牛源性大肠杆菌O157:H7的分离鉴定及其耐酸性比较研究

为比较大肠杆菌（Escherichia coli）O157:H7产毒菌株耐受盐酸和乳酸的差异性,首先采集309 份牛粪及牛肉样,进行菌株分离鉴定,接着采用多重聚合酶链式反应（polymerase chain reaction,PCR）方法检测分离株及其他收集菌株的4 种毒力基因（eae、hly、stx1、stx2）,进而对携带毒力基因的产毒菌株分别进行盐酸和乳酸应激实验。结果表明：共分离鉴定出8 株大肠杆菌O157菌株,样品阳性检出率为2.59%;毒力基因检测表明,8 株菌株均不携带stx1和stx2基因,其中6 株菌株携带eae及hly基因;所有产毒菌株耐酸性实验结果表明,盐酸或乳酸处理2 h后20 株产毒菌株存活菌数均显著下降（P＜0.05）,但下降程度呈现明显的菌株差异性,同一菌株对盐酸、乳酸呈现明显的耐受差异。     

酸应激对大肠杆菌O157:H7生物菌膜形成的影响

本实验探究较长时间酸应激对大肠杆菌O157:H7生物菌膜形成的影响。首先采用微孔板联合结晶紫染色法比较大肠杆菌O157:H7不同菌株黏附性能差异,分析不同黏附力菌株菌膜形成曲线,进而选择代表菌株采用平板计数法分析在较长时间酸应激时其菌膜的形成规律,最后采用共聚焦激光扫描显微镜（confocal laser scanning microscope,CLSM）比较黏附力不同的菌株在酸性环境下菌膜形态结构变化。结果表明,14 株菌株黏附能力有差异;不同黏附力菌株均在2 h开始产生黏附现象,但菌膜形成曲线有明显差异。以中等黏附力菌株ATCC43895作为代表菌株进行酸应激实验,结果表明pH值越低菌膜形成量越少,pH值相同时乳酸对浮游菌数和菌膜形成的抑制效应显著高于盐酸（P＜0.05）。CLSM观察结果表明,成膜能力较强的菌株J29和较弱的菌株CICC21530在中性和酸性培养液中均能形成一定结构的生物菌膜,但前者的菌膜形成量多于后者,酸性条件对成膜过程有抑制作用。提示乳酸能有效抑制大肠杆菌O157:H7菌膜形成过程,可为食品实际加工中该菌菌膜的消除技术提供科学思路。     

乙酰辅酶A羧化酶对乳酸片球菌耐酸性能的影响

通过对来源于山西老陈醋固态酿造过程中的乳酸菌分离株的耐醋酸性能分析,筛选得到了醋酸耐受性较优的乳酸片球菌（Pediococcus acidilactici）AAF3-3,该菌株对其他种类的酸压力（乳酸和盐酸）也表现出一定的耐受性。利用定量实时聚合酶链反应（qRT-PCR）对该菌株中乙酰辅酶A羧化酶基因（acc）的转录水平进行分析发现,其在醋酸、乳酸和盐酸下的转录水平分别是无压力条件下的4.32、2.02和1.13倍,均出现了明显上调。进一步构建该基因的过表达菌株并进行耐酸性能分析。结果表明,与对照菌株相比,acc过表达菌株在3种酸压力下的生长性能和存活性能均有所提高,其中,3%（V/V）醋酸条件下培养24 h时过表达菌株的相对活菌数是对照菌株的24倍。这表明乙酰辅酶A羧化酶在乳酸片球菌AAF3-3耐受不同种类的酸压力中发挥着重要作用。     

