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.     

Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces

This work aimed to investigate the influence of physicochemical properties and prior mode of growth (planktonic or sessile culture) on attachment of 13 Campylobacter jejuni strains and 5 Campylobacter coli strains isolated from chicken samples to three abiotic surfaces: stainless steel, glass and polyurethane. Water contact angle and zeta potential measurements indicated that the strains varied with respect to surface hydrophobicity (17.6 ± 1.5 to 53.0 ± 2.3°) and surface charge (−3.3 ± 0.4 to −15.1 ± 0.5 mV). Individual strains had different attachment abilities to stainless steel and glass (3.79 ± 0.16 to 5.45 ± 0.08 log cell cm⁻²) but did not attach to polyurethane, with one exception. Attachment of Campylobacter to abiotic surfaces significantly correlated with cell surface hydrophobicity (P ≤ 0.007), but not with surface charge (P ≥ 0.507). Cells grown as planktonic and sessile culture generally differed significantly from each other with respect to hydrophobicity and attachment (P < 0.05), but not with respect to surface charge (P > 0.05). Principal component analysis (PCA) clustered strains into three groups (planktonic culture) and two groups (sessile culture) representing those with similar hydrophobicity and attachment. Of the four highly hydrophobic and adherent strains, three were C. coli suggesting that isolates with greater hydrophobicity and adherence may occur more frequently among C. coli than C. jejuni strains although this requires further investigation using a larger number of strains. Assignment of pulsed-field gel electrophoresis profiles to PCA groups using Jackknife analysis revealed no overall relationship between bacterial genotypes and bacterial attachment. No relationship between serotype distribution and bacterial attachment was apparent in this study.     

Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and Non-O157 Strains and Their Tolerance to Sanitizers Commonly Used in the Food Processing Environment

Shiga toxin-producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride-based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination.     

Modulation of cell surface hydrophobicity and attachment of bacteria to abiotic surfaces and shrimp by malaysian herb extracts

The use of simple crude water extracts of common herbs to reduce bacterial attachment may be a cost-effective way to control bacterial foodborne pathogens, particularly in developing countries. The ability of water extracts of three common Malaysian herbs (Andrographis paniculata, Eurycoma longifolia, and Garcinia atroviridis) to modulate hydrophobicity and attachment to surfaces of five food-related bacterial strains (Bacillus cereus ATCC 14576, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 10145, Salmonella Enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923) were determined. The bacterial attachment to hydrocarbon assay was used to determine bacterial hydrophobicity. Staining and direct microscopic counts were used to determine attachment of bacteria to glass and stainless steel. Plating on selective media was used to determine attachment of bacteria to shrimp. All extracts were capable of either significantly ( P < 0.05) increasing or decreasing bacterial surface hydrophobicity, depending on the herb extract and bacteria combination. Bacterial attachment to all surfaces was either significantly (P < 0.05) increased or decreased, depending on the herb extract and bacteria combination. Overall, hydrophobicity did not show a significant correlation (P > 0.05) to bacterial attachment. For specific combinations of bacteria, surface material, and plant extract, significant correlations (R > 0.80) between hydrophobicity and attachment were observed. The highest of these was observed for S. aureus attachment to stainless steel and glass after treatment with the E. longifolia extract (R = 0.99, P < 0.01). The crude water herb extracts in this study were shown to have the potential to modulate specific bacterial and surface interactions and may, with further work, be useful for the simple and practical control of foodborne pathogens.     

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.     

Effects of physicochemical surface characteristics of Listeria monocytogenes strains on attachment to glass

Seven strains of Listeria monocytogenes frequently involved in foodborne disease (epidemic strains) and 14 sporadic strains were examined to compare the attachment and subsequent biofilm growth on glass slides at 37 degrees C. Epidemic strains at 3 h incubation had significantly higher attachment values than sporadic strains (P<0.001), but subsequent biofilm growth over 24 h was not dependent on initial attachment. To better understand this phenomenon, the surface hydrophobicity and charge, as well as the extracellular carbohydrate content of the 21 L. monocytogenes strains were studied to determine if these surface characteristics had an effect on bacterial attachment to glass. Hydrophobicity was measured by the bacterial adherence to hydrocarbon (BATH) and polystyrene adherence methods. Hydrophobicity values obtained with the BATH method were linearly correlated with those from the polystyrene adherence method (r=0.64, P<0.001), but no correlation was found between hydrophobicity and bacterial attachment to glass. Hydrophobicity and surface charge measured as electrophoretic mobility (EM) were correlated (r=0.77, P<0.001); however, there was no correlation between the degree of attachment and surface charge. Colorimetric measurements of the total extracellular carbohydrates revealed that attached cells produced significantly (P<0.05) higher levels than planktonic cells after a 3 h time period. Analysis of co-variance (Nested ANCOVA) furthermore demonstrated that total carbohydrates produced by planktonic cells had a significant positive effect on 24 h biofilm growth (P=0.006). This is the first report to indicate that the ability of a L. monocytogenes strain to produce high levels of extracellular carbohydrates may increase its ability to form a biofilm. Genetic studies targeting carbohydrate synthesis pathways of L. monocytogenes will be required to fully understand the importance of this observation.     

