Roles of outer membrane protein W (OmpW) on survival,morphology, and biofilm formation under NaCl stresses in Cronobacter sakazakii

Cronobacter sakazakii is an important foodborne pathogen associated with rare but severe infections through consumption of powdered infant formula. Tolerance to osmotic stress in Cronobacter has been described. However, the detailed factors involved in tolerance to osmotic stress in C. sakazakii are poorly understood. In this study, roles of outer membrane protein W (OmpW) on survival rates, morphologic changes of cells, and biofilm formation in C. sakazakii under different NaCl concentrations between wild type (WT) and OmpW mutant (ΔOmpW) were determined. The survival rates of ΔOmpW in Luria-Bertani medium with 3.5% or 5.5% NaCl were reduced significantly, and morphological injury of ΔOmpW was significantly increased compared with survival and morphology of WT. Compared with biofilm formation of the WT strain, biofilms in ΔOmpW were significantly increased in Luria-Bertani with 3.5% or 5.5% NaCl using crystal violet staining assay after 48 and 72 h of incubation. Detection of biofilms using confocal laser scanning microscopy and scanning electron microscopy further confirmed the changes of biofilm formation under different NaCl stresses. This study demonstrates that OmpW contributes to survival of cells in planktonic mode under NaCl stresses, and biofilm formation is increased in ΔOmpW in response to NaCl stress.     

高通量转录组测序技术在克罗诺杆菌毒力基因研究中的应用进展

克罗诺杆菌(Cronobacter)是一种兼性厌氧的革兰氏阴性菌, 会引起婴幼儿脑膜炎等多种疾病, 严重危害人类健康。高通量转录组测序技术是一种具体测定各基因表达量的测序方法, 不仅可以全面研究全新研究全新的转录本, 还能获得更准确、详细的研究结果。本文综述了近年来高通量转录组测序技术在克罗诺杆菌新毒力因子相关基因的发现、毒力基因表达量与菌株毒力间的变化关系, 以及不同时间段上毒力基因表达量的差异方面的研究成果, 并展望该技术在克罗诺杆菌毒力研究中的应用前景, 以期为克罗诺杆菌的研究方法、防控防治和临床治疗提供意见和建议。     

Short communication: Roles of outer membrane protein W on survival,cellular morphology,and biofilm formation of Cronobacter sakazakii in response to oxidative stress

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H₂O₂. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.     

克罗诺杆菌主要毒力因子及致病机理研究进展

综述了克罗诺杆菌肠毒素、外膜蛋白、基因Inv、关键酶RpfF等主要的毒力因子在克罗诺杆菌致病过程中的作用,并从菌毛的黏附作用、克罗诺杆菌对铁的吸收作用、对唾液酸的利用能力、生物膜对克罗诺杆菌的保护作用和黏附能力的影响、外排系统对克罗诺杆菌在宿主胃肠道中存活能力的影响、耐干燥能力等几个方面总结了该病原菌的致病机理。     

蓝光对阪崎肠杆菌的杀菌及机制研究

本文以食源性致病菌阪崎肠杆菌为对象,研究了医学领域抗细菌感染蓝光的杀菌作用,并首次对其杀菌机制进行了探究。结果表明,当蓝光剂量大于30 J/cm~2时,对阪崎肠杆菌具有显著杀菌作用,照射剂量达240 J/cm~2时,杀菌率超过8 log 10 CFU。亚致死剂量(0～30 J/cm~2)蓝光照射下,单线态氧(~1O_2)探针SOSG开始出现绿色荧光信号,显示细胞内1O2涌现并造成细胞外壁微小孔洞;活性氧物质(ROS)也迅速产生并逐渐增大至5 a.u.;随后脂质氧化标志物丙二醛(MDA)逐渐增加,显示细胞内产生脂质氧化。上述胞内物质变化导致细胞外膜受损,30 J/cm~2蓝光下细胞外膜渗透性增加了48.96%;此外脂肪酰基谱测定揭示,不饱和脂肪酸C18:2,C16:1和C18:1含量减少并逐渐消失,很可能是细胞外膜损伤的重要原因之一。本研究确证了蓝光下细菌胞内~1O_2、ROS及脂质氧化物的动态变化,更重要是的,揭示了细胞外膜损伤是蓝光杀菌的重要方式之一,因为细菌脂质尤其是不饱和脂肪酸是蓝光杀菌重要靶标,本研究将有助于蓝光杀菌机制的深入探究。     

Environmental factors influencing the inactivation of Cronobacter sakazakii by high hydrostatic pressure

