A field study of the microbiological quality of fresh produce of domestic and Mexican origin

An Economic Evaluation of PulseNet: A Network for Foodborne Disease Surveillance.

A Field Study of the Microbiological Quality of Fresh Produce

BackgroundEscherichia coli O157:H7 (E. coli O157:H7) have frequently been associated with food borne illness and are considered as most serious of known food borne pathogens leading to severe illnesses and high mortality rates in humans. Most of outbreaks were traced to raw meat and raw milk consumption, as well as to dairy products such as yogurt and cheese derived from raw milk.ResultsOut of 200 samples examined, 40 (20%) and 7 (3.5%) of the samples were positive to E. coli and E. coli O157:H7 respectively. The highest isolation of E. coli was from cheese (40%), followed by raw milk (32%), yogurt (25.71%), beef (13.84%), and pasteurized milk (0%). Among E. coli O157:H7 isolates, the highest isolation was from raw milk (12%) followed by cheese (5.71%) and meat (3.07%). However, no E. coli O157:H7 was isolated from pasteurized milk and yogurt. Antibiotic susceptibility profile showed that E. coli was resistant for vancomycin (89.74%), ampicillin (76.92%) and streptomycin (69.23%). The analysis showed that, 92.5% of isolates showed multidrug resistance comprising 2–4 antimicrobials.ConclusionThe occurrence of E. coli O157:H7 and its multiple antibiotic resistant profiles shows a risk for public health and food safety as well as animal production. These findings stress the need for an integrated control of E. coli O157:H7 from farm production to consumption of food of animal origin.

Diseases development due to food is a major threat to consumers; especially in countries where sanitation facilities are poor. E. coli O157:H7 strain causes major outbreaks among foodborne diseases. The current work investigated the prevalence of E. coli O157:H7 and their antibiogram profile in raw milk samples. A total of 180 samples were collected from milk shops in Kohat, Pakistan. The isolates were initially identified on colony morphology on different Media and later through biochemical tests. Isolates were also screened to differentiate E. coli O157:H7 from other fecal E. coli via sorbitol MaCconkey agar. Out of 180 samples 134 (74.4%) milk samples were positive for E. coli and 46 (25.6%) milk samples were negative. Antibiotic susceptibility test was performed by using antibiotics of different classes. Most of the isolates showed susceptibility to ciprofloxacin (82.5%) (80.4%), gentamicin (82.5%) (80.5%) and Oxytetracycline (85.5%) (85.6%) respectively and slightly resistant to Tetracycline (55.8%) (48.2%), Imepenam (52.6%) (42.8%), Ceftriaxone (45.5%) (40.6%), Trimethoprim (54.4%) (36.6%), Chloramphenicol (38.6%) (30.5%), and shows resistance to Amikacin (25.5%) (18.4%) and Cloxacillin (10.5%) (4.5%) respectively. The high frequency of E. coli O157:H7 in milk samples indicate poor hygiene during milking, transportation and milk storage. The awareness against spreading of this pathogen is necessary among stakeholders at different levels of milk handling. Keywords: E. coli O157:H7; Antibiotic resistance, Milk

The aims of this study were to determine the minimal inhibition concentration of 20 different antibiotics on cattle and slaughterhouse wastewater Escherichia coli O157, including both Shiga toxigenic E. coli O157 (STEC O157) and non-Shiga toxigenic strains (non-STEC O157) by the Epsilometer test, and to determine the antibiotic resistance gene profiles of the isolates by PCR. A total of 102 cattle and slaughterhouse wastewater E. coli O157 isolates including 96 E. coli O157:H7+ (81 non-sorbitol fermenting [NSF] STEC O157:H7, 12 NSF non-STEC O157:H7, and three sorbitol fermenting [SF] non-STEC O157:H7) and six non-STEC O157:H7- isolated from 744 cattle and slaughterhouse wastewater samples collected within a 2-year period were assessed. Of 93 NSF E. coli O157:H7 isolates, 19 were resistant to tetracycline and sulfamethoxazole, 14 to trimethoprim, 13 to cefoxitin, 11 to streptomycin, 10 to ampicillin, eight to chloramphenicol, six to cephalothin, four to cefaclor, four to aztreonam, and four to nalidixic acid. In six of the E. coli O157:H7- isolates, tetracycline resistance was detected while five of them were also resistant to ampicillin, sulfamethoxazole, and trimethoprim. In PCR analysis, 26.0 % (25/96) of the NSF E. coli O157:H7+ and all of the E. coli O157:H7- isolates harbored one or more antibiotic resistance genes. While tetA, tetB, tetC, strA, strB, and sulI genes were detected from a number of the isolates, tetD, tetE, tetG, cmlA, floR, sulII, aadA, and ampC genes were not detected in any of the isolates. Results suggest a high antibiotic resistance in E. coli O157:H7+/H7- cattle and wastewater isolates. The majority of our resistant isolates, antibacterial resistance genes did not correlate with observed phenotypic resistance. Other resistance traits and regulatory factors that mediate antibiotic resistance should be included in further antimicrobial resistance investigations.

