Characterization of Shiga toxigenic Escherichia coli isolated from foods

The aim of this study was to characterize Shiga toxigenic Escherichia coli (STEC) by PCR using strains isolated from ham, beef, and cattle in Colombia. A total of 189 E. coli strains were tested for the presence of the uidA, stx1, and stx2 genes, and identification was confirmed by the automated PCR BAX system for E. coli O157:H7. Genes encoding Shiga-like toxins (stx) were found in eight (6.06%) of 132 strains previously isolated from minced beef; four (50%) of these strains yielded amplification products for both toxin genes (stx1 and stx2), and four (50%) yielded products only for the stx2 toxin. None of the strains analyzed were positive by PCR for the presence of the single base-pair mutation in the uidA gene from E. coli O157:H7; these results were confirmed by the BAX system analysis. A multiplex PCR assay was standardized for the three genes. Results from this study confirmed previous data about the low prevalence of E. coli O157:H7 and Shiga-like toxins in Colombia and is the first known report of the prevalence of non-O157 enterohemorrhagic E. coli in this country.     

Attachment of shiga toxigenic Escherichia coli to beef muscle and adipose tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (Sr) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. Sr values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and Sr values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.     

Effect of preservatives on Shiga toxigenic phages and Shiga toxin of Escherichia coli O157:H7

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through the induction of the integrated bacteriophages that encode the toxin genes. These phages might be the principal means for the dissemination and release of Shiga toxins. We evaluated the effect of three common food preservatives, potassium sorbate, sodium benzoate, and sodium propionate, on the propagation of the phages and Shiga toxins. We tested each preservative at four concentrations, 1, 1.25, 2.5, and 5 mg/ml, both on free phages and on lysogenic phages in bacteria. We also evaluated the expression of a lambdoid phage, which was exposed to increasing concentrations of preservatives, by measuring β-galactosidase activity from SPC105, a transductant strain. Furthermore, we tested the effect of the preservatives on cytotoxigenic activity of Shiga toxin on Vero cells. We detected an increase of the inhibitory effect of the phage lytic activity, both in lysogenic and free phages, as the preservative concentration increased. However, the inhibition was higher on the lysogenic phages release than on free phages. Sodium benzoate and potassium sorbate were about equal at inhibiting phages; they were more effective than sodium propionate. A significant decrease of lacZ expression, encoded in a lambda phage, was observed. We also found a reduction in Shiga toxin titer caused by exposure of E. coli O157:H7 to 5 mg/ml sodium benzoate or potassium sorbate. These results imply that these three preservatives, used to inhibit microbial spoilage of foods, also act to inhibit lytic activity and dispersion of a phage carrying the gene encoding powerful Shiga cytotoxins. Also notable was the inactivation of Shiga toxin activity, although this effect was detected using concentrations of preservatives greater than those allowed by the Argentine Food Code.     

Shiga toxigenic Escherichia coli show strain dependent reductions under dry-fermented sausage production and post-processing conditions

Dry-fermented sausages (DFS) are considered possible risk products regarding Shiga toxigenic Escherichia coli (STEC). We have compared the reduction of 11 E. coli isolates of various serogroups in salami during the sausage production process and during post-process measures including storage, heating and freezing. The 11 E. coli isolates, mainly STEC, included enterohaemorrhagic E. coli (EHEC) outbreak strains linked to DFS along with apathogenic E. coli. During sausage production, there was a statistically significant difference in reduction between the E. coli strains ranging from 1.3 to 2.4 log?? (p<0.001). When sausages were subjected to post-process heat treatment of 43 °C for 24 h, a total reduction of more than 5 log?? was obtained for all E. coli isolates. Freezing and thawing of DFS with subsequent storage for 1 month at 16 or 20 °C generally contributed to large E. coli reductions with the latter conditions giving an average additional 3.9 log?? reduction, with a range from 3.4 to 4.4 log??. The combination of freezing and 1 month of storage gave higher reductions compared with storage for 2 months for all examined temperatures. No systematic differences in survival of E. coli of different serogroups were detected for the different post-process measures. The reductions were also similar to those of apathogenic control isolates. Isolates showing higher survival during the ripening process did not have a lower reduction when exposed to post-process stress like storage, heating and freezing. The ability of the isolates to survive in salami was also compared with their survival at equivalent conditions in a tryptic soy broth (TSB) model. There was a low and not significant correlation (p>0.1) between the reductions of E. coli in salami and in the TSB broth model. Results based on broth models and/or single or surrogate strains must therefore be interpreted with caution. The EHEC reducing post-processing measures tested can easily be implemented in DFS production with marginal influence on the quality of the sausages.     

