The evaluation of the microbial safety of fresh ready‐to‐eat vegetables produced by different technologies in Italy

Aims: The study was performed to evaluate the safety of whole and RTE vegetables and to investigate the effectiveness of different preventive strategies for the quality assurance of RTE vegetables collected from three Italian production systems. Producer 1, applied a strict system in compliance with GAP‐ GMP – HACCP, Producer 2 used chlorine disinfection at a second washing step, and Producer 3 using a physical microbial stabilization. Methods: During the period 2005–2007, a total of 964 samples including whole vegetables and RTE salads, collected from three different producers in central Italy, were analysed to quantify the aerobic mesophilic count (AMC) and Escherichia coli, and for the presence of Salmonella spp, Listeria monocytogenes, E. coli O157:H7, hepatitis A virus and Norovirus (NoV). Results: None of the whole vegetable samples were positive for L. monocytogenes, E. coli O157:H7, HAV and NoV; however, a low prevalence of Salmonella was found. No pathogens were detected with cultural methods in any of the RTE vegetables analysed, only two RTE samples were positive for L. monocytogenes with PCR, but were not confirmed by the cultural method. The median values of AMC in RTE vegetables measured 24 h after packaging were statistically different among the 3 producers (5·4 × 10⁶, 1·5 × 10⁷ and 3·7 × 10⁷ CFU g^?1, respectively; P = 0·011). The lowest level was detected in Producer 1. Conclusion: The products that were processed applying rigorously GAP, GMP and HACCP showed a better microbiological quality than those processed with chemical or physical stabilization. Study Significance and Impact: The results of the study evidenced the efficacy of GAP, GMP and HACCP in improving microbiological quality of whole and RTE vegetables.     

Environmental Factors Influencing Human Viral Pathogens and Their Potential Indicator Organisms in the Blue Mussel, Mytilus edulis: the First Scandinavian Report

This study was carried out in order to investigate human enteric virus contaminants in mussels from three sites on the west coast of Sweden, representing a gradient of anthropogenic influence. Mussels were sampled monthly during the period from February 2000 to July 2001 and analyzed for adeno-, entero-, Norwalk-like, and hepatitis A viruses as well as the potential viral indicator organisms somatic coliphages, F-specific RNA bacteriophages, bacteriophages infecting Bacteroides fragilis, and Escherichia coli. The influence of environmental factors such as water temperature, salinity, and land runoff on the occurrence of these microbes was also included in this study. Enteric viruses were found in 50 to 60% of the mussel samples, and there were no pronounced differences between the samples from the three sites. E. coli counts exceeded the limit for category A for shellfish sanitary safety in 40% of the samples from the sites situated in fjords. However, at the site in the outer archipelago, this limit was exceeded only once, in March 2001, when extremely high levels of atypical indole-negative strains of E. coli were registered at all three sites. The environmental factors influenced the occurrence of viruses and phages differently, and therefore, it was hard to find a coexistence between them. This study shows that, for risk assessment, separate modeling should be done for every specific area, with special emphasis on environmental factors such as temperature and land runoff. The present standard for human fecal contamination, E. coli, seems to be an acceptable indicator of only local sanitary contamination; it is not a reliable indicator of viral contaminants in mussels. To protect consumers and get verification of “clean” mussels, it seems necessary to analyze for viruses as well. The use of a molecular index of the human contamination of Swedish shellfish underscores the need for reference laboratories with high-technology facilities.     

Frequency of indicator bacteria,Salmonella and diarrhoeagenic Escherichia coli pathotypes on ready‐to‐eat cooked vegetable salads from Mexican restaurants

The presence of coliform bacteria, faecal coliforms, Escherichia coli, diarrhoeagenic E. coli pathotypes (DEP) and Salmonella were determined in ready‐to‐eat cooked vegetable salads (RECS) from restaurants in Pachuca city, Mexico. The RECS were purchased from three types of restaurants: national chain restaurants (A), local restaurants (B) and small restaurants (C). Two restaurants for each A and B, and three for C, were included. Forty RECS samples were purchased at each A and B restaurant and 20 at each C restaurant. Of the overall total of 220 analysed samples, 100, 98·2, 72·3, 4·1 and 4·1% had coliform bacteria, faecal coliforms, E. coli, DEP and Salmonella, respectively. Identified DEP included enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC). The EPEC, ETEC and STEC were isolated each from 1·4% of samples. No E. coli O157:H7 were detected in any STEC‐positive samples. The analysis of Kruskal–Wallis anova and median test of microbiological data showed that the microbiological quality of RECS did not differ between the different restaurants (P > 0·05).

