Virulence markers of Escherichia coli strains isolated from traditional cheeses made from unpasteurised sheep milk in Slovakia

Numerous virulence factors including adhesins, host cell surface-modifying factors, invasins, toxins, and secretion systems are involved in Escherichia coli pathogenicity mechanisms. Strains of the same pathotype are genetically similar and carry the same virulence determinants which are ideal targets for the determination of the pathogenic potential of any given E. coli isolate.We examined 95 strains of E. coli isolated from two kinds of traditional Slovak cheeses made from raw sheep milk in course of one year. We tested those strains with the goal to determine some of 11 selected genes of virulence factors which at least partially represent all frequent pathotypes of E. coli. Markers of VTEC (VT1, VT2, VT2e), ETEC (ST I, ST II, LT I), EPEC (EaeA), EIEC (Einv), EAEC (Eagg), and UPEC (CNF1, CNF2) were examined using PCR assays. The result of our testing was the determination of nine potentially pathogenic strains from 95 isolates (9.72%). We did not prove VT1 and VT2 factors as well as Einv and Eagg in any strains. We detected ST I in two strains, ST II and LT I in one strain, and VT2e in one strain. In this study most frequent virulence markers were CNF 1 (three strains) and EaeA (three strains).     

A Turkey survey of hygiene indicator bacteria and Yersinia enterocolitica in raw milk and cheese samples

In this research, a total of 200 dairy (raw milk, cheese) samples obtained from Ankara, were examined for the presence of Yersinia spp., total coliform and Escherichia coli. As expected, raw milk 55% (55/100) were significantly contaminated with Yersinia spp., than cheese samples 14% (14/100). Y. enterocolitica was the most commonly isolated species, and was recovered from 47.3% in raw milk 35.7% in cheese samples. The other Yersinia spp. were identified as Y. frederiksenii (31.0%, 21.4%), Y. kristensenii (12.7%), Y. intermedia (7.2%, 7.1%) and atypical Yersinia spp. (1.8%, 35.7%) in raw milk and cheese samples, respectively. All the samples of cheese examined were negative for Y. kristensenii. All Y. enterocolitica strains tested gave negative results in the autoagglutination tests and crystal violet binding test.Furthermore total coliform bacteria and E. coli were investigated in these samples. According to the analysis results, most of the samples containing Yersinia spp.were found high number of viable count cell total coliform than E. coli. Total coliform bacteria and E. coli, were detected (3.0 × 10⁴ cfu/ml and 1.5 × 10⁴ cfu/ml) in four milk samples containing Y. enterocolitica while, they were detected (2.5 × 10⁴ cfu/ml and 1.0 × 10⁴ cfu/ml) in eight milk samples containing Y. enterocolitica. In the present study, total coliform bacteria and E. coli were detected >1.1 × 10⁵ cfu/g in six cheese samples containing Y. enterocolitica and atypical Yersinia spp. The results indicate that Yersinia spp. is more likely to be isolated from foods with a high level of coliforms than from foods with low coliform counts.     

Good agricultural practices in a Brazilian produce plant

A Brazilian produce plant was evaluated according to good agricultural practices (GAPs) using a checklist. The agricultural water was sampled for total and faecal coliform and Escherichia coli counts. Equipment/handler sanitary facilities, the handling of agrochemicals and pesticides, records of the composting process, prior analysis of the irrigation water for the identification of heavy metals and pesticide residues, inadequate temperature control of the raw material storage room, qualified person to manage the operations and periodic training programmes on food manipulation, hygiene and microbiology, were the main critical points found during the inspection. Microbiological analyses of the irrigation water gave counts between 0.3 and 1.2 log MPN/ml for total and faecal coliforms, respectively and absence of E. coli. The results are discussed and the necessary corrections suggested in order to improve process safety.     