Biotic and Abiotic Variables Affecting Internalization and Fate of Escherichia coli O157:H7 Isolates in Leafy Green Roots

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is one of the notorious foodborne pathogens causing high mortality through the consumption of contaminated food items. The food safety risk from STEC pathogens could escalate when a group of bacterial cells aggregates to form a biofilm. Bacterial biofilm can diminish the effects of various antimicrobial interventions and enhance the pathogenicity of the pathogens. Therefore, there is an urgent need to have effective control measurements. Bacteriophages can kill the target bacterial cells through lytic infection, and some enzymes produced during the infection have the capability to penetrate the biofilm for mitigation compared to traditional interventions. This study aimed to characterize a new Escherichia phage vB_EcoS-UDF157lw (or UDF157lw) and determine its antimicrobial efficacy against E. coli O157:H7. Phage characterization included biological approaches, including phage morphology, one-step growth curve, stability tests (pH and temperature), and genomic approaches (whole-genome sequencing). Later, antimicrobial activity tests, including productive infection against susceptible bacterial strains, in vitro antimicrobial activity, and anti-biofilm, were conducted. UDF157lw is a new member of the phages belonging to the Rogunavirus genus, comprising a long and non-contractile tail, isolated from bovine feces and shares close genomic evolutionary similarities with Escherichia phages vB_EcoS-BECP10 and bV_EcoS_AKS96. When used against E. coli O157:H7 (ATCC35150), phage UDF157lw exhibited a latent period of 14 min and a burst size of 110 PFU per infected cell. The phage remained viable in a wide range of pH values (pH 4-11) and temperatures (4-60°C). No virulence genes, such as stx, lysogenic genes, and antibiotic resistance genes, were found. Phage UDF157lw demonstrated high infection efficiencies against different E. coli O157:H7 and generic E. coli strains. In addition, UDF157lw encoded a unique major tail protein (ORF_26) with prominent depolymerase enzyme activity against various E. coli O157:H7 strains, causing large plaque sizes. In contrast to the phage without encoding depolymerase gene, UDF157lw was able to reduce the 24-h and 48-h E. coli O157:H7 biofilm after 1-h phage treatment. The findings of this study provide insights into a new member of the Rogunavirus phages and demonstrate its antimicrobial potential against E. coli O157:H7 in vitro.

Many community gardens in large cities worldwide grow vegetables; however, no information regarding the levels of sanitary indicator bacteria and prevalence of foodborne pathogens in vegetables grown in urban community gardens is available. To evaluate the microbiological quality of vegetables from urban community gardens in Korea, 530 samples (nine types of vegetable, including Chinese cabbage, lettuce, radish leaves, spinach, mustard leaves, crown daisy, leek, Korean cabbage, and chicory) were collected at 11 urban community gardens in Seoul, Korea from September through October 2012. The levels of total aerobic bacteria, Escherichia coli, total coliforms, Salmonella spp. Listeria monocytogenes, and E. coli O157:H7 were evaluated quantitatively and/or qualitatively. The mean numbers of total aerobic bacteria and coliforms were 6.3 log CFU/g (range 3.8-8.1 log CFU/g) and 4.3 log CFU/g (range 2.1-6.4 log CFU/g), respectively. Total coliforms were detected on 67% of whole vegetables. Chicory showed the highest number of total aerobic bacteria and coliforms, whereas the lowest number of coliforms was detected on leeks. E. coli was detected on 2.3% of whole vegetables, including lettuce, radish leaves, mustard leaves, and chicory; however, foodborne pathogenic bacteria were not detected on any of the vegetable samples using this highly sensitive and validated procedure. Based on these findings, the presence of coliforms and E. coli demonstrates that opportunity for improvement of microbiological safety exists throughout the produce production chain, although no major foodborne pathogens were present in vegetables grown in urban community gardens.