Effect of acid tolerance response (ATR) on attachment of Listeria monocytogenes Scott A to stainless steel under extended exposure to acid or/and salt stress and resistance of sessile cells to subsequent strong acid challenge

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate (ca. 10? CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly (p<0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment.     

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.     

Identification and biochemical characteristics of yeast microflora of Rokpol cheese

The inhibiting characteristics of lactic acid bacteria on Shiga toxin-producing Escherichia coli (STEC) O157:H7 (three strains, clinically isolated) was investigated by using a batch fermentation system. The species such as Lactobacillus casei strain Shirota or L. acidophilus YIT 0070 exert growth inhibitory and bactericidal activities on STEC. The pH value and undissociated lactic acid (U-LA) concentration of the culture medium of STEC cocultured with L. casei or L. acidophilus dramatically lowered or increased, respectively [corrected], when compared with those of the control culture. The cytotoxic properties of U-LA on STEC strain 89020087 analyzed in vitro was divided into two phases, i.e., the bacteriostatic phase (between 3.2 to 62 mM) and the bactericidal phase (over 62 mM). These data suggest that the bactericidal effect of Lactobacillus on STEC depends on its lactic acid production and pH reductive effect.     

Attachment of Salmonella spp. and Listeria monocytogenes to stainless steel,rubber and polytetrafluorethylene: the influence of free energy and the effect of commercial sanitizers

Bacteria and material surfaces were characterized with respect to their hydrophobicity and surface free energy using the contact angle method. Salmonella strains showed higher hydrophobicity and lower surface free energies than Listeria monocytogenes strains. Polytetrafluorethylene was the most hydrophobic material (and had the lowest surface free energy), followed by rubber and stainless steel. Bacteria attached in higher numbers to the more hydrophobic materials. Bacterial adherence could not be correlated with surface free energies or contact angles of bacteria, although L. monocytogenes strains attached in higher numbers than Salmonella strains to all of the materials tested. The cleaning of materials with commercial sanitizers resulted in a decrease of their contact angles (and an increase of their surface free energies), accompanied by a reduction in the number of adhered bacteria in comparison with the standard conditions. The degree of reduction in bacterial adherence varied with the bacteria, the substrate material and the sanitizer tested. Quaternary ammonium compounds were more effective against Salmonella attachment than L. monocytogenes attachment. Diethylenetriamine showed similar efficacy against attachment of both bacteria. Polytetrafluorethylene showed the greatest reduction in attachment after being washed with commercial sanitizers. It is concluded that stainless steel is less adherent than rubber or polytetrafluorethylene and should be preferred in the food industry when possible. On the other hand, polytetrafluorethylene seems to be more easily sanitized. Since effectiveness of sanitizers in the reduction of bacterial adherence was dependent upon the bacteria and the materials studied, the use of mixtures of sanitizers would help to control bacterial adherence in the food industry.     

Attachment of Listeria monocytogenes on ready-to-eat meats

Five individual strains of Listeria monocytogenes and a mixed cocktail of all five were studied for attachment on frankfurters, ham, bologna, and roast beef relative to their cell surface characteristics. The ratio of strongly attached (sessile) L. monocytogenes cells compared with total (sessile and planktonic) attached cells on ready-to-eat meats was also determined. Because bacterial cell surfaces were characterized by net negative charge and hydrophobicity, electrostatic interaction chromatography and cationized ferritin methods were chosen to study net negative charge distribution on the bacterial cell surface, whereas hydrophobic interaction chromatography and contact angle measurement were used to examine the cell surface hydrophobicity. No differences (P > 0.05) were observed in cell surface charge or cell surface hydrophobicity among strains. Approximately 84 to 87% L. monocytogenes were found to attach strongly to ready-to-eat meats within 5 min. No differences (P > 0.05) were found among strains or among meats. Micrographs observed from scanning electron microscopy showed no differences among the strains but showed a difference in age of cells (mixed culture) in terms of surface negative charge distribution. More surface negatively charged sites were observed at 0 and 7 days and much fewer at 3 days during storage of washed, harvested cells in buffer at 4 degrees C (aged cells under cold and nutrient deprivation), indicating a possible change in cell surface properties. Because no difference in strains was observed, the contact angle measurement study was carried out with the five-strain mixed culture. The surface hydrophobicity increased in frankfurters, decreased in roast beef, and was unchanged in ham and bologna as a result of inoculation.     

