Cristobal Chaidez

Occurrence of Cryptosporidium and Giardia in irrigation water and its impact on the fresh produce industry

Abstract In Mexico, relatively few studies have examined the presence of protozoan parasites from surface waters with irrigation and wash-water applications in the fresh produce industry. In this study, the presence of Cryptosporidium oocysts and Giardia cysts in surface water for irrigation, washing and disinfecting applications was evaluated. In addition, the occurrence of Cryptosporidium oocysts and Giardia cysts was determined in wash-water tanks at selected packinghouses. The occurrence of both Cryptosporidium and Giardia was determined by an immunofluorescent technique. Forty-eight percent of the surface water samples tested positive for Cryptosporidium oocysts and 50% tested positive for Giardia cysts. Concentration ranges for Cryptosporidium oocysts and Giardia cysts were 17 – 200 and 17 – 1633 per 100 litres, respectively. Sixteen percent of the wash-water tanks tested positive for Cryptosporidium oocysts and 83% were positive for Giardia cysts with concentrations ranging from 1 – 133 oocysts and 100 – 533 cysts per 100 litres, respectively. The presence of protozoan parasites in surface water for irrigation and packinghouse operations suggests that there may be a risk of contamination of fresh produce as protozoan oocysts/cysts might come in contact with and attach to crop surfaces posing a risk of infection to consumers who eat these products.

Risk assessment of cryptosporidium and giardia in water irrigating fresh produce in Mexico.

A rise in foodborne outbreaks in the United States associated with fresh produce has resulted in increased concerns with the importation of fruits and vegetables. Mexico is a major exporter of produce to the United States, particularly tomatoes and bell peppers. A quantitative microbial risk assessment (QMRA) was conducted to evaluate the public health impact of protozoan-laden water irrigating produce in Culiacan, Sinaloa, Mexico. Specifically, a QMRA was conducted to address the human health impact associated with consumption of tomatoes, bell peppers, cucumbers, and lettuce irrigated with water contaminated with Cryptosporidium and Giardia. Yearly infection risks were estimated and assumed a 120-day exposure in a given year. Annual risks range from 9 x 10(-6) for Cryptosporidium at the lowest concentration associated with bell peppers to almost 2 x 10(-1) for exposure to Giardia on lettuce at the highest detected concentration. With the relatively high number of illnesses resulting from produce-related outbreaks, addressing pre- and postharvest points of contamination for fruits and vegetables consumed raw should be a food industry priority. This research shows how QMRA can be used to interpret microbial contamination data for public health significance and subsequently provide the foundation for guideline development.

Impact of prescribed cleaning and disinfectant use on microbial contamination in the home.

Aims:  To identify and quantify the presence of Escherichia coli, Staphylococcus aureus, Salmonella, hepatitis A and norovirus in households and to assess the effect of chlorine and quaternary ammonium–based disinfectants following a prescribed use.

Comparison of the disinfection efficacy of chlorine-based products for inactivation of viral indicators and pathogenic bacteria in produce wash water.

Outbreaks of pathogenic bacteria infections associated with the consumption of fresh produce has occurred with increased frequency in recent years. This study was undertaken to determine the efficacy of three commonly used disinfectants in packing-houses of Culiacan, Mexico (sodium hypochlorite [NaOCl], trichlor-s-triazinetrione [TST] and thrichlormelamine [TCM]) for inactivation of viral indicators and pathogenic bacteria inoculated onto produce wash water. Each microbial challenge consisted of 2 L of water containing approximately 8 log10 bacterial CFU ml−1, and 8 log10 viral PFU ml−1 treated with 100 and 300 mg l−1 of total chlorine with modified turbidity. Water samples were taken after 2 min of contact with chlorine-based products and assayed for the particular microorganisms. TST and NaOCl were found to effectively reduce for bacterial pathogens and viral indicators 8 log10 and 7 log10, respectively (α = 0.05). The highest inactivation rate was observed when the turbidity was low and the disinfectant was applied at 300 mg l−1. TCM did not show effective results when compared with the TST and NaOCl (P < 0.05). These findings suggest that turbidity created by the organic and inorganic material present in the water tanks carried by the fresh produce may affect the efficacy of the chlorine-based products.

Detection and characterization of hepatitis A virus and Norovirus in estuarine water samples using ultrafiltration – RT-PCR integrated methods

Aims:  Waterborne outbreaks of hepatitis A and Norovirus disease have been reported and associated with contaminated water supply in various countries. However, in Mexico, there are no studies that report HAV and NV presence in water. This study reports the application of ultrafiltration and RT‐nested PCR methods to concentrate and identify these viruses.

