Impact of cold and cold-acid stress on poststress tolerance and virulence factor expression of Escherichia coli O157:H7

The effect of extended cold or cold-acid storage of Escherichia coli O157:H7 on subsequent acid tolerance, freeze-thaw survival, heat tolerance, and virulence factor (Shiga toxin, intimin, and hemolysin) expression was determined. Three E. coli O157:H7 strains were stressed at 4 degrees C in TSB or pH 5.5 TSB for 4 weeks. The acid (TSB [pH 2.0] or simulated gastric fluid [pH 1.5]) tolerance, freeze-thaw (-20 degrees C to 21 degrees C) survival, and heat (56 degrees C) tolerance of stressed cells were compared with those of control cells. The beta-galactosidase activities of stressed and control cells containing a lacZ gene fusion in the stx2, eaeA, or hlyA gene were determined following stress in TSB or pH 5.5 TSB at 37 degrees C and in the exponential and stationary phases. Cold and cold-acid stresses decreased acid tolerance (P < 0.05), with a larger decrease in acid tolerance being observed after cold stress than after cold-acid stress (P < 0.05). Cold stress increased freeze-thaw survival for all three strains (P < 0.05). Prior cold or cold-acid stress had no effect on virulence factor production (P > 0.05), although growth in acidic media (pH 5.5) enhanced eaeA and hlyA expression (P < 0.05). These results indicate that the prolonged storage of E. coli O157:H7 at 4 degrees C has substantial effects on freeze-thaw tolerance but does not affect subsequent virulence gene expression.     

Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments

Acid-adapted bacterial cells are known to have enhanced tolerance to various secondary stresses. However, a comparison of heat tolerance of acid-adapted and acid-shocked cells of Escherichia coli O157:H7 has not been reported. D - and z -values of acid-adapted, acid-shocked, and control cells of an unusually heat-resistant strain (E0139) of E. coli O157:H7, as well as two other strains of E. coli O157:H7, were determined based upon the number of cells surviving heat treatment at 52, 54 or 56°C in tryptic soy broth (pH 7·2) for 0, 10, 20 or 30 min. The unusual heat tolerance of E. coli O157:H7 strain E0139 was confirmed. D -values for cells from 24-h cultures were 100·2, 28·3, and 6·1 min at 52, 54 and 56°C, respectively, with a z -value of 3·3°C. The highest D -values of other E. coli O157:H7 strains were 13·6 and 9·2 min at 52 and 54°C, respectively, whereas highest D -values of non-O157:H7 strains were 78·3 and 29·7 min at 52 and 54°C. D -values of acid-adapted cells were significantly higher than those of unadapted and acid-shocked cells at all temperatures tested. In a previous study, we observed that both acid-adapted cells and acid-shocked cells of strain E0139 had enhanced acid tolerance. This suggests that different mechanisms protect acid-adapted and acid-shocked cells against subsequent exposure to heat or an acidic environment. The two types of cells should be considered separately when evaluating survival and growth characteristics upon subsequent exposure to different secondary stress conditions.     

Sublethal sanitizer stress and adaptive response of Escherichia coli O157:H7

The effect of sublethal exposure to peroxyacetic acid (PAA) sanitizer on adaptation to peroxidative stress and development of thermal cross-resistance was investigated in Escherichia coli O157:H7. Acute sublethal PAA sanitizer exposure was used to represent a contact scenario. Cultures were grown in Trypticase soy-yeast extract broth. Acute treatment cultures were pretreated with 0.1% PAA, then all cultures were challenged at either 80 mM H202 or 54 degrees C. Acute and peroxide control cultures showed substantially increased peroxidative tolerance (D80mM > 2 h) versus negative control cultures not exposed to sanitizer (D80mM = 0.19+/-0.03 h). The inactivation rate of the acetic acid control (D80mM = 0.21+/-0.05 h) was similar to the negative control rate. Acute (D54 degrees C = 0.55+/-0.07 h) cultures did not exhibit increased thermal resistance versus the control (D54 degrees C = 0.54+/-0.07 h). Thermal injury was determined as difference in D54 degrees C value (deltaD54 degrees c) obtained on pyruvate and deoxycholate media. Thermal-induced injury was not observed in either control (deltaD54 degrees C = 0.04 h) or acute (deltaD54 degrees C = 0.05 h) cultures.     