基于免疫磁分离的7种产志贺毒素大肠埃希氏菌快速检测方法的评价

目的 研究基于免疫磁分离的七种产志贺毒素大肠埃希氏菌快速检测方法的灵敏度与特异性。方法 将大肠埃希氏菌O157：H7和大肠埃希氏菌O103不同稀释度的菌悬液,用免疫磁珠富集后,检测其携带毒力基因stx1、stx2 和黏附基因eae以及O157:H7和O103的抗原基因。同时,对菌悬液进行活菌计数,进行灵敏度研究。对8株携带stx1、stx2、eae基因的目标菌菌悬液,以及25株非目标菌的标准菌株及分离菌株的菌悬液,用免疫磁珠富集后,检测其携带毒力基因stx1、stx2 和黏附基因eae以及抗原基因,进行特异性研究。结果 本方法检测大肠埃希氏菌O157：H7的stx1、stx2、eae以及抗原基因的灵敏度为10_²CFU/mL,检测大肠埃希氏菌O103抗原基因的灵敏度为10_³CFU/mL. 8株目标菌检测结果与其携带的基因一致,没有假阴性,包容性达到100%。25株目标菌检测结果与其携带的基因一致,未发现有假阳性,排他性达到100%。结论 方法具有良好的灵敏性及特异性,适用于食品中七种产志贺毒素大肠埃希氏菌的快速检测。     

Bacterial attachment to immobilized extracellular matrix proteins in vitro

Meat surfaces are contaminated with bacteria during slaughter and processing. Understanding bacterial attachment properties to specific structures of meat could result in more targeted interventions to improve its safety and quality. However, the influence of temperatures relevant to abattoir environments on bacterial attachment to specific meat structures is not known. In this study, the effect of temperature and protein concentration on attachment of 10 Escherichia coli and seven Salmonella strains to extracellular matrix (ECM) proteins (collagen I, fibronectin, collagen IV and laminin) was measured using crystal violet stain and epifluorescence microscopy assays. By crystal violet assay, only five of 17 strains showed significant attachment to any ECM protein and only one strain attached to all proteins. Strains that attached at all tested temperatures (4, 25, 37°C) were E. coli M23Sr and M23 (collagen I); E. coli M23Sr (fibronectin); E. coli M23Sr, O157:H12 and M23, (collagen IV); and E. coli M23Sr, O157:H12, O78:K80:H1, O26:H11 and M23 (laminin). A higher proportion of strains attached to basement membrane proteins (laminin and collagen IV) than to interstitial proteins (collagen I and fibronectin). Highest attachment levels occurred at 4°C for collagen I and at 25°C for the other three proteins. Generally, the attachment levels of Salmonella strains to all ECM proteins were lower than for E. coli. No significant effect was found for concentration of collagen I, fibronectin and collagen IV, but was for higher laminin concentration. A strong positive correlation was found between results of both the crystal violet and epifluorescent methods (r≥0.905, p<0.05). This study demonstrated that attachment properties to ECM proteins displayed distinct variation among strains, that temperature highly influenced attachment and that protein concentration had a minor effect.     

Penicillium camemberti and Penicillium roqueforti enhance the growth and survival of Shiga toxin-producing Escherichia coli O157 under mild acidic conditions

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 10(8) CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application: This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.     

Shiga toxin-producing Escherichia coli in beef heifers grazing an irrigated pasture

Shiga toxin-producing Escherichia coli (STEC) produce toxins that have been associated with several human illnesses. E. coli O157:H7 is the most well-studied STEC and was first associated with consumption of improperly cooked ground beef in 1982. E. coli O157:H7 is not the only foodborne STEC because other STEC serotypes are also associated with human illnesses. The objective of this study was to assess prevalence of STEC in 23 yearling beef (Angus) heifers grazing an irrigated grass pasture in spring (April), summer (July), fall (October), and winter (December) of 1999. A total of 86 fecal samples were rectally collected and were subjected to microbiological testing for the presence of STEC. Nine E. coli isolates from five heifers (one in spring and fall and three in winter) were toxic to Vero cells. Of these isolates, four were E. coli O157:H7, two belonged to the serogroup O6, one O39:NM, one O113:H-, and the final isolate was untypable. The STEC prevalence rate in our herd ranged from 4% (spring) to 15% (winter). Based on detecting both O157:H7 and non-O157:H7 STEC in our heifers, it is clear that screening fecal samples should not be limited to E. coli O157:H7. Identification of STEC-positive cattle prior to slaughter should help in reducing the risk of beef contamination with such foodborne pathogens if pre- and/or postharvest control measures are applied to such animals.     