Comparison of the microbiologic quality of point-of-use (POU)-treated water and tap water

Activated carbon filtration devices placed on household faucets are used to improve the taste and odour of tap water. However, there has been a concern that the growth of bacteria capable of causing opportunistic infections in these devices might present a public health risk. The water quality from point-of-use (POU) water activated carbon treatment devices and that of tap water with POU-connections and tap water without POU devices were compared. Heterotrophic plate count (HPC) bacteria, total and faecal coliforms, and acid-fast organisms (Mycobacteria spp.), as well as, the opportunistic bacterial pathogens Aeromonas hydrophila, Plesiomonas shigelloides, and Pseudomonas aeruginosa were enumerated. The highest concentration of bacteria was found in POU-treated water. P. aeruginosa, acid-fast organisms, and total coliforms were present in 38.5, 43.8, and 82.4% of the samples, respectively. HPC bacteria were present in all of the POU-treated water samples, with concentrations ranging from 102 to 107 colony forming units/mL. Neither faecal coliforms nor P. shigelloides were recovered from any samples. Tap water with a POU-connection also had higher numbers of bacteria than tap water samples. It was concluded that tap water without POU devices had lower numbers of A. hydrophila, acid-fast organisms, HPC bacteria, P. aeruginosa and coliforms than POU-treated water, and tap water with a POU-connection. The use of POU-devices may amplify the numbers of bacteria present in the tapwater by promoting biofilm formation. Based on a daily ingestion of two liters of POU treated water, A. hydrophila and P. aeruginosa had a probability of less than 10 − 6 of colonizing the gut; however, annual risks could be as much as 100-fold greater.

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.

Virulence profiling of Shiga toxin-producing Escherichia coli recovered from domestic farm animals in Northwestern Mexico

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic enteric pathogen that causes human gastrointestinal illnesses. The present study characterized the virulence profiles of O157 and non-O157 STEC strains, recovered from domestic animals in small rural farms within the agricultural Culiacan Valley in Mexico. Virulence genes coding for adhesins, cytotoxins, proteases, subtypes of Shiga toxin (Stx), and other effectors were identified in the STEC strains by PCR. The genotyping analysis revealed the presence of the effectors nleA, nleB, nleE, and nleH1-2, espK, and espN in the O157:H7 and O111:H8 STEC strains. Furthermore, the genes encoding the autoagglutinating adhesin (Saa) and subtilase (SubA) were exclusively identified in the O8:H19 eae-negative strains. The adhesin (iha) and the silent hemolysin (sheA) genes were detected in 79% of the O157 and non-O157 strains. To examine the relative toxicities of the STEC strains, a fluorescent Vero cell line, Vero-d2EGFPs, was employed to measure the inhibition of protein synthesis by Stx. Analysis of culture supernatants from serotype O8:H19 strains with the stx gene profile stx1a, stx2a, and stx2c and serotypes O75:H8 and O146:H8 strains with the stx gene profile stx1a, stx1c, and stx2b, resulted in a significant reduction in the Vero-d2EGFP fluorescent signal. These observations suggest that these non-O157 strains may have an enhanced ability to inhibit protein synthesis in Vero cells. Interestingly, analysis of the stx2c-positive O157:H7 strains resulted in a high fluorescent signal, indicating a reduced toxicity in the Vero-d2EGFP cells. These findings indicate that the O157 and non-O157 STEC strains, recovered in the Culiacan Valley, display distinct virulence profiles and relative toxicities in mammalian cells and have provided information for evaluating risks associated with zoonotic STEC in this agricultural region in Mexico.

Antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli O157 and Non-O157 recovered from domestic farm animals in rural communities in Northwestern Mexico

BackgroundAntimicrobial resistance in Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 is a matter of increasing concern. The aim of the present study was to investigate the antimicrobial resistance profiles of STEC O157 and non-O157 recovered from feces of domestic farm animals in the agricultural Culiacan Valley in Northwestern Mexico.FindingsAll of the examined STEC strains showed susceptibility to five antimicrobials, ceftazidime, ceftriaxone, ciprofloxacin, nalidixic acid, and trimethoprim-sulfamethoxazole. However, resistance to the four antimicrobials, ampicillin, cephalothin, chloramphenicol, and kanamycin was commonly observed. Interestingly, non-susceptibility to cephalothin was predominant among the examined STEC strains, corresponding to 85 % (22/26) of the O157:H7 from cattle, sheep and chicken and 73 % (24/33) of the non-O157 strains from cattle and sheep. Statistical analyses revealed that resistance to ampicillin was significantly correlated to 38 % (10/26) of STEC O157:H7 strains from multiple animal sources. Another significant correlation was found between serotype, source, and antimicrobial resistance; all of the O20:H4 strains, recovered from sheep, were highly resistant to tetracycline. Multidrug resistance profiles were identified in 42 % (22/53) of the non-susceptible STEC strains with clinically-relevant serotypes O8:H9, O75:H8, O146:H21, and O157:H7.ConclusionsSTEC O157 and non-O157 strains, recovered from domestic farm animals in the Culiacan Valley, exhibited resistance to classes of antimicrobials commonly used in Mexico, such as aminoglycosides, tetracyclines, cephalosporins and penicillin but were susceptible to fluoroquinolones, quinolones, and sulfonamides. These findings provide fundamental information that would aid in the surveillance of antimicrobial resistance in an important agricultural region in Northwestern Mexico.