Significance and Impact of the Study

This is the first report regarding microbiological quality and Salmonella, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC) isolation from ready‐to‐eat cooked vegetable salads from Mexican restaurants. Ready‐to‐eat cooked vegetable salads could be an important factor contributing to the endemicity of EPEC, ETEC and STEC, and Salmonella caused gastroenteritis in Mexico.     

Detection of human food-borne and zoonotic viruses on irrigated, field-grown strawberries

This study evaluated the presence of pathogenic human and zoonotic viruses on irrigated, field-grown strawberries. Norovirus genogroup I, rotavirus, and swine hepatitis E virus genogroup 3 were detected on strawberries, and irrigation water is suspected as the contamination origin.     

Environmental Escherichia coli: ecology and public health implications—a review

Escherichia coli is classified as a rod‐shaped, Gram‐negative bacterium in the family Enterobacteriaceae. The bacterium mainly inhabits the lower intestinal tract of warm‐blooded animals, including humans, and is often discharged into the environment through faeces or wastewater effluent. The presence of E. coli in environmental waters has long been considered as an indicator of recent faecal pollution. However, numerous recent studies have reported that some specific strains of E. coli can survive for long periods of time, and potentially reproduce, in extraintestinal environments. This indicates that E. coli can be integrated into indigenous microbial communities in the environment. This naturalization phenomenon calls into question the reliability of E. coli as a faecal indicator bacterium (FIB). Recently, many studies reported that E. coli populations in the environment are affected by ambient environmental conditions affecting their long‐term survival. Large‐scale studies of population genetics revealed the diversity and complexity of E. coli strains in various environments, which are affected by multiple environmental factors. This review examines the current knowledge on the ecology of E. coli strains in various environments with regard to its role as a FIB and as a naturalized member of indigenous microbial communities. Special emphasis is given on the growth of pathogenic E. coli in the environment, and the population genetics of environmental members of the genus Escherichia. The impact of environmental E. coli on water quality and public health is also discussed.     

Physical, chemical and microbiological quality of ice used to cool drinks and foods in Greece and its public health implications

Ice used for direct human consumption or to preserve foods and cool down drinks can be contaminated with pathogenic microorganisms and may potentially become a vehicle for consumer’s infection. To evaluate physical, chemical and microbiological quality of commercial ice and ice used for fish and seafood, 100 ice samples collected at 10 different retail points in the region of Epirus were studied. The following microbiological parameters were determined: Total coliforms, fecal coliforms, Salmonella spp., Shigella spp., Yersinia spp., Escherichia coli, Campylobacter sp., Vibrio cholerae, Aeromonas spp., Pseudomonas aeruginosa and Clostridium perfringens.E. coli was detected in 22% and coliforms were detected in 31% of samples. Samples in which coliforms were detected fail to meet the microbiological criteria specified by the drinking water legislation.Aeromonas spp., Shigella spp., Campylobacter sp. and V. cholerae were not detected. Spore forms of C. perfringens were prevalent at 35% and the psychotropic bacterium’s P. aeruginosa and Yersinia spp. were found only at three samples each.The presence of large numbers of coliforms as well as of other pathogenic strains suggested that commercial ice and ice used to make cool drinks or in preservation of fish and seafood may represent a potential hazard to the consumer. In view of the results reported herein, it is highly recommended that national regulatory guidelines should be established for the production of ice as long as regular inspections.     

Examination of indigenous microbiota and survival of Escherichia coli 0157 and Salmonella in a paper milling environment

Aims: A public beach was frequently cited for health advisories because of high Escherichia coli levels, the source suspected to be a paper mill located upstream. This investigation sought to confirm whether or not the paper mill was the pollution source, and to characterize the risk to recreational bathers imposed by the source. Methods and Results: Quantification of E. coli in river water collected at incremental distances showed that paper mill effluent caused elevated E. coli levels in beach samples. Samples collected throughout the mill were variably positive for heterotrophic bacteria, total coliforms and E. coli, but negative for pathogenic E. coli O157 and Salmonella. Escherichia coli O157 or Salmonella spiked into mill samples (4·2 log₁₀ or 5·6 log₁₀ CFU per 100 ml, respectively) fell below detection levels within 14–24 h in raw (unaltered) samples, while in heat‐sterilized replicates, the counts remained at initial levels or increased over 36 h. Conclusions: Pathogenic E. coli O157 and Salmonella were not isolated from paper mill samples. The absence of native bacteria allowed the survival of pathogens, while their presence accelerated pathogen decline. Significance and Impact of the Study: The co‐existence of paper mill and swimming beach may be reasonable for now in spite of the limitations of an E. coli‐based assay for beach water.     