盐应激对大肠杆菌O157:H7存活和毒力基因表达的影响

探讨不同盐应激水平对大肠杆菌O157:H7存活和毒力基因表达的影响,并分析两者之间的相关性,选取本实验室收集的3株产毒大肠杆菌O157:H7菌株(CICC21530、95和109),于不同NaCl添加量(0、6、12、18 g/100 mL)胰蛋白胨大豆肉汤中应激不同时间,进行细菌培养计数及实时聚合酶链反应检测毒力基因表达情况。结果显示,盐应激显著抑制了3株大肠杆菌O157:H7的存活(P0.05),抑制效应存在菌株差异,菌株CICC21530 NaCl添加量越高抑制越明显,而菌株95和109则呈现波动性变化。大肠杆菌O157:H7毒力基因表达的变化也与菌株、NaCl添加量有关。较高NaCl添加量时,3株菌存活数显著降低的同时,毒力基因表达量却显著增加(P0.05),其中菌株CICC21530和菌株95的18 g/100 mL NaCl处理组毒力基因表达量最高,菌株109的12 g/100 mL NaCl处理组毒力基因表达量最高。结果表明盐应激时大肠杆菌O157:H7存活与毒力基因表达的变化不完全一致,存活菌数下降的同时,毒力却会增强,提示在实际含盐食品风险评估中,不仅要关注存活菌量,还需重视残存菌的毒力水平,从而更科学全面地评估大肠杆菌O157:H7的安全风险。     

酸应激对1株乳酸乳球菌质膜F_1F_0-ATPase活性的影响

评价10株乳酸乳球菌的酸应激能力,选用应激能力较高的菌株,确定其最佳酸应激条件。将4 h、10 h、20 h培养的该菌菌体在此条件下应激,以硫酸亚铁钼蓝比色法测定应激前后的质膜F_1 F_0-ATPase活性。结果表明,菌株KLDS 4.0312具有较高应激能力,应激后耐酸性可提高7542倍,其最佳酸应激条件为pH4.5,30 min。4 h、10 h、20 h培养的该菌菌体应激后,质膜F_1F_0-ATPase活性分别提高56.68%、11.05%、5.16%。     

产PrtS蛋白酶嗜热链球菌的筛选及其生长特性研究

从商业发酵剂中初步分离出8株疑似嗜热链球菌,与实验室保存的26株嗜热链球菌,共收集到34株菌。通过PCR反应获得具有PrtS基因的一株菌,利用FSDA培养基确定该菌株具有PrtS蛋白酶活性,经生理生化实验和16S rDNA序列同源性分析该菌株为嗜热链球菌,命名为Streptococcus thermophiles 922。该菌株在LM17培养基中培养10 h时,活菌数达到9.68 log cfu/mL;在脱脂乳培养基中,产酸速度快,产酸量较高。实验结果为深入探究PrtS蛋白酶对嗜热链球菌生长和发酵性能影响提供基础。     

出血性大肠杆菌O157:H7国内分离株耐酸性的研究

EHEC O157：H7难以控制的原因之一是该菌对酸的耐受性。将国内6株EHEC O157：H7在不同pH的LB溶液中振荡培养，每隔1h计数，实验结果表明了6株菌耐受时间相比较存在差异，菌株97063较其它菌株在pH2．5条件下，耐受时间长超过了29h。本实验旨在对EHEC O157：H7的国内分离株耐酸性进行研究，为控制EHEC O157：H7的感染提供科学依据。检验检疫部门、食品卫生部门等应高度重视对酸性食品中大肠杆菌O157：H7的检验。     

耐酸和酸敏感酒酒球菌突变株酸胁迫下膜脂肪酸差异分析

利用气质联用(GC)法测定了两株不同酸耐受表型酒酒球菌(Oenococcus oeni)突变株在酸胁迫及无酸胁迫条件下不同生长时期的细胞膜脂肪酸种类与含量，并对两株菌中引起膜脂肪酸差异关键基因fabF在酸压力响应中的功能进行异源验证。结果表明，pH 3.0条件下在培养3 h、36 h及108 h的酸耐受突变株b1和酸敏感突变株b2的2-己基环丙烷-1-癸酸(C_19:1)含量均显著高于pH 4.8条件下的C_19:1含量(P<0.05)；培养3 h及36 h的菌株b1的2-己基-环丙烷辛酸(C_17:1)、C_19:1含量及总不饱和脂肪酸与总饱和脂肪酸比值均显著高于菌株b2(P<0.05)。此外，pH 3.0条件下培养36 h时，仅在突变株b1中检测到C_17:1；培养108 h时，菌株b1的C_17:1含量约高于菌株b2的7倍。酸胁迫条件下，过表达菌株b1、b2的fabF基因的重组植物乳杆菌(Lactobacillus plantarum)L1、L2的稳...     