Influence of acid adaptation on the tolerance of Escherichia coli O157:H7 to some subsequent stresses

Three stains of Escherichia coli O157:H7, including ATCC 43889, ATCC 43895, and 933, were first subjected to acid adaptation at a pH of 5.0 for 4 h. Thermal tolerance at 52 degrees C and survival of the acid-adapted as well as the nonadapted cells of E. coli O157:H7 in the presence of 10% sodium chloride, 0.85% bile salt, or 15.0% ethanol were investigated. Results showed that the effect of acid adaptation on the survival of E. coli O157:H7 varied with the strains and types of subsequent stress. Acid adaptation caused an increase in the thermal tolerance of E. coli O157:H7 ATCC 43889 and ATCC 43895, but no significant difference in the thermal tolerance was noted between acid-adapted and nonadapted cells of E. coli O157:H7 933. Although the magnitude of increase varied with strains of test organisms, acid adaptation generally led to an increase in the tolerance of E. coli O157:H7 to sodium chloride. On the other hand, the susceptibility of acid-adapted cells of the three strains of E. coli O157:H7 tested did not show a significant difference from that of their nonadapted counterparts when stressed with bile salt. The acid-adapted cells of E. coli O157:H7 ATCC 43889 and ATCC 43895 were less tolerant than the nonadapted cells to ethanol, whereas the tolerance of adapted and nonadapted cells of E. coli O157:H7 933 showed no significant differences.     

Effect of nutrient starvation on the resistance of Escherichia coli O157:H7 to subsequent heat stress

The resistance of three strains of Escherichia coli O157:H7 in their stationary growth phase to starvation (24 h in water at 37 degrees C) followed by a heat treatment (56 degrees C for up to 90 min) was determined. Starvation was found to increase significantly the resistance of two strains (NCTC 12079; eae+, VT1+, VT2+, and ATCC 43889 eae+, VT2+) but not the remaining strain (ATCC 43890 eae+, VT1+). Strain NCTC 12079 (only one tested) was shown to retain all of the three virulence factors after the two stresses. De novo protein synthesis was shown to be required for heat resistance. Evidence using an rpoS mutant indicated a central role for this gene in inducing heat resistance after a starvation stress. It is hoped that this work will contribute to more accurate risk assessments in certain food processing operations.     

Proteome analysis of virulence factor regulated by autoinducer-2-like activity in Escherichia coli O157:H7

Many pathogenic bacteria, including Escherichia coli O157:H7, can control gene expression in a cell density-dependent manner by producing small signaling molecules (autoinducers) in a process known as quorum sensing. In this study, the effects of the autoinducer-2-like activity on the expression of proteins, including virulence factors, in E. coli O157:H7 were characterized by proteomic analysis. Compared with the control, E. coli O157:H7 strains in the presence of autoinducer-2-like activity exhibited elevated virulence by more rapidly forming cell aggregates on epithelial cells and rapidly killing the nematode Caenorhabditis elegans, the surrogate host. Two-dimensional gel electrophoresis revealed 18 proteins that were upregulated by autoinducer-2-like activity and 4 proteins that were down-regulated. These proteins were further characterized by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and are involved in the metabolic process, adaptation and protection, cell motility, secretion, envelope biogenesis, and protein translation. These results indicate that the newly identified proteins are associated with the control of virulence in E. coli O157:H7 and that these proteins can be potential targets for the development of antibiotics and other antimicrobial agents.     

Growth and processing conditions affecting acid tolerance inEscherichia coli O157:H7

