双组分调控系统及其对细菌诱导性耐酸响应调控机理的研究进展

双组分调控系统（two-component regulatory system,TCS）是维持细菌在压力环境中存活的重要结构。食品加工过程极易产生高渗、弱酸等压力环境,双组分系统能够帮助细菌感受外部环境的胁迫,及时动员体内对抗机制,这一过程容易产生耐酸、耐渗透压、耐高温甚至是高毒性的菌株,威胁食品安全。本文主要对TCS的结构组成、识别信号及调控作用等进行概述,并与细菌诱导耐酸响应（acid tolerance response,ATR）中的酸休克蛋白、细胞膜系统和氨基酸代谢等产生机制进行联系,综述了细菌在酸性条件下通过TCS响应信号分子激发ATR的具体过程。     

乙酰丙酸对沙门氏菌诱导性耐酸响应

本实验探究了鼠伤寒沙门氏菌（Salmonella typhimurium）和德尔卑沙门氏菌（S. Derby）在pH值为6.0、5.4的乙酰丙酸、L-乳酸、醋酸和盐酸条件下酸适应后产生的耐酸性。结果表明,酸化剂对沙门氏菌抑菌效果为：乙酰丙酸＞醋酸＞L-乳酸＞盐酸。pH 5.4时,沙门氏菌的诱导耐酸显著高于pH 6.0（P＜0.05）;与其他两种有机酸处理组相比,乙酰丙酸诱导的细菌耐酸性最弱且不同pH值之间差异显著（P＜0.05）。有机酸处理的沙门氏菌细胞内pH值与对照组相比存在差异,其中pH 6.0时乙酰丙酸处理组的鼠伤寒沙门氏菌的细胞内pH值显著高于德尔卑沙门氏菌（P＜0.05）。结果表明沙门氏菌经pH 6.0和pH 5.4的酸适应后会产生耐酸性,而细胞内pH值的变化和诱导耐酸密切相关。     

基于转录组学分析酸胁迫影响鼠伤寒沙门氏菌耐酸能力的机理

目的：探究酸胁迫和非酸胁迫条件下鼠伤寒沙门氏菌（Salmonella typhimurium）的转录组反应,分析差异基因（differentially expressed genes,DEGs）表达水平,阐明酸胁迫影响鼠伤寒沙门氏菌耐酸反应（acid tolerance response,ATR）的相关代谢通路。方法：对鼠伤寒沙门氏菌进行酸胁迫处理,利用转录组测序技术和生物信息学分析相关DEGs,并通过实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）进行验证。结果：经酸胁迫后,共筛选到683 个DEGs,其中上调343 个,下调340 个。其中涉及细胞运动、氨基酸代谢、细胞膜组成等通路上调能够使鼠伤寒沙门氏菌快速适应酸环境;碳水化合物代谢相关通路上调能够为鼠伤寒沙门氏菌快速适应酸环境提供更多的能量,与此同时,嘧啶代谢等能量代谢通路下调能够使鼠伤寒沙门氏菌降低能量消耗以维持上述的必需代谢过程;细菌应激调控相关通路上调赋予鼠伤寒沙门氏菌交叉保护抗性;鞭毛、外膜蛋白、脂多糖等毒力相关基因表达上调增强了鼠伤寒沙门氏菌的毒力。real-time PCR验证结果与转录组测序分析表达趋势一致。结论：酸胁迫显著提高了鼠伤寒沙门氏菌的耐酸能力,其中与代谢和细胞过程相关的通路发挥主要作用,本研究结果为进一步了解该菌的酸胁迫反应及更好地控制其在食品中的污染提供了理论依据。     

沙门氏菌和单增李斯特菌诱导性耐酸响应机制的研究进展

沙门氏菌和单增李斯特菌被认为是肉制品中最重要的食源性致病菌。它们在弱酸环境下会发生强烈的诱导性耐酸响应,同时诱导产生高毒、耐酸、耐渗透压的高危菌株,是影响消费者健康安全的重大隐患。本文主要从沙门氏菌和单增李斯特菌产生诱导耐酸的发现过程、诱导耐酸响应的危害、产生诱导耐酸的影响因素方面进行概述,进一步从pH值稳态系统、应激蛋白分子的调控及细胞膜组成和流动性调控的角度分析了产生诱导耐酸响应的分子机制。     