云南撒尼地区乳饼及酸乳清中优良乳酸菌的筛选和发酵性能研究

选取SDC4-1、MDC3-1、WDC3-1 3株从云南撒尼地区乳饼和酸乳清中分离鉴定的乳酸菌株,用耐酸、耐胆盐实验筛选其中的优势菌株;并用产酸能力、后酸化、产乙醛能力、蛋白水解酶测定和β-半乳糖苷酶测定等实验研究优势菌株的发酵性能。3株菌均能耐酸,其中MDC3-1耐酸能力最高,达254%,WDC4-1最低,为52%,但仅SDC4-1可耐受胆盐,在0.5%的胆盐中,4 h后的存活率为81%,SDC4-1是优势菌。SDC4-1发酵性能实验中,SDC4-1菌株具有很好的凝乳状态,发酵酸奶口感风味浓郁,在贮藏过程中活菌数含量高,冷藏15 d后活菌数达到了9.79 lg CFU/g,远超国家标准。产酸较快,13 h时,酸度已达82.0°T,后酸化性好,产乙醛能力较好,蛋白水解能力强,产β-半乳糖苷酶的量高,达7.7μg/mol,能保证酸奶在冷藏过程中品质稳定,因而筛选出具有优良发酵特性的菌株SDC4-1可以为发酵食品提供更广泛的素材。     

Effect of acid shock with hydrochloric, citric, and lactic acids on the survival and growth of Salmonella typhi and Salmonella typhimurium in acidified media

The effect of acid shock with hydrochloric, citric, or lactic acid on the survival and growth of Salmonella Typhi and Salmonella Typhimurium in acidified broth was evaluated. Salmonella serovars were acid shocked (1 h at 35 degrees C) in Trypticase soy broth acidified with hydrochloric, citric, or lactic acid at pH 5.5. Unshocked cells were exposed to the same media that had been neutralized before use to pH 7.0. Shocked and unshocked cells were inoculated into broth acidified with hydrochloric acid (pH 3.0), citric acid (pH 3.0), or lactic acid (pH 3.8), and growth and survival ability were evaluated. The acid shock conferred protection to Salmonella against the lethal effects of low pH and organic acids. The adaptive response was not specific to the anion used for adaptation. The biggest difference in reduction of survival between shocked and unshocked strains (approximately 2 log CFU/ml) was observed when the microorganisms were shocked with lactic acid and then challenged with citric acid. Salmonella Typhi was more tolerant of citric acid than was Salmonella Typhimurium, but Salmonella Typhimurium had higher acid tolerance in response to acid shock than did Salmonella Typhi. The acid shock decreased the extension of the lag phase and enhanced the physiological state values of Salmonella Typhi and Salmonella Typhimurium when the pH of growth was 4.5. This increased ability to tolerate acidity may have an important impact on food safety, especially in the case of Salmonella Typhi, given the very low infectious dose of this pathogen.     

The Acetic Acid Tolerance Response induces cross-protection to salt stress in Salmonella typhimurium

Salmonella typhimurium induces an Acid Tolerance Response (ATR) upon exposure to mildly acidic conditions in order to protect itself against severe acid shock. This response can also induce cross-protection to other stresses such as heat and salt. We investigated whether both the acetic acid induced and lactic acid induced ATR in S. typhimurium provided cross-protection to a salt stress at 20 degrees C. Acid-adapted cells were challenged with both a sodium chloride (NaCl) and potassium chloride (KCl) shock and their ability to survive ascertained. Acetic acid adaptation provided cells with protection against both NaCl and KCl stress. However, lactic acid adaptation did not protect against either osmotic stressor and rendered cells hypersensitive to NaCl. These results have implications for the food industry where hurdle technology means multiple sub-lethal stresses such as mild pH and low salt are commonly used in the preservation of products.     