Prevalence of virulence genes in Escherichia coli isolated from food in Casablanca (Morocco)

This study was undertaken to determine the frequency of potentially pathogenic Escherichia coli (E. coli) strains among E. coli isolated from Casablanca, Morocco. The E. coli strains were isolated from ground beef (n = 140), turkey (n = 200), sausage (n = 120), seafood (n = 60), domestic water (n = 35) and well water (n = 50). The prevalence of E. coli was 48%, 45%, 35.5%, 30%, 8.3%, 0%, for well water, ground beef, turkey, sausage, sea food and domestic water, respectively. Two hundreds E. coli isolates were tested for the presence of 17 virulence genes associated with strains causing intestinal and extra-intestinal infections. The virulence genes included stx1, stx2, lt, st, hlyA, aggA, saa, astA, iucD, cnf1, eaeA, bfpA, ial, ipaH, afa, pap and sfa. PCR showed that 37% (74) of E. coli isolates carried one or more of these virulence genes. No virulence genes were found in E. coli strains isolated from sea food samples. In contrast, 10% of the ground beef samples, 18% of the turkey samples, 17.5% of sausage samples and 6% of well water contained specific factors for intestinal E. coli pathogens.     

Histamine contents and histamine-forming bacteria in natto products in Taiwan

Thirty-six natto products imported from Japan and three domestic natto products sold in the supermarkets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH and aerobic plate count (APC) in all samples ranged from 4.9 to 7.3 and 7.8 to 11.2 log CFU/g, respectively. None of these samples contained total coliform and E. coli. Although the average content for each of the nine biogenic amines in all samples was less than 5 mg/100 g, seven of them (17.9%) had histamine content greater than 5 mg/100 g, allowable limit suggested by the US Food and Drug Administration for scombroid fish and/or product. Four histamine-producing bacterial strains capable of producing 13.4 ppm to 17.5 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Bacillus subtilis (two strains) and Staphylococcus pasteuri (two strains) by 16S rDNA sequencing with PCR amplification. To our knowledge, this is the first report to demonstrate the occurrence of histamine-forming bacteria in natto products.     

Pre-processed bovine milk quality in Trinidad: Prevalence and characteristics of bacterial pathogens and occurrence of antimicrobial residues in milk from collection centres

The study was conducted to assess the impact of the changes in the milk collection system in Trinidad (from twice daily collection to once, introduction of chilling facilities to the collection centres and transportation of milk to the processing plant in insulated truck instead of in metal churns at ambient temperature) on the microbial load and antimicrobial residue quality of the milk as well as the temperature and pH of milk, using standard methods. The presence of antimicrobial residues was detected using the Delvo test kit. Of a total of 266 milk samples from churns, the mean ± sd temperature and log₁₀ ± sd TAPC per ml was 20.36 ± 7.91 °C and 6.3 ± 1.09 respectively. For 20 milk samples from the chillers, the mean temperature and log₁₀ ± sd TAPC per ml was 15.10 ± 2.73 °C and 7.04 ± 0.33 respectively compared with corresponding values for 36 samples collected from the truck, 11.64 ± 4.22 °C and 7.11 ± 0.62 respectively (P < 0.05; X²). The mean TAPC, staphylococcal and E. coli counts per ml of milk from churns were significantly (P < 0.05; X²) higher for milk at low temperature (0–20 °C) compared with milk at high temperature (>30 °C). Eight (4.2%) of 192 milk samples tested contained antimicrobial residues. Of 168 S. aureus isolates tested, 24 (14.3%) were enterotoxigenic while 53 (45.3%) of 117 isolates tested exhibited resistance to various antimicrobial agents while of 386 isolates of E. coli tested, 3 (0.8%) were O157 strain and 129 (64.5%) of 200 isolates exhibited resistance to antimicrobial agents. It was concluded that despite the changes, the microbial load of milk was still high suggesting poor sanitary practices at the farm level. The detection of antimicrobial residues agents coupled with toxigenic S. aureus and E. coli isolates could pose health hazards to consumers.     

