study, we report the genome of bacteriophage Av-05 and its sequence analysis, which has strong lytic activity against Escherichia coli O157:H7 strains and several Salmonella serotypes. The analysis revealed that the phage Av-05 genome consists of 120,938 bp, containing 209 putative open reading frames (ORFs) and 9 tRNAs.

Occurrence and Survival of Pathogenic Microorganisms in Irrigation Water

The consumption per capita of fresh vegetables has increased in the last years in the USA and other countries, which has contributed to the increase of gastroenteritis outbreaks attributed to contaminated fruits and vegetables. Fresh produce can incorporate pathogenic microorganisms thru the process of irrigation, harvesting, postharvest processing and distribution. Most microorganisms use irrigation water and/or soil as a vehicle of transport (Beuchat, 1995; Bhagwat, 2003). Untreated water is most likely to transmit several microorganisms, which may include pathogenic strains of Escherichia coli, Salmonella, Listeria, protozoa and viruses (Diaz et al., 1999). Studies in different countries indicate that the use of untreated water for irrigation of vegetables is the practice most related to fresh produce safety issues (Mongeet al., 1996; Diaz et al., 1999; Tyrrel y Quinton, 2003). Surface water may pose a risk of contamination if its source is unknown. Water is used for multiple issues in diverse agricultural activities including application of fertilizers, washing and disinfecting produce. Therefore, water has to meet the chemical and microbiological requirements before its use (Siller et al., 2002). The quality of water is based on the amount of indicator microorganisms. The major source of coliform contamination when vegetables are grown is probably the irrigation water (Okafoet al., 2003). There are critical factors that need to be monitored to ensure safe water supply. All water sources must be examined periodically for microbiological determination, the results must be recorded and existing problems corrected, for example bathing and grazing animals nearby water resources which should be prohibited to prevent fecal contamination and reduce risks to human health from consuming contaminated fresh produce. Frequently, contamination is associated with the application of irrigation water and the type of crop. Studies have proved that flooding irrigation represents the greatest possibility of contamination if it’s used on produce having direct contact with the soil while the sprinkler irrigation technique provides a rapid means to contaminate the product if the water is contaminated; On the other hand, the drip irrigation technique has represented the lowest risk of contamination of produce (Siller-Cepeda et al., 2009). Lettuce, radishes, carrots are vegetables most likely to become contaminated due to their direct contact with soil and water which can possibly contain bacteria (Okafoet al., 2003). It is well recognized that fecal indicator bacteria may be transported and be a source of contamination of water sources (Tyrrel and Quinton, 2003).

Effect of river water exposition on adhesion and invasion abilities of Salmonella Oranienburg and Saintpaul

Abstract This study was performed to evaluate in vitro the adherence and invasiveness capacity of Salmonella Oranienburg and Saintpaul (isolated from river water) exposed to laboratory and river water growth conditions and inoculated into epithelial HEp-2 cell. Results showed that Salmonella Oranienburg and Salmonella Saintpaul showed lower ability to adhere and invade epithelial HEp-2 cells under both growth conditions as compared to Salmonella Typhimurium reference strain. S. Oranienburg adhesion capacity was not affected by the growth conditions, while S. Saintpaul exposed to river water significantly (p < 0.05) decreased its adhesion capacity by 75.7 %. On the contrary, S. Oranienburg exposed to river water reduced its invasion efficiency by 80 %, whereas S. Saintpaul showed no differences between growth conditions. In conclusion, this study suggests that the exposure to non-host conditions, such as river water, adversely affects the adhesion and invasiveness of Salmonella serotypes differently, impacting on their ability to re-enter a new host.