渗透压胁迫对耐盐乳酸菌发酵特性的影响

采用传统分离培养法从新疆伊犁昭苏县土壤样品中分离筛选耐盐乳酸菌,通过形态观察及分子生物学技术对其进行鉴定,并探究高盐胁迫（7%、8% NaCl）下其生长速率、产酸性能、耐酸、耐胆盐胁迫能力的变化。结果表明,筛选得到4株耐盐乳酸菌,其中菌株a、b、c被鉴定为海氏肠球菌（Enterococcus hirae）,菌株d被鉴定为植物乳杆菌（Lactobacillus plantarum）。在高盐胁迫（7%、8% NaCl）下,L. plantarum d的生长特性、产酸及耐酸性能最好,但耐胆盐能力最差,E. hirae a、b、c的耐胆盐胁迫能力更佳;在10%和12%的NaCl含量下胁迫3 h,4株菌的存活率均能维持在36%以上,其中E. hirae b的耐盐能力最佳,存活率最高。     

Genotypic diversity of stress response in Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus

Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum are three closely related species which are widespread in food and non-food environments, and are important as starter bacteria or probiotics. In order to evaluate the phenotypic diversity of stress tolerance in the L. plantarum group and the ability to mount an adaptive heat shock response, the survival of exponential and stationary phase and of heat adapted exponential phase cells of six L. plantarum subsp. plantarum, one L. plantarum subsp. argentoratensis, one L. pentosus and two L. paraplantarum strains selected in a previous work upon exposure to oxidative, heat, detergent, starvation and acid stresses was compared to that of the L. plantarum WCFS1 strain. Furthermore, to evaluate the genotypic diversity in stress response genes, ten genes (encoding for chaperones DnaK, GroES and GroEL, regulators CtsR, HrcA and CcpA, ATPases/proteases ClpL, ClpP, ClpX and protease FtsH) were amplified using primers derived from the WCFS1 genome sequence and submitted to restriction with one or two endonucleases. The results were compared by univariate and multivariate statistical methods. In addition, the amplicons for hrcA and ctsR were sequenced and compared by multiple sequence alignment and polymorphism analysis. Although there was evidence of a generalized stress response in the stationary phase, with increase of oxidative, heat, and, to a lesser extent, starvation stress tolerance, and for adaptive heat stress response, with increased tolerance to heat, acid and detergent, different growth phases and adaptation patterns were found. Principal component analysis showed that while heat, acid and detergent stresses respond similarly to growth phase and adaptation, tolerance to oxidative and starvation stresses implies completely unrelated mechanisms. A dendrogram obtained using the data from multilocus restriction typing (MLRT) of stress response genes clearly separated two groups of L. plantarum strains from the other species but there was no correlation between genotypic grouping and grouping obtained on the basis of the stress response pattern, nor with the phylograms obtained from hrcA and ctsR sequences. Differences in sequence in L. plantarum strains were mostly due to single nucleotide polymorphisms with a high frequency of synonymous nucleotide changes and, while hrcA was characterized by an excess of low frequency polymorphism, very low diversity was found in ctsR sequences. Sequence alignment of hrcA allowed a correct discrimination of the strains at the species level, thus confirming the relevance of stress response genes for taxonomy.     

Induced expression of the heat shock protein genes uspA and grpE during starvation at low temperatures and their influence on thermal resistance of Escherichia coli O157:H7

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37 degrees C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52 degrees C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37 degrees C, for 24 h at 10 degrees C, and for 2 days at 5 degrees C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5 degrees C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37 degrees C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.     

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.     

Relationship between the effects of stress induced by human bile juice and acid treatment in Vibrio cholerae