Bactericidal efficacy of peracetic acid in combination with hydrogen peroxide against pathogenic and non pathogenic strains of Staphylococcus spp., Listeria spp. and Escherichia coli

The aim of this research was to evaluate the bactericidal efficacy of peracetic acid in combination with hydrogen peroxide (PAHP) against pathogenic and non pathogenic strains of Staphylococcus spp., Listeria spp. and Escherichia coli. Killing experiments were performed according to the European standard suspension test UNE-EN-1276. The PAHP was tested at five concentrations from 0.05% to 0.4% v/v and three exposure times (10, 20 and 30 min) using sterile semi-skim milk, orange juice, liquid egg and chocolate milk shake as interfering substances. Staphylococcus spp. showed more resistance at low concentrations of disinfectant than strains of E. coli and Listeria spp. Egg was the organic matter with greatest interfering capacity and orange juice was the one with least interfering capacity. However PAHP was effective (reductions of >5 log CFU/ml) at concentrations from 0.1% and 10 min of exposure in all cases. No statistical differences were found between pathogenic and non pathogenic strains in the same group. Consequently, when the utilization of pathogenic strains is not possible, they may be substituted by non pathogenic strains of the same group, obtaining similar reductions.     

Antimicrobial activity of essential oils against Escherichia coli O157:H7 in organic soil

Soil can be a significant source of preharvest contamination of produce by pathogens. Demand for natural pesticides such as essential oils for organic farming continues to increase. We examined the antimicrobial activity of several essential oils against Escherichia coli O157:H7 in soil. Two essential oils (cinnamaldehyde and eugenol), two bio-pesticides (Ecotrol and Sporan) containing essential oils, and an organic acid (acetic acid) at 0.5%, 1.0%, 1.5% and 2.0%, were mixed with organic sandy soil and inoculated with five different strains of E. coli O157:H7. Soils were incubated at room temperature (22 °C) and samples obtained at 1, 7 and 28 days were enumerated to determine survival. E. coli O157:H7 populations in soil were reduced by up to 5 log cfu/g after 24 h incubation at room temperature with 2% cinnamanaldehyde, Ecotrol, Sporan or vinegar. Reduction in E. coli O157:H7 by eugenol was not significantly different from control. Overall, E. coli O157:H7 strain 4406 was the most sensitive of all the five strains tested and cinnamaldehyde was superior to other treatments in reducing E. coli O157:H7 in soil. In general, increases in essential oil concentrations corresponded to reduced survival of E. coli O157:H7 with all oils used in this study. The results suggest that oils can reduce potential contamination of fresh organic produce inadvertently contaminated by soil.     

Effects of lactic acid and lauricidin on the survival of Listeria monocytogenes,Salmonella enteritidis and Escherichia coli O157:H7 in chicken breast stored at 4 °C

Lauricidin and lactic acid were evaluated for their effects on growth and survival of Listeria monocytogenes (L55), Salmonella enteritidis (S552) and Escherichia coli O157:H7 (E19) inoculated onto raw chicken breast. Fresh, raw chicken breasts were purchased immediately after slaughter and transported on ice to the laboratory within 20 min. Each chicken breast was decontaminated by briefly dipping in 70% ethanol and passed through a flame of a Bunsen burner and then allowed to cool. The decontaminated Chicken breast was dipped in TSB broth, at room temperature (25 °C) for 15 min, containing approximately log 9 CFU/ml of L. monocytogenes, S. enteritidis or E. coli O157:H7. Initial counts of L. monocytogenes, S. enteritidis or E. coli O157:H7 counts in chicken breast immediately after dipping in TSB broth were in the range of log 7–log 8 CFU/g. After inoculation, the chicken breasts were kept at room temperature for 20 min to allow attachment. Each inoculated chicken breast (25 °C) was dipped in 0 (control – sterile water), 0.5%, 1%, 1.5% or 2% of lauricidin (w/v) or lactic acid (v/v) for 10, 20 or 30 min and then individually placed in oxygen-permeable polyethylene bags. Breasts were subjected to microbiological analyses after treatment (day 0) and after storage for 2, 5, 7, 10 and 14 d at 4 °C. Initial counts of L. monocytogenes, S. enteritidis and E. coli O157:H7, in chicken breast treated with lauricidin decreased by 2.90, 1.31 and 2.27 log CFU/g, respectively. Lauricidin was more effective in reducing L. monocytogenes population than S. enteritidis and E. coli O157:H7 population. Dipping chicken breast in lauricidin for 30 min caused a significant reduction of L. monocytogenes, S. enteritidis and E. coli O157:H7 population compared to 10 and 20 min dipping. Initial L. monocytogenes, S. enteritidis and E. coli O157:H7 counts on chicken breast treated with lactic acid decreased by 1.97, 1.71 and 2.59 log CFU/g, respectively. Lactic acid caused a higher reduction in initial S. enteritidis and E. coli O157:H7 counts compared to lauricidin.     