Role of Lactic Acid Bacteria as a Biosanitizer To Prevent Attachment of Listeria monocytogenes F6900 on Deli Slicer Contact Surfaces

The study was conducted to evaluate the attachment of three lactic acid bacteria (LAB) strains and their combination in a cocktail, to stainless steel coupons from a deli slicer, and their ability to inhibit the attachment of Listeria monocytogenes. In a previous study, three LAB strains, Pediococcus acidilactici, Lactobacillus amylovorus, and Lactobacillus animalis, were isolated from ready-to-eat meat and exhibited antilisterial effect. In the study reported here, hydrophobicity tests were determined according to the method of microbial adhesion to solvent. The attachment of the cells was evaluated on stainless steel coupons from deli slicers. Extracellular carbohydrates were determined with a colorimetric method. Based on these tests, L. animalis exhibited the greatest hydrophobicity (26.3%), and its adherence increased sharply from 24 to 72 h, whereas L. amylovorus yielded the lowest hydrophobicity (3.86%) and was weakly adherent. Although P. acidilactici had moderate hydrophobicity (10.1%), it adhered strongly. The attached LAB strains produced significantly (P < 0.05) higher total carbohydrates than their planktonic counterparts did, which is an important characteristic for attachment. Three conditions were simulated to evaluate the ability of the LAB cocktail (10(8) CFU/ml) to competitively exclude L. monocytogenes (10(3) CFU/ml) on the surface of the coupons. The coupons were pretreated with the LAB cocktail for 24 h prior to the addition of L. monocytogenes, simultaneously treated with the LAB cocktail and L. monocytogenes, or pretreated with L. monocytogenes 24 h prior to the addition of the LAB cocktail. The LAB cocktail was able to reduce the attachment L. monocytogenes significantly (P < 0.05). The LAB cocktail indicated potential attachment on stainless steel and bacteriostatic activity toward L. monocytogenes attached on stainless steel, which indicates a possible role for LAB as a biosanitizer in the food industry.     

大肠杆菌O157:H7与假单胞菌混合菌膜形成时交互作用及其对毒力基因表达的影响

以大肠杆菌O157:H7和食品中常见腐败菌假单胞菌为对象,研究两种菌混合培养时菌体泳动能力和混合菌膜形成时的交互作用,进一步采用反转录实时定量聚合酶链式反应分析大肠杆菌O157:H7代表菌株在混合菌膜形成时毒力基因（stx1、stx2、hly和eae）表达变化。菌体泳动性结果显示,3 株大肠杆菌O157:H7泳动性均低于4 株假单胞菌（P＜0.05）,两种菌混合培养时假单胞菌的泳动能力受到显著抑制。选择性培养计数发现混合菌膜形成72 h时,两种菌未发生相互促进,其中4 株假单胞菌的菌膜形成均受到3 株大肠杆菌O157:H7显著抑制（P＜0.05）。选取大肠杆菌O157:H7 CICC21530菌株分析毒力基因表达,结果发现混合菌液单位体积、混合菌膜单位面积4 种毒力基因表达分别低于单种菌液、单种菌膜（P＜0.05）,进一步的单位菌数结果亦然,表明混合培养和混合菌膜形成时假单胞菌抑制了大肠杆菌O157:H7毒力基因表达;单位菌数结果还发现菌膜中4 种毒力基因表达均高于浮游菌（P＜0.05）,表明形成菌膜后菌体毒力增强。本研究可为揭示食源性致病菌和腐败菌混合菌膜形成的交互作用及进一步风险评估和风险防控提供科学依据。     

Relationship of cell surface charge and hydrophobicity to strength of attachment of bacteria to cantaloupe rind