The impact of growth conditions (anaerobiosis, growth phase, NaCl, pH, and temperature) on the development of acid tolerance in Escherichia coli O157:H7 was investigated directly (D_pH1.15) and indirectly by monitoring the specific activity of acid phosphatase. Anaerobic growth of O157:H7 strain 43895 in synthetic rumen fluid resulted in earlier development of acid tolerance than aerobic growth. However, stationary-phase cells of both aerobic and anaerobic cultures had an equivalent degree of acid tolerance that was greater than that achieved in log-phase cultures grown anaerobically. These results are consistent with the growth-phase regulation of acid tolerance by the stationary-phase sigma factors³⁸. The addition of NaCl (1%) also enhanced acid tolerance of log-phase but not stationary-phase cells of strain 43895. Growth temperature influenced the acid tolerance with progressively greater D_pH1.15values obtained at 15, 25, and 37°1C, in both log and stationary phase. Therefore, the influence of temperature on the subsequent survival and acid tolerance of E. coli O157:H7 strain 43895 in ground beef was evaluated. Numbers of strain 43895 decreased c. 1.14 log₁₀cfu g^-1in inoculated ground beef stored at 4°1C, whereas numbers remained essentially unchanged during storage at -20°C. While pre-incubation at 15°1C for 4 h prior to storage at 4 or -20°C did not influence survival, the acid tolerance of E. coli O157:H7 survivors was significantly decreased (P<0.10001). These results indicate that the processing temperature can influence acid tolerance in E. coli O157:H7.     

大肠杆菌O157:H7耐酸性及酸胁迫下菌体形态变化的初步研究

目的 对大肠杆菌O157:H7 耐酸性进行初步探讨。方法 通过稀释涂布平板计数法观察大肠杆菌O157:H7 在不同的pH 生长条件下的存活能力和利用电镜扫描法观察其在酸胁迫下菌体形态的变化。结果 当pH 为5.0 到7.0 之间时, 大肠杆菌O157:H7 生长状况良好, 当pH 小于4.0 时, 其生长受到抑制, 特别是pH 降到2.0 以下时, 大肠杆菌O157:H7 完全不能生长, 由此可以说明大肠杆菌O157:H7 耐酸性能力较强, 可以抵御酸性环境的影响。扫描电镜图显示大肠杆菌O157:H7 在不同pH 的环境条件下其菌体形态会作出相应的变化,随着培养基的酸性增强, 其细胞形态从长杆菌体形态变成了短杆状或钝圆形态。结论 本研究测定不同酸性条件下大肠杆菌O157:H7 的生存和繁殖能力的研究, 有利于帮助人们进一步了解其耐酸性, 从而为制定防治方案和措施提供参考。     

Increased acid tolerance of Escherichia coli O157:H7 as affected by acid adaptation time and conditions of acid challenge

Three strains of Escherichia coli O157:H7, ATCC 43889, 43895 and 933 were subjected to acid adaptation in Tryptic Soy Broth (pH 5.0) for 1, 2, 3, 4 and 6 h. Acid tolerance of the adapted cells was determined in subsequent acid challenge at pH 3.0, 4.0 and 5.0 (acidified with HCl) and in the presence of lactic, acetic or propionic acid. It was found that acid adaptation increased acid tolerance of the E. coli O157:H7 strains tested and was dependent on strain, acid adaptation time and pH of the challenge. Among the acid adaptation times tested, 4 h of adaptation enabled the test organism, regardless of strains, to exhibit the most pronounced acid adaptation response which was most marked at pH 3.0, followed by pH 4.0 and 5.0. The extent of increased acid tolerance varied with the strains of E. coli O157:H7 and challenge of organic acid. The 4-h acid-adapted cells of ATCC 43889 and 933 showed an increase in acid tolerance in the presence of lactic, acetic and propionic acids. An increase in tolerance was also noted with ATCC 43895 in the presence of acetic and lactic acid, but not in the presence of propionic acid.     

纺织品中大肠杆菌O157:H7的检测方法

为建立一种灵敏、特异、快速、高效地鉴定纺织品基质中大肠杆菌O157:H7的方法,筛选出大肠杆菌O157:H7特有的rfbE基因保守序列,设计PCR特异性引物和荧光双标记探针,结合免疫磁珠技术,集成创新开发一种目标菌低浓度、不可培养情况下的样品中高效、快速富集分离大肠杆菌O157:H7的技术,建立了一种针对纺织品基质的免疫磁珠富集-实时荧光定量PCR方法,其检测下限可达8 CFU/mL。利用该方法对收集到的30份阳性样品进行鉴定,鉴定结果与传统方法结果100%吻合。结果表明,新建方法稳定性强,实现了纺织品中大肠杆菌O157:H7的快速灵敏鉴定。     

Effect of pH-dependent,stationary phase acid resistance on the thermal tolerance of Escherichia coli O157:H7