Ecology of coliphages in southern California coastal waters

Aims: This study aims to investigate the ecology of coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of coliphages in coastal waters. Methods and Results: Water samples from three locations in California’s Newport Bay watershed were tested for the presence of coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The coliphages isolated on the environmental hosts had a broad host‐range relative to the coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Conclusions: Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host‐range may enable the proliferation of coliphages in the aquatic environment. Significance and Impact of the Study: Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host‐range of coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic environment.     

Modulation of virulence and antibiotic susceptibility of enteropathogenic Escherichia coli strains by Enterococcus faecium probiotic strain culture fractions

The increasing rate of antimicrobial resistance drastically reduced the efficiency of conventional antibiotics and led to the reconsideration of the interspecies interactions in influencing bacterial virulence and response to therapy. The aim of the study was the investigation of the influence of the soluble and cellular fractions of Enterococcus (E.) faecium CMGB16 probiotic culture on the virulence and antibiotic resistance markers expression in clinical enteropathogenic Escherichia (E.) coli strains.The 7 clinical enteropathogenic E. coli strains, one standard E. coli ATCC 25,922 and one Bacillus (B.) cereus strains were cultivated in nutrient broth, aerobically at 37 °C, for 24 h. The E. faecium CMGB16 probiotic strain was cultivated in anaerobic conditions, at 37 °C in MRS (Man Rogosa Sharpe) broth, and co-cultivated with two pathogenic strains (B. cereus and E. coli O28) culture fractions (supernatant, washed sediment and heat-inactivated culture) for 6 h, at 37 °C. After co-cultivation, the soluble and cellular fractions of the probiotic strain cultivated in the presence of two pathogenic strains were separated by centrifugation (6000 rpm, 10 min), heat-inactivated (15 min, 100 °C) and co-cultivated with the clinical enteropathogenic E. coli strains in McConkey broth, for 24 h, at 37 °C, in order to investigate the influence of the probiotic fractions on the adherence capacity and antibiotic susceptibility. All tested probiotic combinations influenced the adherence pattern of E. coli tested strains. The enteropathogenic E. coli strains susceptibility to aminoglycosides, beta-lactams and quinolones was increased by all probiotic combinations and decreased for amoxicillin-clavulanic acid. This study demonstrates that the plurifactorial anti-infective action of probiotics is also due to the modulation of virulence factors and antibiotic susceptibility expression in E. coli pathogenic strains.     

Microbiological Analysis of Stuffed Mussels Sold in the Streets

Stuffed mussel is a traditional food that sold by street venders in various countries. In the present study, samples of stuffed mussels were collected from various places in Ankara. The mussels were analyzed to show the microbiological risks for human health. Thirty samples (600 stuffed mussels in total) were collected periodically and microbiological analyses were performed by standard procedures for Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella sp., Clostridium sp. In terms of Salmonella sp., approximately 50% of samples were not suitable for consumption. Besides, in accordance with Turkish Food Codex Microbiological Criteria Announcement in terms of E. coli 30%, in terms of B. cereus 80%, in terms of S. aureus 76.6%, in terms of Clostridium perfringens 13.3% of these samples were not suitable for consumption. The aim of this study is to discuss the microbiological quality of stuffed mussels as a ready-to-eat food according to Turkish Food Codex (TFC). The result of this investigation indicates that stuffed mussels as a street food may constitute a potential health hazard, depending on contamination level and lack of sanitary practices, and therefore, handling practices should require more attention and improvement.     

Fate of manure-borne pathogen surrogates in static composting piles of chicken litter and peanut hulls

The fate of manure-borne pathogen surrogates (gfp-labeled Escherichia coli O157:H7 and Listeria innocua and avirulent Salmonella Typhimurium) in the field was monitored at both sub-surface (30 cm from surface) and surface sites of static composting piles (3.5-m base diameter) composed of chicken litter and peanut hulls. Despite exposure to elevated temperatures, Salmonella was detected by enrichment culture in sub-surface samples following 14 days of composting. In surface samples, pathogen surrogates were detected in the summer after 4 days of composting by enrichment culture only, whereas E. coli O157:H7 and L. innocua remained detectable by direct plating (>2log₁₀ cfu/g) up to 28 days in piles composted during the fall and winter. All three types of bacteria remained detectable by enrichment culture in surface samples composted for 56 days during the winter.     

