Salmonella typhimurium resistance on pulsed electric fields associated with membrane fluidity and gene regulation

Effects of different growth temperatures on cytoplasmic membrane fluidity and phospholipids phase transition temperature (T_m) of Salmonella typhimurium and resistance to pulsed electric field (PEF) inactivation, as well as the expression of stress-related genes and fatty acid biosynthesis-associated genes were investigated. Results indicated that the PEF resistance of S. typhimurium increased as growth temperature increased. S. typhimurium cultivated at 10 °C exhibited the lowest PEF resistance with the reduction of 4.23 log₁₀ CFU/mL, while the reduction of 2.10 log₁₀ CFU/mL was found in S. typhimurium cultivated at 45 °C under the same PEF treatment, due to the up-regulation of the expression of fabA gene, which was characterized by the lowest T_m of membrane phospholipids and the greatest membrane fluidity. Although the expression of alternative sigma factors were altered by growth temperature, these genes were not essential for S. typhimurium to develop PEF resistance, suggesting that the PEF resistance modified by growth temperature could be caused by alterations in membrane fluidity.Industrial relevancePulsed electric fields (PEF) treatment has been widely applied in nonthermal pasteurization and increasingly focused on synergistic combinations with other techniques such as thermal treatment, sonication and antibacterial agents to improve the efficacy of PEF to inactivate micro-organisms. Our results indicated that S. typhimurium cultivated at relatively lower temperature was easily inactivated by PEF, due to the up-regulation of the expression of fabA gene, which was characterized by the lowest phase transition temperature of cytoplasmic membrane phospholipids and the greatest membrane fluidity. Therefore, the underlying mechanism of alterations in PEF resistance of S. typhimurium induced by growth temperature was explored to achieve better understanding of microbial inactivation by PEF.     

沙门菌属特异性检测靶点碱基差异位点的识别及其血清型决定力

以美国国家生物技术信息中心(NCBI)已公布的28株沙门菌(14个血清型)全基因组序列为研究对象,对前期研究获得的7个沙门菌属特异性检测靶点在不同血清型间的单核苷酸多态性进行比较分析,结果表明,S9和S69为碱基差异位点最多的两个靶点,具有沙门菌分子血清分型的潜在能力。随后,通过分析S9和S69在沙门菌不同血清型菌株中的差异位点,分别绘制两个血清分型靶点的差异位点表,从而获得了这两个靶点同时用于14个沙门菌血清型的快速分子血清分型的差异位点组合。在这些组合中,同一血清型具有相同的差异位点,不同血清型可以通过差异位点得以区分。最后,采集食品样品192份,用国标方法获得疑似沙门菌21株;利用血清分子分型靶点S9和S69进行快速分型,并同时用传统玻片凝集反应鉴定方法进行验证,两种方法鉴定结果的符合率为100%。鉴定结果为:21株沙门菌共包括4个不同血清型,其中肠炎沙门菌10株、鼠伤寒沙门菌7株、鸡沙门菌2株、纽波特沙门菌2株。基因组序列分析结果和分离株分型结果均表明,沙门菌特异分子检测靶点S9和S69相结合具备沙门菌的分子血清分型能力,以差异位点表为基础建立的血清分型方法有望替代传统的玻片凝集血清分型这一繁杂步骤,从而降低沙门菌血清鉴定的时间与成本,为聚合酶链式反应技术应用于沙门菌分子血清分型提供新思路。     

Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses

The maintenance of bacterial membrane fluidity plays an important role in a variety of cell physiological functions such as nutrient transport, protection from external adverse environments, and cell morphology. The fluidity of membranes is modified in response to several environmental cues, enabling bacterial survival in otherwise unfavorable conditions. Many foodborne bacterial pathogens are able to survive a variety of food preservation treatments used to prevent microbial contamination. These pathogens are able to successfully exploit membrane fluidity-related adaptation strategies under unfavorable conditions, resulting in food hygiene failures. Factors involved in food preservation include pH, temperature, osmotic stress, antimicrobial agents, and high pressure. The fluidity of bacterial membrane lipid bilayer is altered mainly via the adjustment of membrane fatty acid composition. Under undesirable conditions, Gram-negative bacteria alter their membrane fluidity primarily by regulating the ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) and, to a lesser extent, the levels of cyclopropane fatty acids (CFAs), or by cis/trans isomerization. Gram-positive bacteria typically alter their membrane fluidity with changes in fatty acyl chain length or by forming branched-chain fatty acids (BCFAs), besides changes to the ratio of UFA to SFA. This review encompasses various modulators of membrane fluidity, particularly with respect to foodborne pathogens, which often survive even the hostile environments associated with food processing.     