发酵鱼酱酸产GABA乳酸菌的分离筛选及发酵特性

从传统发酵鱼酱酸中筛选出产γ-氨基丁酸（γ-aminobutyric acid，GABA）的乳酸菌并分析其菌株发酵特性。通过薄层色谱法定性、Berthelot比色法定量获得产GABA菌株，并进行耐酸、耐胆盐、氨基酸脱羧酶活性、抑菌性、生长曲线及pH值、产酸速率等菌株发酵特性分析。结果表明：从分离自鱼酱酸不同发酵阶段的387 株乳酸菌中，获得15 株典型产GABA菌株，包括2 株食窦魏斯氏菌（Weissella cibaria）、1 株熊蜂魏斯氏菌（Weissella bombi）、11 株植物乳杆菌（Lactobacillus plantarum）和1 株戊糖乳杆菌（Lactobacillus pentosus）。15 株乳酸菌产GABA能力及发酵特性在主成分分析图上差异显著，其中食窦魏斯氏菌Y113产GABA量为0.239 mg/mL，高于其他菌株；植物乳杆菌Y279和Y64展现出较好的耐酸性、耐胆盐性、抑菌性、无氨基酸脱羧酶活性、生长速率及产酸速率快的特点。鉴于其优良的发酵特性、益生特性及产GABA能力，菌株Y279可作为鱼酱酸工业化、标准化生产的潜在优良菌株。     

耐酸乳酸菌的筛选及生长特性研究

从徐州地区天然发酵泡菜中筛选出一株耐酸能力较强的菌株HLAB0143,该菌株能在pH2.5的条件下生长;通过形态、生理生化和16sDNA序列测定,确认该菌株为干酪乳杆菌;在单因素实验的基础上进行正交实验,确定了该菌株的最佳生长条件,即温度为37℃,初始pH为5,接种量为1%(v/v)。对其耐酸机制进行了初步研究,认为谷氨酸脱羧酶活性是其耐受低酸性环境的机制之一。     

全基因组测序和real-time PCR法检测食源性沙门氏菌parC、gyrA基因突变特征

以45 株食源性沙门氏菌喹诺酮耐药株为对象,采取全基因组测序和实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）法检测parC、gyrA基因突变位点,并对检测方法可靠性、耐药基因突变特征进行评估和分析。首先将4 株沙门氏菌进行二代全基因组测序,根据测序数据分析结果,建立了一种real-time PCR法检测gyrA Asp87Tyr、gyrA Asp87Asn、parC Thr57Ser和parC Ser80Ile这4 个突变位点。将沙门氏菌进行qnrS、qnrA、qnrB的real-time PCR检测,发现有31 株菌未检出qnr基因。以这31 株菌为对象,采取real-time PCR法筛查基因突变位点,结果发现parC Thr57Ser和gyrA Asp87Asn型突变最常见。将real-time PCR阳性的10 株菌扩增parC、gyrA基因全长并测序,real-time PCR检测和测序结果完全吻合,说明了real-time PCR检测的可靠性。全基因组测序和real-time PCR法相结合的方法用于耐药基因突变筛查,既可以发现新的基因突变,又可以快速筛查大样本的主要突变类型,可作为沙门氏菌耐药性研究的一种可靠手段。     

非预包装食品中沙门氏菌快速检测方法的建立

当前沙门氏菌传统的国家标准检测方法不能满足食品安全监管高效性的需求,尤其是针对非预包装食品这种较易腐败变质的食品。研究针对非预包装食品利用聚合酶链式反应（polymerase chain reaction,PCR）分子层析方法得出一个快速、简单、准确的沙门氏菌检测方法。通过对60批普通基质食品（面食制品、乳制品、肉制品）和40批复杂基质食品（凉菜类、生鲜蛋类）模拟不同程度被沙门氏菌污染的实验,采用国标法和PCR分子层析法对比检测。结果显示：普通基质食品PCR分子层析法检测灵敏度更高,可达1 CFU/mL～10 CFU/mL的阳性菌添加浓度;复杂基质食品尤其对生鲜蛋类食品,两种检测方法的灵敏度均较低。故对沙门氏菌的增菌液进行了优化,在沙门氏菌的增菌液缓冲蛋白胨水（buffered peptone water,BPW）中添加一定比例六水氯化镁,即利用提高增菌液渗透压和降低pH值抑制其他肠道菌群的生长的原理,使其检测灵敏度提高,由104CFU/mL的阳性菌添加浓度降低至102CFU/mL的添加浓度,灵敏度提高了100倍;经特异性试验验证,PCR分子层析法具有较强的抗干扰能力,样本体系中存在其他干扰菌株的脱氧核糖核酸（deoxyribonucleic acid,DNA）亦不会影响沙门氏菌的检测。PCR分子层析法检测时间只需22 h,对比需要6 d检测时间的传统国标方法的检测效率提高6倍。     