The ability of pH-dependent, stationary phase acid resistance to cross-protect Escherichia coli O157:H7 against a subsequent lethal thermal stress was evaluated using microbiological media and three liquid foods. Three strains were grown for 18 h at 37°C in acidogenic (TSB+G, final pH 4·6–4·7) and non-acidogenic (TSB-G, final pH 7·0–7·2) media to provide stationary phase cells with and without induction of pH-dependent acid resistance. The cells were then heated in BHI broth (pH 6·0) at 58°C, using a submerged coil apparatus. The TSB+G grown strains had greatly increased heat resistance, with the heating time needed to achieve a five-log inactivation, being increased two- to four-fold. The z -values of TSB+G and TSB-G grown cells were 4·7°C and 4·3°C, respectively. Increases in heat resistance with TSB+G-grown E. coli O157:H7 were also observed using milk and chicken broth, but not with apple juice. However, cross-protection was restored if the pH of the apple juice was increased from 3·5 to 4·5. The data indicate that pH-dependent acid resistance provides E. coli O157:H7 with cross-protection against heat treatments, and that this factor must be considered to estimate this pathogen's thermal tolerance accurately.     

Factors affecting the extreme acid resistance of Escherichia coli O157:H7

When stationary phase Escherichia coli O157:H7 cells were subjected to extreme acid shock (pH 2·0, 6 h, 37°C) cell survival was as great as 10%, but culture conditions greatly affected the acid resistance. Anaerobic cultures were more resistant to extreme acid shock if the glucose concentration of the growth medium was high, acids accumulated, and pH declined. By varying pH and acetate concentration, it was possible to demonstrate a high correlation (R²=0·86) between undissociated acetate and extreme acid resistance. Because dissociated acetate and extreme acid resistance were poorly correlated (R²<0·01), it appeared that the pH effects were being mediated via acetate dissociation. Propionate and butyrate were as effective as acetate, but formate, lactate, benzoate and the uncoupler, carbonylcyanide m -chlorophenylhydrazone (CCCP), were much less effective in promoting extreme acid-resistance. Acetate, propionate, butyrate, benzoate and CCCP all decreased the intracellular pH of E. coli O157:H7, but the correlation between intracellular pH and extreme acid resistance was low (R²<0·01). Cultures grown aerobically only needed half as much acetate to induce extreme acid resistance as those grown anaerobically, and the addition of the reducing agent, cysteine, to anaerobic media made the stationary phase cells less responsive to acetate. An rpoS mutant of E. coli O157:H7 was at least 100-fold more sensitive to acid shock than the wild-type, and large amounts of acetate were needed to promote even a small increase in viability.     

Protective effect of exopolysaccharide colanic acid of Escherichia coli O157:H7 to osmotic and oxidative stress

Many strains of Escherichia coli O157:H7 produce, under stress, an exopolysaccharide (EPS) comprised of colanic acid (CA) and form mucoid colonies on minimal glucose agar (MGA) at ambient temperature. Previous research conducted in our laboratory involving a CA-proficient (W6-13) and a CA-deficient (M4020; wcaD::Ekan(r)) strain of E. coli O157:H7 revealed that CA conferred acid and heat tolerance to E. coli O157:H7. Cells covered with CA were more persistent during acid (pH 4.5, 5.5, and 6.5) and heat (55 and 60 degrees C) treatment. The goal of this research was to study the effect of CA on the fate of E. coli O157:H7 under osmotic and oxidative stress. Cells of W6-13 and M4020 were exposed to various concentrations of NaCl (0.5, 1.5, and 2.5 M) and H2O2 (0, 10, and 20 mM) in minimal glucose broth (MGB) at 22 degrees C. Viable counts of E. coli O157:H7 were determined within 48 h of the osmotic stress and 3 h of the oxidative stress. The results suggest that cells of E. coli O157:H7 deficient in CA production are more susceptible than its wild-type parent to NaCl ( P< 0.05) and H2O2 (P< or = 0.05). This indicates that CA plays a role in protecting E. coli O157:H7 from osmotic and oxidative stress.     