Characteristics of Batzos cheese made from raw,pasteurized and/or pasteurized standardized goat milk and a native culture

Experimental cheeses were made from raw (R), raw with starter (RS), pasteurized with starter (PS) and standardized, pasteurized with starter (PSS) goat milk to study the influence of the starter and pasteurization on the quality of Batzos cheese. Lactococcus lactis subsp lactis strains from raw milk cheese used as starter proliferated significantly (P < 0.05) only in PSS cheese. Heat treatment lowered (P < 0.05) the levels of most microbial groups. However, TC and LAB counts were higher (P < 0.05) in PSS cheese and this was accompanied by lower (P < 0.05) pH; thus, a higher rate of decrease of undesirable microorganisms in PSS cheese was recorded. Degradation of αs-casein was in R > RS and in PS > PSS, while a small reduction of β-casein during ripening and storage was recorded. The proportions of aminoacids and lipolysis products increased throughout ripening and storage.     

Prevalence,genetic diversity and antimicrobial susceptibility of Listeria monocytogenes isolated from open-air food markets in Greece

A total of 210 food samples originating from milk products, ready-to-eat salads, raw meat and raw meat products purchased in ten open-air market places in Thessaloniki, Greece, were analyzed for the presence of Listeria monocytogenes. Thirty (14.3%) contained L. monocytogenes with the highest prevalence in raw meat (27.5%), raw meat products (18%) and cheese (8%). The strains were susceptible to 16 antimicrobials as determined by microbroth dilution, except one strain which displayed resistance to tetracycline (MIC > 32 μg/ml). This strain carried the tetracycline resistance gene tet(M). Pulsed-field gel electrophoresis (PFGE) revealed a low genetic diversity among the isolates, irrespective of their origin. This suggests that dominant L. monocytogenes clones are widespread in different food product types in open-air food markets in Greece. The high prevalence of L. monocytogenes in these products indicates that appropriate hygienic measures and periodic bacteriological controls are also necessary in open-air food markets to reduce contamination with food-borne pathogens. Greek specialties made with raw meat and raw milk may contain L. monocytogenes and should not be consumed by persons at risk.     

Occurrence of aflatoxin M1 in raw milk in South Korea using an immunoaffinity column and liquid chromatography

The level of aflatoxin M₁ (AFM₁) contamination in raw milk produced in South Korea was investigated using immunoaffinity column chromatography and high performance liquid chromatography with fluorescence detector. A total of 100 raw milk samples were collected from 100 cattle ranches located in three different provinces of South Korea. Forty eight out of 100 raw milk samples contained AFM₁ at low level (0.002–0.08 μg/L) with mean value of 0.026 μg/L. Among the AFM₁ contaminated samples, 29 raw milk samples contained only traceable amount of AFM₁ below the limit of LOQ, 0.02 μg/L. None of samples exceeded the maximum level (0.5 μg/L) of Korean regulation for AFM₁ in milk. The limit of detection was 0.002 μg/L. The result of recovery test with 0.5 μg/L AFM₁ in raw milk sample was 96.3% (SD 3.6, n = 5). This is the first pioneering study to investigate the level of AFM₁ in raw milk used in dairy industries in South Korea.     