The cantaloupe melon has been associated with outbreaks of Salmonella infections. It is suspected that bacterial surface charge and hydrophobicity may affect bacterial attachment and complicate bacterial detachment from cantaloupe surfaces. The surface charge and hydrophobicity of strains of Salmonella, Escherichia coli (O157:H7 and non-O157:H7), and Listeria monocytogenes were determined by electrostatic and hydrophobic interaction chromatography, respectively. Initial bacterial attachment to cantaloupe surfaces and the ability of bacteria to resist removal by washing with water were compared with surface charge and hydrophobicity. Whole cantaloupes were submerged in inocula containing individual strains or in cocktails containing Salmonella, E. coli, and L. monocytogenes, either as a mixture of strains containing all three genera or as a mixture of strains belonging to a single genus, for 10 min. Inoculated cantaloupes were dried for 1 h in a biosafety cabinet and then stored for up to 7 days at 4 degrees C. Inoculated melons were washed with water, and bacteria still attached to the melon surface, as well as those in the wash water, were enumerated. Initial bacterial attachment was highest for individual strains of E. coli and lowest for L. monocytogenes, but Salmonella exhibited the strongest attachment on days 0, 3, and 7. When mixed-genus cocktails were used, the relative degrees of attachment of the three genera ware altered. The attachment of Salmonella strains was the strongest. but the attachment of E. coli was more extensive than that of L. monocytogenes on days 0, 3, and 7. There was a linear correlation between bacterial cell surface hydrophobicity (r2 = 0.767), negative charge (r2 = 0.738), and positive charge (r2 = 0.724) and the strength of bacterial attachment to cantaloupe surfaces.     

Attachment of Escherichia coli O157:H7 to abiotic surfaces of cooking utensils

We examined the attachment of enterohemorrhagic Escherichia coli O157:H7 to abiotic surfaces of cooking utensils. When the cell suspension in 0.85% NaCl (about 100 cells/mL, 10 mL) was contacted with various abiotic surfaces (square pieces, 25 cm2) at 25 °C for 20 min, the number of attached cells varied depending on the types of abiotic materials. The pathogen well attached to stainless steel (about 50 cells/25 cm2), pure titanium (35 to 45 cells/25 cm2), and glass (about 20 cells/25 cm2), but little attached to aluminum foil and plastics, irrespective of strains used. Fewer cells (below 10 cells/25 cm2) attached to stainless steel, pure titanium, and glass surfaces conditioned with aseptically sliced beef (sirloin) and autoclaved beef tallow at 25 °C for 20 min, but bovine serum albumin did not reduce the number of attached cells. The cells grown at 15 °C to the stationary phase (OD660 = about 2.8) less attached to the abiotic surfaces than those grown at 25 °C and 37 °C. When we pretreated the cells at 37 °C for 2 h with 50 μM N-hexanoyl-L-homoserine lactone (HHL), the number of cells attached to stainless steel was reduced by 70%. The number of cells attached to cooking utensils seemed to change depending on types of abiotic materials, adhesion of beef tallow to abiotic surfaces, growth temperature of the pathogen, and HHL-producing bacteria.     

Synergistic effect of steam and lactic acid against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilms on polyvinyl chloride and stainless steel

This study was designed to investigate the individual and combined effects of steam and lactic acid (LA) on the inactivation of biofilms formed by Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes on polyvinyl chloride (PVC) and stainless steel. Six day old biofilms were developed on PVC and stainless steel coupons by using a mixture of three strains each of three foodborne pathogens at 25°C. After biofilm development, PVC and stainless steel coupons were treated with LA alone (immersed in 0.5% or 2% for 5s, 15s, and 30s), steam alone (on both sides for 5, 10, and 20s), and the combination of steam and LA. The numbers of biofilm cells of the three foodborne pathogens were significantly (p<0.05) reduced as the amount of LA and duration of steam exposure increased. There was a synergistic effect of steam and LA on the viability of biofilm cells of the three pathogens. For all biofilm cells of the three foodborne pathogens, reduction levels of individual treatments ranged from 0.11 to 2.12 log CFU/coupon. The combination treatment of steam and LA achieved an additional 0.2 to 2.11 log reduction compared to the sum of individual treatments. After a combined treatment of immersion in 2% LA for 15s or 30s followed by exposure to steam for 20s, biofilm cells of the three pathogens were reduced to below the detection limit (1.48 log). From the results of this study, bacterial populations of biofilms on PVC coupons did not receive the same thermal effect as on stainless steel coupons. Effectiveness of steam and LA may be attributed to the difference between Gram-negative and Gram-positive characteristics of the bacteria studied. The results of this study suggest that the combination of steam and LA has potential as a biofilm control intervention for food processing facilities.