Prevalence of Shiga toxin-producing Escherichia coli in beef

Over the past two decades, many human illness outbreaks were attributed to consumption of undercooked beef products containing Shiga toxin-producing Escherichia coli (STEC). The illnesses included mild or bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome (HUS). Tracing these outbreaks to O157 and an increasing number of non-O157 STEC strains suggests that beef safety concerns will continue to rise and may negatively affect the beef industry. To effectively address these concerns, it is critical to evaluate the role of beef in STEC infections. In this review, published reports on beef contamination were evaluated to assess prevalence rates and health risks of STEC isolates. Global testing of beef showed wide ranges of prevalence rates of O157 (from 0.01% to 54.2%) and non-O157 (from 1.7% to 62.5%) STEC. Of the 155 STEC serotypes found in beef, 31 and 25 are known to cause HUS and/or other illnesses, respectively.     

产志贺毒素突变株的毒力分析

对2株食物中毒病人体内分离的产志贺毒素突变株EC130和EC169进行毒力分析。EC130和EC169携带stx基因但不能正常表达志贺毒素,具有eae基因和hly基因,仍具有一定毒力。初步探讨了产志贺毒素突变株不能正常表达志贺毒素的机理,高产志贺毒素的对照株携带Q933基因,而EC130和EC169不携带Q933基因。结果表明,单一采用志贺毒素作为检测靶标,容易造成产志贺毒素突变株漏检,今后在检测食品中产志贺毒素株时应增加检验eae基因和hly基因。     

Surface roughness of stainless steel influences attachment and detachment of Escherichia coli O157

Determining the influence of surface roughness on Escherichia coli O157 attachment to and detachment from stainless steel (SS) is important for controlling this foodborne pathogen. The aim of this study was to evaluate the interactions of six E. coli strains (four O157:H7, one O157:H12, and one O1:H7) with SS type 304 finishes of various surface roughness: 2B (unpolished surface), 4 (common food grade SS), and 8 (polished smooth surface). In attachment assays (exposure to cell suspensions with periodic swirling), bacteria were enumerated by epifluorescence microscopy, and in detachment assays a blotting technique and atomic force microscopy (AFM) were used. Attachment data suggest that E. coli attach in greater numbers to significantly smoother SS8; however, detachment assays and AFM data suggest cells are more easily removed from this finish. Conversely, attachment to SS2B was lower, and AFM data suggest that E. coli O157 may adhere more strongly to this finish. Attachment and detachment data for SS4 was variable, suggesting complex attachment mechanisms to this type of SS. SS4 is the most common material used in food processing facilities. The data from this study indicate that bacterial interactions with SS4 are complex and less easily predicted than those with SS of other finishes, including 2B and 8. These differences in bacterial attachment may be of concern to the food industry and warrant further investigation.     

Prevalence of Shiga toxin-producing Escherichia coli in beef cattle

A large number of Shiga toxin-producing Escherichia coli (STEC) strains have caused major outbreaks and sporadic cases of human illnesses, including mild diarrhea, bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome. These illnesses have been traced to both O157 and non-O157 STEC. In a large number of STEC-associated outbreaks, the infections were attributed to consumption of ground beef or other beef products contaminated with cattle feces. Thus, beef cattle are considered reservoirs of STEC and can pose significant health risks to humans. The global nature of the human food supply suggests that safety concerns with beef will continue and the challenges facing the beef industry will increase at the production and processing levels. To be prepared to address these concerns and challenges, it is critical to assess the role of beef cattle in human STEC infections. In this review, published reports on STEC in beef cattle were evaluated to achieve the following specific objectives: (i) assess the prevalence of STEC in beef cattle, and (ii) determine the potential health risks of STEC strains from beef cattle. The latter objective is critically important because many beef STEC isolates are highly virulent. Global testing of beef cattle feces revealed wide ranges of prevalence rates for O157 STEC (i.e., 0.2 to 27.8%) and non-O157 STEC (i.e., 2.1 to 70.1%). Of the 261 STEC serotypes found in beef cattle, 44 cause hemolytic uremic syndrome and 37 cause other illnesses.     

Isolation of shiga toxigenic Escherichia coli from raw beef in Palestine

Shiga toxigenic Escherichia coli (STEC) isolated from raw beef samples in northern Palestine during a 1-year period were characterized for virulence genes by a polymerase chain reaction (PCR) assay and screened for their antibiotic resistance. STEC was identified in 44 (14.7%) of 300 raw beef samples. Twelve (27.3%) of the STEC isolates were serotype O157. Nine of those were isolated during summer. The majority of STEC isolates (70.5%) harbored both stx1 and stx2 genes, while the others harbored either stx1 or stx2. High levels of resistance against different antimicrobial agents were detected. Resistance to at least three drugs was found in 55% of the isolates.     

Attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel as influenced by exopolysaccharide production, nutrient availability, and temperature

The influence of exopolysaccharide (EPS) production, nutrient availability, and temperature on attachment and biofilm formation by Escherichia coli O157:H7 strains ATCC 43895 (wild type) and 43895-EPS (extensive EPS-producing mutant) on stainless steel coupons (SSCs) was investigated. Cells grown on heated lettuce juice agar and modified tryptic soy agar were suspended in phosphate-buffered saline (PBS). SSCs were immersed in the cell suspension (10(9) CFU/ml) at 4 degrees C for 24 h. Biofilm formation by cells attached to SSCs as affected by immersing in 10% tryptic soy broth (TSB), lettuce juice broth (LJB), and minimal salts broth (MSB) at 12 and 22 degrees C was studied. A significantly lower number of strain 43895-EPS cells, compared to strain ATCC 43895 cells, attached to SSCs during a 24-h incubation (4 degrees C) period in PBS suspension. Neither strain formed a biofilm on SSCs subsequently immersed in 10% TSB or LJB, but both strains formed biofilms in MSB. Populations of attached cells and planktonic cells of strain ATCC 43895 gradually decreased during incubation for 6 days in LJB at 22 degrees C, but populations of strain 43895-EPS remained constant for 6 days at 22 degrees C, indicating that the EPS-producing mutant, compared to the wild-type strain, has a higher tolerance to the low-nutrient environment presented by LJB. It is concluded that EPS production by E. coli O157:H7 inhibits attachment to SSCs and that reduced nutrient availability enhances biofilm formation. Biofilms formed under conditions favorable for EPS production may protect E. coli O157:H7 against sanitizers used to decontaminate lettuce and produce processing environments. Studies are under way to test this hypothesis.     

检测产志贺毒素大肠杆菌的菌落PCR方法

针对产志贺毒素大肠杆菌的毒力基因stx 设计特异性引物,并建立一种菌落PCR 方法。菌落PCR 模拟实验证实,该方法特异性强,能良好的扩增出O157 的stx1 和stx2 基因,而普通大肠杆菌、蜡样芽孢杆菌、金黄色葡萄球菌则无PCR 扩增产物。应用分子检测初筛、选择性培养、菌落PCR 相结合的方法,检测实际食品样品,分离检测到一株携带 stx1 的产志贺毒素大肠杆菌。本实验建立的菌落PCR 方法可应用于食品检验。     

Molecular surveillance of shiga toxigenic Escherichia coli O157 by PulseNet USA

PulseNet USA is the national molecular subtyping network system for foodborne disease surveillance. Sixty-four public health and food regulatory laboratories participate in PulseNet USA and routinely perform pulsed-field gel electrophoresis of Shiga toxigenic Escherichia coli isolated from humans, food, water, and the environment on a real-time basis. Clusters of infection are detected in three ways within this system: through rapidly alerting the participants in the electronic communication forum, the PulseNet Web conference; through cluster analysis by the database administrators at the coordinating center at the Centers for Disease Control and Prevention of the patterns uploaded to the central server by the participants; and by matching profiles of strains from nonhuman sources with recent human uploads to the national server. The strengths, limitations, and scope for future improvements of PulseNet are discussed with examples from 2002. In that year, notices of 30 clusters of Shiga toxigenic E. coli O157 infections were posted on the Web conference, 26 of which represented local outbreaks, whereas four were multistate outbreaks. Another 27 clusters were detected by central cluster detection performed at the Centers for Disease Control and Prevention, of which five represented common source outbreaks confirmed after finding an isolate with the outbreak pattern in the implicated food. Ten food isolates submitted without suspicion of an association to human disease matched human isolates in the database, and an epidemiologic link to human cases was established for six of them.     