Effects of Ractopamine HCl on Escherichia coli O157:H7 and Salmonella In Vitro and on Intestinal Populations and Fecal Shedding in Experimentally Infected Sheep and Pigs

The effects of the β-agonist ractopamine, approved for use in finishing swine and cattle to improve carcass quality and performance, were examined on two important foodborne pathogens, Escherichia coli O157:H7 and Salmonella. Ractopamine, administered to sheep before and after oral inoculation with E. coli O157:H7, increased (P < 0.01) fecal shedding and tended to increase (P = 0.08) cecal populations of the challenge strain. Pigs receiving ractopamine in the diet and then experimentally infected with Salmonella Typhimurium, had decreased (P < 0.05) fecal shedding and fewer (P = 0.05) liver samples positive for the challenge strain of Salmonella. Pure cultures of E. coli O157:H7 (used in the present sheep study), E. coli O157:H19 (isolated from pigs with postweaning diarrhea), Salmonella Typhimurium (used in the present pig study), and Salmonella Choleraesuis were incubated with varying concentrations of ractopamine to determine if ractopamine has a direct effect on bacterial growth. No differences in growth rate were observed for either strain of E. coli or for Salmonella Typhimurium when incubated with increasing concentrations of ractopamine. The growth rate for Salmonella Choleraesuis was increased with the addition of 2.0 μg ractopamine/ml compared with the other concentrations examined. Collectively, these results indicate that ractopamine may influence gut populations and fecal shedding of E. coli O157:H7 and Salmonella. Because ractopamine is currently approved to be fed to finishing cattle and swine immediately before slaughter, any potential for decreasing foodborne pathogens has exciting food safety implications. Mention of trade names, proprietary products, or specific equipment does not constitute a guarantee or warranty by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Self-Purificatory Ganga Water Facilitates Death of Pathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

Concern over the prevalence of active pharmaceutical agents and subsequent occurrence of antimicrobial resistance in the environment is increasing. Incorruptible ability of Ganga water was evaluated using fresh, 8-year-old, and 16-year-old Ganga water samples spiked with pathogenic Escherichia coli serotype O157:H7. Survival of E. coli O157:H7 over the course of the experiment was 3, 7, and 15 days for fresh, 8-year-old, and 16-year-old Ganga waters, respectively. On the contrary, in Milli Q water the decline in viable count of E. coli O157:H7 up to 30 days was only 2 log units. Survival of E. coli O157:H7 was greater in boiled water compared with water after passage through a 0.2-μm-pore-size membrane filter, indicating involvement of heat-labile agents influencing survival of E. coli O157:H7 in Ganga water, which seems to indicate the role of antimicrobial peptides. Functional diversity of Ganga water’s native microbial community structure as assessed with Biolog Eco plates was not affected even in the presence of a 5-fold log units higher pathogenic load of E. coli O157:H7. These findings suggest that Ganga water has certain novel antimicrobial attributes, besides its remarkable fluidity, which may provide a much-needed basis for the development of new antimicrobial compounds.     

<Emphasis Type="Italic">Campylobacter</Emphasis> spp., <Emphasis Type="Italic">Salmonella</Emphasis> spp., Verocytotoxic <Emphasis Type="Italic">Escherichia coli</Emphasis>, and Antibiotic Resistance in Indicator Organisms in Wild Cervids

Faecal samples were collected, as part of the National Health Surveillance Program for Cervids (HOP) in Norway, from wild red deer, roe deer, moose and reindeer during ordinary hunting seasons from 2001 to 2003. Samples from a total of 618 animals were examined for verocytotoxic E. coli (VTEC); 611 animals for Salmonella and 324 animals for Campylobacter. A total of 50 samples were cultivated from each cervid species in order to isolate the indicator bacterial species E. coli and Enterococcus faecalis/E. faecium for antibiotic resistance pattern studies. Salmonella and the potentially human pathogenic verocytotoxic E. coli were not isolated, while Campylobacter jejuni jejuni was found in one roe deer sample only. Antibiotic resistance was found in 13 (7.3%) of the 179 E. coli isolates tested, eight of these being resistant against one type of antibiotic only. The proportion of resistant E. coli isolates was higher in wild reindeer (24%) than in the other cervids (2.2%). E. faecalis or E. faecium were isolated from 19 of the samples, none of these being reindeer. All the strains isolated were resistant against one (84%) or more (16%) antibiotics. A total of 14 E. faecalis -strains were resistant to virginiamycin only. The results indicate that the cervid species studied do not constitute an important infectious reservoir for either the human pathogens or the antibiotic resistant microorganisms included in the study.     