Shiga toxin-producing Escherichia coli in food: Incidence,ecology, and detection strategies

Commensal Escherichia coli are commonly utilized for investigating the genetic and biochemical requirements of microorganisms, and have served in a wide variety of applications. Pathogenic E. coli known as Shiga toxin (Stx)-producing E. coli (STEC) are associated with various food products including ground beef. These pathogens are present in a wide range of environments, and have caused numerous foodborne outbreaks and recalls. These outbreaks and the increased awareness of STEC have led to certain STEC serotypes to be declared adulterants in non-intact raw meat. Various STEC detection methods have been investigated, and numerous cultural and molecular-based detection methods continue to be modified to meet regulatory requirements. However, STEC serotypes may possess certain characteristics that lead to bias in the likelihood of a certain serotype being detected in an assay. Understanding the characteristics of these STEC serotypes will provide means for optimizing the detection platforms, and as a result limit foodborne illness and recalls caused by STEC due to enhanced cultural and molecular detection capabilities.     

Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure

Plate counting, viability test, pulse field gel electrophoresis (PFGE) and transmission electron microscopy (TEM) were used to investigate the effect of high hydrostatic pressure (HHP) on Salmonella, Escherichia coli, Shigella and Staphyloccocus aureus in raw milk in order to determine the optimal inactivation conditions, and further understand the mechanisms of HHP on pathogens inactivation in food. The results exhibited that 300 Mpa treatment with 30 min duration at 25 °C was the optimal condition for Salmonella, E. coli, Shigella and S. aureus inactivation. Damage of the cell wall, cell membrane and cytoplasmic components by high pressure treatment can be observed in TEM micrographs. The injured cells could not be recovered, the growth rate of survivors was much lower than that of the untreated cells. PFGE showed neither corresponding DNA bands with same molecular weight nor DNA bands with same brightness could be found in the lanes between HHP treated pathogens and untreated ones. The results indicated that HHP processing can be applied to inactivate pathogens in food, the inactivation is mainly due to cell membrane damage, cell wall rupture and chromosome DNA degradation.     

Inactivation of Salmonella,E. coli and Listeria monocytogenes in phosphate-buffered saline and apple juice by ultraviolet and heat treatments

Two strains of Escherichia coli (K-12 and O157:H7), Salmonella (enteritidis and typhimurium) and Listeria monocytogenes (AS-1 and M24-1) were individually suspended in phosphate-buffered saline (PBS) and apple juice prior to exposure to UV radiation (220–300 nm) and heating at 55 °C. The calculated decimal reduction times (D value, min) varied with suspending medium and mode of inactivation. The AS-1 and M24-1 strains of L. monocytogenes were found to be most resistant to UV in PBS (0.28–0.29 min) while the AS-1 strain was most resistant in juice (1.26 min). The AS-1 strain of L. monocytogenes and E. coli O157:H7 were most heat resistant when suspended in PBS (4.41 min) and juice (4.43 min), respectively. Results obtained from this study may be used by apple juice processors in selecting appropriate organisms for UV irradiation or heat treatment lethality validations.     

Effect of organic acids,hydrogen peroxide and mild heat on inactivation of Escherichia coli O157:H7 on baby spinach

Minimally processed baby spinach contaminated with Escherichia coli O157:H7 has been associated with multiple outbreaks of foodborne illnesses recently. Chlorinated water is widely used to wash vegetables commercially, but this washing procedure has limited efficacy and can lead to the formation of carcinogenic substances. This study was conducted to determine the effects of organic acids and hydrogen peroxide alone and in binary combinations with or without mild heat (40 and 50 °C) on the inactivation of Escherichia coli O157:H7 on baby spinach. Baby spinach leaves were dip-inoculated with E. coli O157:H7 to a level of 6 log CFU/g and stored at 4 °C for 24 h before treatment. Individual washing solutions (1% and 2% lactic acid [LA], citric acid [CA], malic acid [MA], tartaric acid [TA], acetic acid [AA], hydrogen peroxide [H₂O₂] as well as binary combinations of LA, CA, MA and H₂O₂ at final concentrations of 1% were used to decontaminate spinach leaves at 22, 40 or 50 °C for 2–5 min to test their efficacy in reducing E. coli O157:H7. Chlorinated water (200 ppm free chlorine) decreased the population of E. coli O157:H7 on baby spinach by only 1.2–1.6 log CFU/g, which was not significantly different from DI water washing. Washing with 1% LA at 40 °C for 5 min was the most effective treatment achieving a 2.7 log reduction of E. coli O157:H7 which is significantly higher than chlorine washing. Washing with LA + CA or LA + HP at 40 °C for 5 min was equally effective against E. coli O157:H7, resulting in a 2.7 log reduction of E. coli O157:H7. The application of mild heat significantly enhanced the efficacy of washing solutions on the inactivation of E. coli O157:H7. There was, however, no significant difference between treatments at 40 °C for 5 min and 50 °C for 2 min. The results suggested that the use of organic acids in combination with mild heat can be a potential intervention to control E. coli O157:H7 on spinach.     