Validation of pepperoni process for control of Shiga toxin-producing Escherichia coli

The objective of this study was to compare the survival of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 during pepperoni production. Pepperoni batter was inoculated with 7 log CFU/g of a seven-strain STEC mixture, including strains of serotypes O26, O45, O103, O111, O121, O145, and O157. Sausages were fermented to pH ≤4.8, heated at 53.3°C for 1 h, and dried for up to 20 days. STEC strains were enumerated at designated intervals on sorbitol MacConkey (SMAC) and Rainbow (RA) agars; enrichments were completed in modified EC (mEC) broth and nonselective tryptic soy broth (TSB). When plated on SMAC, total E. coli populations decreased 2.6 to 3.5 log after the 1-h heating step at 53.3°C, and a 4.9- to 5-log reduction was observed after 7 days of drying. RA was more sensitive in recovering survivors; log reductions on it were 1.9 to 2.6, 3.8 to 4.2, and 4.6 to 5.3 at the end of cook, and at day 7 and day 14 of drying, respectively. When numbers were less than the limit of detection by direct plating on days 14 and 20 of drying (representing a 5-log kill), no more than one of three samples in each experiment was positive by enrichment with mEC broth; however, STEC strains were recovered in TSB enrichment. Freezing the 7-day dried sausage for 2 to 3 weeks generated an additional 1- to 1.5-log kill. Confirmation by PCR revealed that O103 and O157 had the greatest survival during pepperoni productions, but all serotypes except O111 and O121 were occasionally recovered during drying. This study suggests that non-O157 STEC s trains have comparable or less ability than E. coli O157 to survive the processing steps involved in the manufacture of pepperoni. Processes suitable for control of E. coli O157 will similarly inactivate the other STEC strains tested in this study.     

Attachment of Listeria monocytogenes to an austenitic stainless steel after welding and accelerated corrosion treatments

Austenitic stainless steels, widely used in food processing, undergo microstructural changes during welding, resulting in three distinctive zones: weld metal, heat-affected zone, and base metal. This research was conducted to determine the attachment of Listeria monocytogenes in these three zones before and after exposure to a corrosive environment. All experiments were done with tungsten inert gas welding of type 304 stainless steel. The four welding treatments were large or small beads with high or low heat. After welding, all surfaces were polished to an equivalent surface finish. A 10-microl droplet of an L. monocytogenes suspension was placed on the test surfaces. After 3 h at 23 degrees C, the surfaces were washed and prepared for scanning electron microscopy, which was used to determine attachment of L. monocytogenes by counting cells remaining on each test surface. In general, bacteria were randomly distributed on each surface type. However, differences in surface area of inoculum due to differences in interfacial energy (as manifested by the contact angle) were apparent and required normalization of bacterial count data. There were no differences (P > 0.05) in numbers of bacteria on the three surface zones. However, after exposure to the corrosive medium, numbers of bacteria on the three zones were higher (P < 0.05) than those on the corresponding zones of noncorroded surfaces. For the corroded surfaces, bacterial counts on the base metal were lower (P < 0.05) than those on heat-affected and weld zones.     

Food as a vehicle for transmission of Shiga toxin-producing Escherichia coli

Contaminated food continues to be the principal vehicle for transmission of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) to humans. A large number of foods, including those associated with outbreaks (alfalfa sprouts, fresh produce, beef, and unpasteurized juices), have been the focus of intensive research studies in the past few years (2003 to 2006) to assess the prevalence and identify effective intervention and inactivation treatments for these pathogens. Recent analyses of retail foods in the United States revealed E. coli O157:H7 was present in 1.5% of alfalfa sprouts and 0.17% of ground beef but not in some other foods examined. Differences in virulence patterns (presence of both stx1 and stx2 genes versus one stx gene) have been observed among isolates from beef samples obtained at the processing plant compared with retail outlets. Research has continued to examine survival and growth of STEC in foods, with several models being developed to predict the behavior of the pathogen under a wide range of environmental conditions. In an effort to develop effective strategies to minimize contamination, several influential factors are being addressed, including elucidating the underlying mechanism for attachment and penetration of STEC into foods and determining the role of handling practices and processing operations on cross-contamination between foods. Reports of some alternative nonthermal processing treatments (high pressure, pulsed-electric field, ionizing radiation, UV radiation, and ultrasound) indicate potential for inactivating STEC with minimal alteration to sensory and nutrient characteristics. Antimicrobials (e.g., organic acids, oxidizing agents, cetylpyridinium chloride, bacteriocins, acidified sodium chlorite, natural extracts) have varying degrees of efficacy as preservatives or sanitizing agents on produce, meat, and unpasteurized juices. Multiple-hurdle or sequential intervention treatments have the greatest potential to minimize transmission of STEC in foods.     