Semi-quantitative food safety risk profile of the Australian red meat industry

This study investigated the impact of storage temperature and duration on the fate of Escherichia coli O157:H7 on commercially packaged lettuce salads, and on product quality. Fresh-cut Romaine and Iceberg lettuce salads of different commercial brands were obtained from both retail and wholesale stores. The packages were cut open at one end, the lettuce salad inoculated with E. coli O157:H7 via a fine mist spray, and resealed with or without an initial N(2) flush to match the original package atmospheric levels. The products were stored at 5 and 12 °C until their labeled "Best If Used By" dates, and the microbial counts and product quality were monitored periodically. The results indicate that storage at 5 °C allowed E. coli O157:H7 to survive, but limited its growth, whereas storage at 12 °C facilitated the proliferation of E. coli O157:H7. There was more than 2.0 log CFU/g increase in E. coli O157:H7 populations on lettuce when held at 12 °C for 3 d, followed by additional growth during the remainder of the storage period. Although there was eventually a significant decline in visual quality of lettuce held at 12 °C, the quality of this lettuce was still fully acceptable when E. coli O157:H7 growth reached a statistically significant level. Therefore, maintaining fresh-cut products at 5 °C or below is critical for reducing the food safety risks as E. coli O157:H7 grows at a rapid, temperature-dependent rate prior to significant quality deterioration. Specific information regarding the effect of temperature on pathogen growth on leafy greens is needed to develop science-based food safety guidelines and practices by the regulatory agencies and produce industry. Temperature control is commonly thought to promote quality of leafy greens, not safety, based at least partially on a theory that product quality deterioration precedes pathogen growth at elevated temperatures. This prevalent attitude results in temperature abuse incidents being frequently overlooked in the supply chain. This study demonstrates that human pathogens, such as E. coli O157:H7, can grow significantly on commercially packaged lettuce salads while the product's visual quality is fully acceptable. Packaged fresh-cut salads are marketed as "ready-to-eat" while lacking an effective pathogen kill step during their preparation. Thus, maintaining storage temperature at 5 °C or below is critical to prevent pathogen proliferation and mitigate food safety risks should pathogen contamination inadvertently occur during crop growth or postharvest fresh-cut processing.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE+E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 109 CFU of a pool of three stx-negative LEE+E. coli O157:H7 strains (vaccine group) or three stx-negative LEE- non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx+ LEE+E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ.IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Novel strategies of essential oils, chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Our objectives were to evaluate the effects of transportation and lairage on fecal shedding of Escherichia coli O157 (E. coli O157), total Escherichia coli, and total coliforms in feedlot cattle, and the relationships between E. coli O157 prevalence and total E. coli population. The study was a randomized complete block design with a split-plot including 2 treatments: a nontransported group, which remained in its pen at all times, and a transported group, which was transported for 1 h in a trailer and subsequently unloaded in a different pen. The experiment was repeated on 3 different days (blocking factor) with 20 steers/d (10 steers/treatment, 60 total). Fecal samples were taken pretransport (h 0) and after 4 and 28 h, lairage from freshly voided fecal pats were taken from each animal. One gram of feces was transferred to a PBS tube, serially diluted, and plated onto Petrifilm for enumeration of total coliforms. Another sample (1 g) was added to gram-negative broth containing cefixime, cefsulodin, and vancomycin, and subjected to immunomagnetic separation. Resulting beads were plated onto MacConkey agar with sorbitol, cefixime, and tellurite. Nonsorbitol fermenting colonies were selected and tested for indole production and O157 antigen agglutination. Results were confirmed using an API 20E kit. Prevalence of E. coli O157 was transient across blocks. E. coli O157 prevalence revealed no treatment × sampling time interaction (P = 0.179) or sampling time effect (P = 0.937), but a tendency for a treatment effect (P = 0.092). Numbers of E. coli and other coliforms did not change across blocks. No effect of treatment (P > 0.7) was observed on total E. coli concentrations or total coliforms. However, tendencies for treatment × sampling time interactions were observed on both populations (P < 0.08), as well as a tendency for a sampling time effect on total E. coli (P = 0.087) and an effect on total coliforms (P = 0.004). Prevalence of E. coli O157 was not correlated with the concentration of total E. coli (P = 0.954). Results suggest that shedding of E. coli O157 and coliforms can vary within a period of 29 h. Greater statistical power and pathogen quantification, as well as hide sampling and stress-related measurements, are needed to be able to conclude on the effects of transport stress on E. coli O157 prevalence and the changes undergone in pathogen shedding patterns after transportation.

Escherichia coli O157:H7 is one of the pathogenic bacteria causing foodborne disease. The use of lytic bacteriophages can be a good solution to overcome the disease. This study is aimed at isolating lytic bacteriophages from environmental sewage with E. coli O157:H7 bacterial cells. The sample used in this study was eight bacteriophages, and the technique used in identifying E. coli O157:H7 carriers of the stx1 and stx2 genes was PCR. The double layer plaque technique was used to classify bacteriophages. Plaque morphology, host specificity, and electron micrograph were used to identify the bacteriophages. The result obtained plaque morphology as a clear zone with the largest diameter size of 3.5 mm. Lytic bacteriophage could infect E. coli O157:H7 at the highest titer of 10 × 108 PFU/mL. Bacteriophages have been identified as Siphoviridae and Myoviridae. Phage 3, phage 4, and phage 8 could infect Atypical Diarrheagenic E. coli 1 (aDEC1) due to their host specificity. The Friedman statistical tests indicate that lytic bacteriophage can significantly lyse E. coli O157:H7 (p = 0.012). The lysis of E. coli O157:H7 by phage 1, phage 2, phage 3, and phage 5 bacteriophages was statistically significant, according to Conover's posthoc test (p < 0.05). The conclusion obtained from this study is that lytic bacteriophages from environmental sewage could lyse E. coli O157:H7. Therefore, it could be an alternative biocontrol agent against E. coli O157:H7 that contaminates food causing foodborne disease.

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 103 E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 102 E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 101 E. coli O104:H4 initial load). The specificity was 100%.