Isolation and identification of histamine-forming bacteria in tuna sandwiches

Forty-three tuna sandwiches sold in diners and convenience store in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, water content, ash content, salt content, total volatile basic nitrogen (TVBN), aerobic plate count (APC), total coliform (TC), and Escherichia coli in all samples ranged from 4.5 to 6.9, 36.8% to 62.8%, 1.11% to 6.55%, 0.10% to 3.90%, 11.2 to 78.0 mg/100 g, <1.0 to 9.7 log CFU/g, <3 to 70,000 MPN/g and <3 to 1000 MPN/g, respectively. The average content of each of the eight biogenic amines in all samples was less than 3 mg/100 g, and only one tuna sandwich sample had histamine content (5.21 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the U.S. Food and Drug Administration. Five histamine-producing bacterial strains, capable of producing 42.1–595.4 ppm of histamine in trypticase soy broth supplemented with 1.0% L-histidine (TSBH), were identified as Hafnia alvei (one strain), Raoultella ornithinolytica (three strains) and Raoultella planticola (one strain), by 16S rDNA sequencing with PCR amplification.     

Biogenic amines content,histamine-forming bacteria,and adulteration of pork and poultry in tuna dumpling products

Nine tuna dumpling products were purchased at nine retail markets in southern Taiwan. Occurrence of biogenic amines, histamine-forming bacteria, and adulteration of pork and poultry were determined. This study showed the high contents of aerobic plate count, total coliform and Escherichia coli in tested tuna dumpling products. Average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g. Fifteen histamine-producing bacterial strains isolated from tested samples produced 8.7–466 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH). Assay of multiplex polymerase chain reaction (PCR) revealed adulteration rates were 88.9% (8/9) and 33.3% (3/9) for pork and poultry, respectively, in tuna dumpling. In addition, six samples of tuna dumpling meat were identified as Thunnus obesus, while other three samples were identified as Thunnus thynnus.     

Sanitization potency of slightly acidic electrolyzed water against pure cultures of Escherichia coli and Staphylococcus aureus,in comparison with that of other food sanitizers

The sanitization potency of slightly acidic electrolyzed water (SAEW) on pure cultures of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was evaluated. The potency was compared with that of strong acidic electrolyzed water (StAEW) and sodium hypochlorite (NaOCl) solution. SAEW (ca. pH 5.8 and 21 mg/l available chlorine concentration; ACC) resulted into >5 log₁₀CFU/ml reduction of E. coli and S. aureus after 90 s of exposure. The relative bacterial reduction potency at each exposure time was in the order StAEW > NaOCl > SAEW and increased with exposure time, with relative effect being 90 s > 60 s > 30 s. The results indicate that SAEW with low ACC and near neutral pH can potentially sanitize E. coli and S. aureus within a short period of exposure presenting a potential replacement to NaOCl solution commonly used in the food industry.     

Histamine contents and histamine-forming bacteria in sufu products in Taiwan

Twelve white and ten brown sufu products sold in the supermarkets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, and aerobic plate count (APC) in all samples ranged from 4.6 to 6.6, 6.2% to 12.0%, and 3.0 to 7.9 log CFU/g, respectively. None of these samples contained total coliform and Escherichia coli. Although the average content for each of the nine biogenic amines in all samples was less than 5 mg/100 g, only one brown sufu sample had histamine content (15.8 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. Two histamine-producing bacterial strains capable of producing 1.33 mg/100 ml and 1.34 mg/100 ml of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Bacillus subtilis by 16S rDNA sequencing with PCR amplification.