Taqman MGB PCR定量检测水产品中沙门氏菌的方法建立

建立采用Taqman MGB实时荧光PCR法快速定量检测水产品中沙门氏菌。根据沙门氏菌fimY基因保守序列,设计引物和Taqman MGB探针,建立Taqman MGB实时PCR定量检测体系。采用本方法对24株共14种血清型沙门氏菌和17株与沙门氏菌亲缘关系比较近,以及在样品中能同时存在的常见食源性致病菌菌株进行PCR扩增。结果显示：所有的沙门氏菌菌株结果均为阳性,而非沙门氏菌菌株检测结果均为阴性,反应特异性为100%。本方法的纯菌最低检测低限为13CFU/ml,样品江瑶贝和蚬子肉中添加肠炎沙门氏菌的最低检测低限为130CFU/ml;香螺肉中添加肠炎沙门氏菌的最低检测低限为1300CFU/ml。定量关系式为y=－3.381418lnx+45.115715,R2= 0.964878。整个实验2h即可完成,可应用于水产品中沙门氏菌污染状况调查及快速检测。     

实时荧光PCR法检测食品中亚利桑那沙门氏菌

沙门氏菌（Salmonella）是食品检测过程中最常见的致病菌之一,亚利桑那沙门氏菌（Salmonella arizonae）又是沙门氏菌中比 较难鉴定的亚种。该实验通过实时荧光聚合酶链式反应（PCR）方法快速准确的检测亚利桑那沙门氏菌和其他沙门氏菌。根据GenBank 公布的亚利桑那沙门氏菌和其他沙门氏菌gud D基因序列, 分别设计引物和Taqman探针。 使用10株不同血清型的沙门氏菌标准菌 株、88株沙门氏菌分离株和29株食品中常见食源性致病菌进行实时荧光PCR特异性实验。结果显示,该实验所设计的引物探针特异 性非常好。 实时荧光PCR灵敏性试验结果表明,检测灵敏度可达到1～10 CFU/mL的添加浓度。 经模拟污染样品验证,所建立的实时 荧光PCR方法与传统方法的检测结果相一致,具有检测周期短、操作简便的优势。     

冷诱导金黄色葡萄球菌环境胁迫耐受性及生物被膜控制

为研究金黄色葡萄球菌经冷诱导后对其他环境胁迫耐受性的变化,将耐冷冻能力较强的分离株S.aureus W3与标准菌株S.aureus CICC10201分别在4 ℃下冷胁迫5 h后,测定其在高温、酸碱、氯化钠、过氧化氢、酒精的胁迫下存活率变化。同时对分离株S.aureus W3的生物被膜形成及控制进行了研究。结果表明,经-20 ℃冷藏8周后,S.aureus W3的存活率显著高于S.aureus CICC10201(p<0.05)。经冷诱导后,S.aureus W3在45~60 ℃环境下存活率提升至18.5%。冷诱导使金黄色葡萄球菌对酸敏感性增强但对碱产生交叉抗性。冷诱导后的两株菌在氯化钠中的存活率均降低,S.aureus W3最低下降了24.09%。另外,冷诱导后金黄色葡萄球菌对酒精的耐受性增强。分离株S.aureus W3在冷诱导前后过氧化氢耐受性没有明显变化(p>0.05),但标准菌株S.aureus CICC10201敏感性增强,存活率显著下降(p<0.05)。分离株S.aureus W3较S.aureus CICC10201具有更强的生物被膜形成能力,运用1% 84消毒水能有效清除其生物被膜。本研究为冷冻食品中金黄色葡萄球菌的安全控制提供理论依据。     

Cell membrane damage induced by lacticin 3147 enhances aldehyde formation in Lactococcus lactis IFPL730

Amino acid catabolism is mainly initiated in Lactococcus lactis by a transamination reaction that leads to the formation of alpha-keto acids. In addition, a novel alpha-keto acid decarboxylase enzyme, rare in lactic acid bacteria, responsible for the conversion of alpha-keto acids into aldehydes has been reported in L. lactis IFPL730. The effect of lacticin 3147-induced cell damage on both amino acid transamination and alpha-keto acid decarboxylation by L. lactis IFPL730 leading to the formation of aldehydes from amino acids was investigated. Cell membrane permeabilization induced by lacticin 3147 facilitated the diffusion of amino acids into the cells and thus, enhanced amino acid transamination and formation of alpha-keto acids. However, alpha-keto acid decarboxylation was not affected by cell membrane permeabilization since decarboxylation of alpha-keto acids in both control and lacticin 3147-treated cells were similar, suggesting that these substrates could freely diffuse inside the cells. Nevertheless, the formation of 2-methylbutyraldehyde from isoleucine was enhanced in lacticin 3147-treated cells. The increase in alpha-keto acids formation rate by L. lactis IFPL730 due to lacticin 3147-induced cell damage, led to a concomitant increase in the subsequent decarboxylation reaction that complete the metabolic pathway to aldehyde production from amino acids. The present study points out to the use of the food grade lacticin 3147 along with L. lactis IFPL730 as a valuable tool in the development of cheese flavour.     