Cross-contamination of lettuce with Escherichia coli O157:H7

Contamination of produce by bacterial pathogens is an increasingly recognized problem. In March 1999, 72 patrons of a Nebraska restaurant were infected with enterohemorrhagic Escherichia coli (EHEC) O157:H7, and shredded iceberg lettuce was implicated as the food source. We simulated the restaurant's lettuce preparation procedure to determine the extent of possible EHEC cross-contamination and growth during handling. EHEC inoculation experiments were conducted to simulate the restaurant's cutting procedure and the subsequent storage of shredded lettuce in water in the refrigerator. All lettuce pieces were contaminated after 24 h of storage in inoculated water (2 x 10(9) CFU of EHEC per 3 liters of water) at room temperature or at 4 degrees C; EHEC levels associated with lettuce increased by > 1.5 logs on the second day of storage at 4 degrees C. All lettuce pieces were contaminated after 24 h of storage in water containing one inoculated lettuce piece (approximately 10(5) CFU of EHEC per lettuce piece) at both temperatures. The mixing of one inoculated dry lettuce piece with a large volume of dry lettuce, followed by storage at 4 degrees C or 25 degrees C for 20 h resulted in 100% contamination of the leaves tested. Microcolonies were observed on lettuce stored at 25 degrees C, while only single cells were seen on leaves stored at 4 degrees C, suggesting that bacterial growth had occurred at room temperature. Three water washes did not significantly decrease the number of contaminated leaves. Washing with 2,000 mg of calcium hypochlorite per liter significantly reduced the number of contaminated pieces but did not eliminate contamination on large numbers of leaves. Temperature abuse during storage at 25 degrees C for 20 h decreased the effectiveness of the calcium hypochlorite treatment, most likely because of bacterial growth during the storage period. These data indicate that storage of cut lettuce in water is not advisable and that strict attention must be paid to temperature control during the storage of cut lettuce.     

抗大肠杆菌O157:H7的卵黄特异性IgY的研究

以大肠杆菌O157:H7为抗原免疫产蛋母鸡,从鸡卵黄中提取免疫球蛋白,建立抗大肠杆菌O157:H7的特异性IgY的效价检测方法,并研究母鸡的免疫应答性,以及抗体的提取方法和体外抑菌效果.研究结果表明,初次免疫后第6d,在卵黄中可以检测到抗大肠杆菌O157:H7 IgY,效价为1:7200;经加强免疫后效价迅速上升,至第44d达到最高效价1:230400;免疫后360 d,效价仍维持在1:7200.用水稀释法、硫酸铵分级盐析和Sephadex G-25凝胶过滤以提取IgY,提纯后IgY的效价是之前的4倍.SDS-PAGE鉴定抗体的纯度,电泳图谱中出现抗体的轻链和重链两条带.体外抑菌实验表明,IgY能抑制大肠杆菌O157:H7的生长.     

Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

Acid tolerance and gad mRNA levels of Escherichia coli O157:H7 grown in foods

We examined the acid tolerance and gad mRNA levels of Escherichia coli O157:H7 (three strains) and nonpathogenic E. coli (strains K12, W1485, and B) grown in foods. The E. coli cells (approximately 30,000 cells) were inoculated on the surface of 10 g of solid food samples (asparagus, broccoli, carrot, celery, cucumber, eggplant, ginger, green pepper, onion, potato, radish, tomato and beef) and in 10 ml of cow's milk, cultured statically at 10-25 degrees C for 1-14 days, and subjected to an acid challenge at 37 degrees C for 1 h in LB medium (pH 3.0). When grown at 20 and 25 degrees C in all foods, except for tomato and ginger, the strains showed a stationary-phase specific acid tolerance. The acid tolerance of the O157 strains changed depending on the types of foods (3-10% survival), but was clearly lower than that of the cells grown in EC medium (more than 90% survival). Tomato and ginger induced relatively high acid tolerances (10-30% survival) in the O157 strains irrespective of the growth phase, probably because of their acidity. No remarkable difference was observed in the acid tolerance between the O157 and nonpathogenic strains grown in all foods. When grown at 10 and 15 degrees C in the foods and EC medium, none of the strains showed the stationary-phase specific acid tolerance. In beef, broccoli, celery, potato and radish, the acid tolerance showed a tendency to decrease with the prolonged cultivation time. In other foods, the acid tolerance was almost constant (about 0.1% survival) irrespective of the growth stage. The mRNA level of glutamate decarboxylase genes (gadA and gadB) correlated to the acid tolerance level when the E. coli cells were grown at 25 degrees C, but was very low even in the stationary phase when the E. coli cells were grown at 15 degrees C or below.