2008—2012年上海市肠炎沙门氏菌分离株毒力基因筛查与ERIC-PCR分型

采集上海地区2008—2012年来自临床病人和市售食品的肠炎沙门氏菌分离株90株;采用聚合酶链式反应(polymerase chain reaction,PCR)方法,对存在于毒力岛SPI和毒力质粒上的9种常见毒力基因携带情况进行了调查;并采用本实验优化的肠杆菌基因间保守重复序列-聚合酶链式反应(enterobacterial repetitive intergenic consensus-PCR,ERIC-PCR)方法对这些分离株进行亚分型。结果显示:毒力基因inv H、sop E、sug R的携带率为100.0%(90/90),iac P、avr A、rhu M、prg K均为98.9%(89/90),而spv B、spv C分别为85.6%(77/90)和78.9%(71/90)。优化的ERIC-PCR体系能够较好地区分8种主要沙门氏菌血清型,共17株菌,辛普森指数(D值)为0.970 6。然而,90株肠炎沙门氏菌分离株却具有一致的ERIC-PCR指纹图谱。在所调查分离株中,毒力基因的携带率较高,尤其是inv H、sop E和sug R,对人类健康存在较大威胁;ERIC-PCR虽然可用于区分沙门氏菌不同血清型,但对肠炎沙门氏菌的分型效果不佳。     

市售猪肉金黄色葡萄球菌的分离及菌株耐消毒剂基因的检测

研究市售猪肉源金黄色葡萄球菌的污染情况,测定季铵盐类消毒剂对分离菌株MIC范围,以及耐消毒剂基因携带情况。采集春夏季与秋冬季市售生猪肉样品576份,采用肉汤稀释法测定苯扎溴铵对分离菌株的MIC,采用PCR技术对分离菌株5种耐消毒剂相关基因(qacA/B、qacC/D、qacG、qacH、qacJ)进行检测。576份样品中分离得到297株金黄色葡萄球菌,总分离率为51.56%;其中,春夏季样品374份,分离出158株;秋冬季样品202份,分离出139株。苯扎溴铵对分离菌株的MIC在0.00125~0.01μg/mL,297株中168株检出携带耐消毒剂基因,总检出率为56.6%,qacA/B、qacC/D、qacG、qacH、qac J的检出率分别为2.36%、19.86%、40.74%、6.73%、0.67%。结果表明猪肉源金黄色葡萄球菌的分离率和菌株消毒剂抗性基因携带率较高,猪肉中分离的金黄色葡萄球菌对季铵盐类消毒剂敏感。本研究为市售猪肉金黄色葡萄球菌的防控及消除提供参考。     

Modelling survival behaviour of Salmonella enterica ser. Enteritidis,Typhimurium and Tennessee on table eggs during storage at different temperatures

Quantitative risk assessment studies on the health risk of Salmonella due to consumption of contaminated table eggs are based on the assumption that Salmonella is inside the egg and that the pathogen belongs to serovar Enteritidis. However different serovars of Salmonella may contaminate the surface of table eggs and spread to other foods at consumer's kitchen due to improper food handling. In the present study the survival behaviour of one strain each from Salmonella enterica serovars Enteritidis, Typhimurium and Tennessee on table egg surface during storage at 4, 8, and 20 °C have been described. Besides, in those cases observed data were subjected for modelling; linear, log-linear tail and Weibull models were compared in terms of model fitting and model performance. Overall, in most cases, inactivation kinetics presented a linear trend on Salmonella behaviour so that Weibull and linear models adequately described observed data. Regarding log-linear tail models, though they presented a better fitting, their adequacy could not be assessed given the lack of data in the tail region. Regarding storage temperatures, 4 °C was predicted to be the most inhibitory temperature for table eggs externally contaminated by a strain of S. enterica serovar Enteritidis. After 28 days of storage, a reduction of 4 log₁₀ cfu/g of eggshell on the S. enterica ser. Enteritidis load was registered at 4 °C. S. enterica ser. Typhimurium and Tennessee showed higher survival rates at all tested temperatures. The results highlighted the importance of keeping constant the storage temperature of table eggs in order to reduce the risk of S. enterica ser. Enteritidis contaminating the surface of table eggs. However this temperature might not be the optimal one in view of S. enterica serovars other than Enteritidis.