Development of a combined immunochromatographic lateral flow assay for accurate and rapid Escherichia coli O157:H7 detection

Escherichia coli O157:H7 is an important pathogenic Bacterium that threatens human health. A convenient, sensitive and specific method for the E. coli O157:H7 detection is necessary. We developed two pairs of monoclonal antibodies through traditional hybridoma technology, one specifically against E. coli O157 antigen and the other specifically against E. coli H7 antigen. Using these two pairs of antibodies, we developed two rapid test kits to specifically detect E. coli O157 antigen and E. coli H7 antigen, respectively. The detection sensitivity for O157 positive E. coli is 1 × 10³ CFU per ml and for H7 positive E. coli is 1 × 10⁴ CFU per ml. Combining these two pairs of antibodies together, we developed a combo test strip that can specifically detect O157: H7, with a detection sensitivity of 1 × 10⁴ CFU per ml, when two detection lines are visible to the naked eye. This is currently the only rapid detection reagent that specifically detects O157: H7 by simultaneously detecting O157 antigen and H7 antigens of E. coli. Our product has advantages of simplicity and precision, and can be a very useful on-site inspection tool for accurate and rapid detection of E. coli O157:H7 infection.     

Identification of free-living amoebas and amoeba-resistant bacteria accumulated in Dreissena polymorpha

To identify the free-living amoeba (FLA) and amoeba-resistant bacteria (ARB) accumulated in zebra mussels and in the water in which they are found, mussels were collected at two locations in the Ebro river basin (North East Spain). FLAs and bacteria were isolated from mussel extracts and from natural water. PCR techniques were used to identify the FLAs and endosymbiont bacteria (Legionella, Mycobacterium, Pseudomonas and cyanobacteria), and to detect Giardia and Cryptosporidium. The most frequently found FLAs were Naegleria spp. The presence of Legionella, Mycobacterium and Pseudomonas inside the FLA was demonstrated, and in some cases both Legionella and Pseudomonas were found together. Differences between FLAs and ARB identified inside the mussels and in the water were detected. In addition, Escherichia coli, Clostridium perfringens, Salmonella spp. and Enterococcus spp. were accumulated in mussels in concentrations unconnected with those found in water. The results show the ability of the zebra mussel to act as a reservoir of potentially pathogenic FLAs, which are associated with potentially pathogenic ARB, although the lack of association between microorganisms inside the mussels and in the water suggests that they are not useful for monitoring microbiological contamination at a specific time.     

Enteric bacteria isolated from acute diarrheal patients in the Republic of Korea between the year 2004 and 2006

In an epidemiological survey of human enterobacterial infections in the Republic of Korea during three years from 2004 to 2006, we isolated 1,784 (6.2%, isolation rate of enteropathogens from stool samples) in 2004, 2,547 (9.5%) in 2005 and 3,506 bacteria (12.3%) from people who visited clinics. Among the isolated bacteria, pathogenic Escherichia coli, especially, EAEC was the most frequently identified pathogen in both urban and rural regions followed by Staphylococcus aureus, Salmonella species, Bacillus cereus, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, and Shigella species. Distinct seasonality was found in V. parahaemolyticus species, while this pathogen showed no age-specific patterns. However, other bacteria, i.e., pathogenic E. coli, S. aureus, Salmonella spp., and B. cereus showed similar seasonality throughout the year, showing a slight increase in the infection rate during the summer months and high prevalence among children under 10 years of age and elder-age people. The antibiotic susceptibility patterns of pathogenic E. coli, Salmonella spp., and S. aureus showed high resistance to penicillins. However, both pathogenic E. coli and Salmonella spp. were susceptible to several cephems, imipenem, and amikacin. Moreover, S. aureus strains resistant to vancomycin were not found. In conclusion, these surveillances can play an important role for the control and prevention to the diseases originated by enteritis bacteria.     

Role of the ArcAB two-component system in the resistance of Escherichia coli to reactive oxygen stress

Background  

The global regulatory system ArcAB controls the anaerobic growth of E. coli, however, its role in aerobic conditions is not well characterized. We have previously reported that ArcA was necessary for Salmonella to resist reactive oxygen species (ROS) in aerobic conditions.     

Surface display of Salmonella epitopes in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus carnosus</Emphasis>

Background  

Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.