食源性产志贺毒素大肠杆菌的分离及菌株特征分析

了解不同食品中产志贺毒素大肠杆菌的流行情况、菌株特征及潜在致病性。方法 对我国不同地区采集的355份食品样品进行产志贺毒素大肠杆菌分离鉴定,对菌株进行stx1/stx2基因分型、eae等毒力基因检测,并对菌株进行多位点序列分型(MLST)分析。结果 355份样品中44份stx2基因阳性,共分离出11株非O157 产志贺毒素大肠杆菌,其中3株携带stx2a亚型,3株携带stx2e亚型,1株携带stx2b亚型,4株不能分型;5株携带ehxA、saa毒力基因,2株携带subA基因,1株携带katP基因;MLST将11株菌分为7个不同的ST型,存在与溶血性尿毒综合症患者肠出血性大肠杆菌分离株(HUS-associated enterohemorrhagic E.coli,HUSEC)及主要流行血清群产志贺毒素大肠杆菌亲缘关系较近的ST型别。结论 我国食品中存在一定程度的非O157产志贺毒素大肠杆菌污染,部分菌株具有潜在致病性,应加强对食品中STEC的监测。     

Attachment and inactivation of Vibrio parahaemolyticus on stainless steel and glass surface

Vibrio parahaemolyticus is an important food-borne pathogen in Asia. Strains of this pathogen are commonly associated with seafood and may attach to abiotic surfaces during food processing. This work investigates the attachment, biofilm formation and inactivation of this pathogen, on stainless steel and glass surfaces. Attachment of V. parahaemolyticus to these abiotic surfaces was influenced by the growth phase, composition of the culture medium, and stress treatments of the bacterial cells, and also by the presence of sugars in the bacterial suspension. Bacterial culture grown in synthetic MM9 significantly attached more than did the tryptic soy broth culture. Attachment was reduced in the bacterial cultures subjected to various stress treatments, such as low-temperature treatment at 4°C, heat shock at 42°C or two-phase acid adaptation at pH 5·8 and 5·0. Sugars in the bacterial suspension significantly inhibited the attachment, while melibiose, raffinose and stachyose were superior to other sugars as attachment inhibitors to a stainless-steel surface. Clinical strains attached better on stainless steel surface than did environmental strains. V. parahaemolyticus did not form a biofilm effectively in the batch-type culture. The bacterial cell density increased and reached a maximum at 6 or 8 h on stainless steel and glass surfaces, respectively, and declined thereafter. The cells attached on stainless-steel surface were readily inactivated by distilled water, sodium hypochlorite or propionic acid.     

An overview of molecular stress response mechanisms in Escherichia coli contributing to survival of Shiga toxin-producing Escherichia coli during raw milk cheese production

The ability of foodborne pathogens to survive in certain foods mainly depends on stress response mechanisms. Insight into molecular properties enabling pathogenic bacteria to survive in food is valuable for improvement of the control of pathogens during food processing. Raw milk cheeses are a potential source for human infections with Shiga toxin-producing Escherichia coli (STEC). In this review, we focused on the stress response mechanisms important for allowing STEC to survive raw milk cheese production processes. The major components and regulation pathways for general, acid, osmotic, and heat shock stress responses in E. coli and the implications of these responses for the survival of STEC in raw milk cheeses are discussed.     

Thermal resistance parameters for Shiga toxin-producing Escherichia coli in apple juice

The purpose of the present study was to determine the heat resistance of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serotypes in comparison to E. coli O157:H7 in single-strength apple juice without pulp. The thermal parameters for stationary-phase and acid-adapted cells of E. coli strains from serogroups O26, O45, O103, O111, O121, O145, and O157:H7 were determined by using an immersed coil apparatus. The most heat-sensitive serotype in the present study was O26. Stationary-phase cells for serotypes O145, O121, and O45 had the highest D(56°C)-value among the six non-O157 serotypes studied, although all were significantly lower (P < 0.05) than that of E. coli O157:H7. At 60°C E. coli O157:H7 and O103 demonstrated the highest D-values (1.37 ± 0.23 and 1.07 ± 0.03 min, respectively). The D(62°C) for the most heat-resistant strain belonging to the serotype O145 was similar (P > 0.05) to that for the most resistant O157:H7 strain (0.61 ± 0.17 and 0.60 ± 0.09 min, respectively). The heat resistance for stationary-phase cells was generally equal to or higher than that of acid-adapted counterparts. Although E. coli O157:H7 revealed D-values similar to or higher than the individual six non-O157 STEC serotypes in apple juice, the z-values for most non-O157 STEC tested strains were greater than those of E. coli O157:H7. When data were used to calculate heat resistance parameters at a temperature recommended in U.S. Food and Drug Administration guidance to industry, the D(71.1°C) for E. coli O157:H7 and non-O157 STEC serotypes were not significantly different (P > 0.05).