The effects of low pH and human bile juice on Vibrio cholerae were investigated. A mild stress condition (exposure to acid shock at pH 5.5 or exposure to 3 mg of bile per ml for 20 min) slightly decreased (by < or = 1 log unit) V. cholerae cell viability. However, these treatments induced tolerance to subsequent exposures to more severe stress. In the O1 strain, four proteins were induced in response to acid shock (ca. 101, 94, 90, and 75 kDa), whereas only one protein (ca. 101 kDa) was induced in response to acid shock in the O139 strain. Eleven proteins were induced in response to bile shock in the O1 strain (ca. 106, 103, 101, 96, 88, 86, 84, 80, 66, 56, and 46 kDa), whereas only one protein was induced in response to bile shock in the O139 strain (ca. 88 kDa). V. cholerae O1 and O139 cells that had been preexposed to mild acid shock were twofold more resistant to pH 4.5 (with times required to inactivate 90% of the cell population [D-values] of 59 to 73 min) than were control cells (with D-values of 24 to 27 min). Likewise, cells that were preexposed to mild bile shock (3 mg/ml) were almost twofold more tolerant of severe bile shock (30 mg/ml; D-values, 68 to 87 min) than were control cells (with D-values of 37 to 43 min). These protective effects persisted for at least 1 h after the initial shock but were abolished when chloramphenicol was added to the culture during the shock. Cells preexposed to acid shock exhibited cross-protection against subsequent bile shock. However, cells preexposed to bile shock exhibited no changes in acid tolerance. Bile shock induced a modest reduction (0 to 20%) in enterotoxin production in V. cholerae, whereas acid shock had no effect on enterotoxin levels. Adaptation to acid and bile juice and protection against bile shock in response to preexposure to acid shock would be predicted to enhance the survival of V. cholerae in hosts and in foods. Thus, these adaptations may play an important role in the development of cholera disease.     

Impact of antibiotic stress on acid and heat tolerance and virulence factor expression of Escherichia coli O157:H7

This study was conducted to determine the effect of antibiotic stress on the virulence factor expression, simulated gastric fluid (SGF; pH 1.5) survival, and heat tolerance (56 degrees C) of Escherichia coli O157:H7. The MIC for three antibiotics (trimethoprim, ampicillin, and ofloxacin) was determined for two E. coli O157:H7 strains (ATCC 43895 [raw hamburger isolate] and ATCC 43890 [fecal isolate]) by the dilution series method. Subsequently, cells were stressed at the MIC of each antibiotic for 4 h, and poststress tolerance and virulence factor production were evaluated. Heat tolerance (56 degrees C) was determined by the capillary tube method, and SGF (pH 1.5) survival was used to assess acid tolerance. Virulence factor expression (stx, hlyA, and eaeA) was evaluated by the creation of lacZ gene fusions and then use of the Miller assay (a beta-galactosidase assay). Stressed and control cells were evaluated in triplicate. The MIC for trimethoprim was 0.26 mg/liter for both strains; for ampicillin, it was 2.05 mg/liter for both strains; and for ofloxacin, it was 0.0256 and 0.045 mg/liter for each strain. Heat tolerance and SGF survival following antibiotic stress decreased when compared with control cells (P < 0.05). Exposure to ofloxacin increased stx and eaeA expression (P < 0.05). Exposure to ampicillin or trimethoprim increased eaeA expression (P < 0.05). hly expression increased following trimethoprim stress (P < 0.05). Antibiotics can increase E. coli O157:H7 virulence factor production, but they do not produce a cross-protective response to heat or decreased pH.     

Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses

The majority of published studies on the adaptive heat or acid tolerance response of Listeria monocytogenes have been performed with a single strain exposed to a single adaptation treatment; however, in food ecosystems, microorganisms commonly exist as multi-species communities and encounter multiple stresses, which may result in "stress hardening". Therefore, the present study evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (T; 46 degrees C) stresses, sequentially or simultaneously within 1.5h, in tryptic soy broth with 0.6% yeast extract (TSBYE). All treatments induced adaptive responses on L. monocytogenes at 57 degrees C, while no such cross-protection was observed at 52 and 63 degrees C. Survivor curves at 57 degrees C appeared convex with profound shoulders determined by a Weibull model. The highest thermotolerance was observed after combined exposure to acid and heat shock (pH-T), followed by exposure to osmotic shock, and by the combination of osmotic with heat shock (NaCl-T). Regarding acid tolerance, prior exposure to low pH, pH-T, or a combination of NaCl, pH and T resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log(10)CFU ml(-1)) than the control cultures. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses (e.g., NaCl-pH-T, NaCl-T and NaCl-pH) resulted in higher survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. The results indicate that combinations and sequences of sublethal hurdles may affect L. monocytogenes acid and heat tolerance, especially in acidic environments with mild heating or in low moisture environments.