Comparative analysis of acid resistance between susceptible and multi-antimicrobial-resistant Salmonella strains cultured under stationary-phase acid tolerance-inducing and noninducing conditions

This study compared acid resistance levels among five antimicrobial-susceptible strains of Salmonella and five strains that were simultaneously resistant to a minimum of six antimicrobial agents. The induction of a stationary-phase acid tolerance response (ATR) was attempted by both transient low-pH acid shock and acid adaptation. For acid shock induction, strains were grown for 18 h in minimal E medium containing 0.4% glucose (EG medium) and exposed to sublethal acid stress (pH 4.3) for 2 h, and subsequently, both shocked and nonshocked cultures were acid challenged (pH 3.0) for 4 h. Acid adaptation was achieved by growing strains for 18 h in tryptic soy broth containing 1.0% glucose (TSB+G), while nonadapted cultures were grown for 18 h in glucose-free tryptic soy broth (TSB-G). Acid-adapted and nonadapted inocula were acid challenged (pH 2.3) for 4 h. Initial (0 h) mean populations of nonchallenged Salmonella were 8.5 to 8.7, 8.4 to 8.8, and 8.2 to 8.3 log CFU/ml for strains grown in EG medium, TSB-G, and TSB+G, respectively. After 4 h of acid challenge, mean populations were 3.0 to 4.8 and 2.5 to 3.7 log CFU/ml for previously acid-shocked susceptible and resistant strains, respectively, while corresponding counts for nonshocked strains were 4.3 to 5.5 log CFU/ml and 3.9 to 4.9 log CFU/ml. Following 4 h of acid exposure, acid-adapted cultures of susceptible and resistant strains had mean populations of 6.1 to 6.4 log CFU/ml and 6.4 to 6.6 log CFU/ml, respectively, while corresponding counts for nonadapted cultures were 1.9 to 2.1 log CFU/ml and 1.8 to 2.0 log CFU/ml, respectively. A low-pH-inducible ATR was not achieved through transient acid shock, while an ATR was evident following acid adaptation, as adapted populations were 4.2 to 4.8 log units larger than nonadapted populations following acid exposure. Although some strain-dependent variations in acid resistance were observed, results from this study suggest no association between susceptibility to antimicrobial agents and the ability of the Salmonella strains evaluated to survive low-pH stress.     

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.     

Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.     

酸适应海德尔堡沙门氏菌对环境胁迫耐受性分析

目的：研究海德尔堡沙门氏菌（Salmonella heidelberg）低pH值酸适应性及酸适应对细胞其他胁迫环境耐受性的影响。方法：海德尔堡沙门氏菌在pH 5.5酸性培养基中生长2 h诱导酸适应性,以pH 3.0酸胁迫培养基及不同温度、NaCl、胆盐、消毒剂环境的存活率评价其耐受性。结果：pH 5.5酸适应2 h后,海德尔堡沙门氏菌在pH 3.0酸胁迫环境的存活率大幅提高。低pH值酸适应也使海德尔堡沙门氏菌在45、50 ℃高温条件下的存活率显著提高,对4 ℃冷藏环境的耐受性显著低于非酸处理菌株,但对－20 ℃冷冻环境的生存无影响。酸适应海德尔堡沙门氏菌对高渗、胆盐胁迫也表现良好的耐受性,5～10 g/100 mL NaCl溶液处理1 h,酸适应海德尔堡沙门氏菌的存活率大于60%,在1～10 g/100 mL胆盐溶液的存活率比对照组高3.8 倍。海德尔堡沙门氏菌酸适应性也使其对消毒剂的敏感性发生变化,酸适应性海德尔堡沙门氏菌在10～25 mmol/L H2O2溶液的存活率显著高于对照菌株,最高达3.1 倍;但对NaClO、乙醇消毒剂的耐受性显著降低,高质量浓度下二者无显著差异。结论：海德尔堡沙门氏菌在低pH值酸性条件生长一段时间会诱导产生酸适应性,还会协同提高其对高温、高渗、胆盐、H2O2胁迫环境的交叉抗性;但对4、－20 ℃低温、NaClO、乙醇等杀菌、保藏措施敏感性提高。     

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.