The effect of High Hydrostatic Pressure (HHP) on the survival of Cronobacter sakazakii was investigated. Deviations from linearity were found on the survival curves and the Mafart equation accurately described the kinetics of inactivation. Comparisons between strains and treatments were made based on the time needed for a 5-log₁₀ reduction in viable count. The ability of C. sakazakii to tolerate high pressure was strain-dependent with a 26-fold difference in resistance among four strains tested. Pressure resistance was greatest in the stationary growth phase and at the highest growth temperatures tested (30 and 37 °C). Cells treated in neutral pH buffer were 5-fold more resistant than those treated at pH 4.0, and 8-fold more sensitive than those treated in buffer with sucrose added (a_w = 0.98). Pressure resistance data obtained in buffer at the appropriate pH adequately estimated the resistance of C. sakazakii in chicken and vegetables soups. In contrast, a significant protective effect against high pressure was conferred by rehydrated powdered milk. As expected, treatment efficacy improved as pressure increased. z values of 112, 136 and 156 MPa were obtained for pH 4.0, pH 7.0 and a_w = 0.98 buffers, respectively. Cells with sublethal injury to their outer and cytoplasmic membranes were detected after HHP under all the conditions tested. The lower resistance of C. sakazakii cells when treated in media of pH 4.0 seemed to be due to a decreased barostability of the bacterial envelopes. Conversely, the higher resistance displayed in media of reduced water activity may relate to a higher stability of bacterial envelopes.     

反式肉桂醛与温和加热结合对复原婴幼儿牛乳中阪崎克罗诺肠杆菌的抑杀作用

研究反式肉桂醛与温和加热结合对复原婴幼儿牛乳中阪崎克罗诺肠杆菌的抑杀作用。将3 种阪崎克罗诺 肠杆菌的混合菌株（浓度约6.6（lg（CFU/mL）））接种于含有不同质量分数反式肉桂醛（0、0.1%、0.2%、0.3% 和0.4%）的复原婴幼儿牛乳样品中,将样品置于25、45、50、55 ℃培养,并在不同的时间点对样品中存活的阪崎 克罗诺肠杆菌涂布计数。为探究反式肉桂醛与温和加热结合的抑杀机制,实验利用LIVE/DEAD?细菌活性检测试 剂盒和场发射扫描电子显微镜探究细胞膜完整性及细胞形态。结果表明：0.4%的反式肉桂醛在25 ℃处理90 min、 45 ℃处理20 min、50 ℃处理10 min及55 ℃处理10 min使复原婴幼儿牛乳中阪崎克罗诺肠杆菌总数降低至检出限 以下。与反式肉桂醛及温和加热单独作用相比,反式肉桂醛结合温和加热对阪崎克罗诺肠杆菌有显著的抑杀效果 （P＜0.05）,并且随温和加热温度的升高及反式肉桂醛质量分数的增加,效果更加明显。温和加热与反式肉桂醛 结合会影响细胞膜的通透性并使细胞破碎瓦解。以上结果表明：反式肉桂醛与温和加热结合有潜力在冲调乳粉过程 中应用,从而降低阪崎克罗诺肠杆菌的感染风险。     

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35−68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.     

吡嗪类化合物抗幽门螺杆菌活性及机制研究

该实验研究了吡嗪类化合物（PYRs）对幽门螺杆菌（Hp）的抗菌活性及作用机制。采用PYRs处理Hp菌液,观察Hp的活性及形态变化;利用反转录-聚合酶链式反应（RT-PCR）分析PYRs对Hp基因及相关毒力蛋白表达的影响;检测PYRs预处理Hp对胃黏膜上皮细胞（GES-1）活性、细胞凋亡的影响。结果表明,PYR-19（2,5-二甲基-3-正戊基吡嗪）可抑制Hp增殖,Hp外膜蛋白基因（BabA）和毒力基因（CagA和VacA）的转录及毒力蛋白（CagA和VacA）的表达,Hp诱导的GES-1细胞炎性细胞因子分泌及对GES-1细胞的损伤,减轻Hp对胃黏膜上皮细胞的损伤能力。     

Inactivation of Cronobacter sakazakii by ultrasonic waves under pressure in buffer and foods

The objective of this research was to characterize the resistance of Cronobacter sakazakii to ultrasonic waves under pressure (manosonication, MS). The D_MS value (decimal reduction time value) of C. sakazakii in standard conditions (35 °C, 117 μm, 200 kPa, citrate-phosphate buffer pH 7.0) was 0.41 min. This value was higher than that of Yersinia enterocolitica (D_MS = 0.19 min) and lower than those of Salmonella enterica serovar Enteritidis (D_MS = 0.61 min), Listeria monocytogenes (D_MS = 0.86 min), and Enterococcus faecium (D_MS = 1.2 min). Strain studied (ATCC 29544, NCTC 8155, 9238, and 9529), growth temperature (10, 20, 30, and 37 °C), and pH of the treatment media (4.0, 5.0, 6.0, and 7.0) did not significantly change C. sakazakii MS resistance. Conversely, entry into stationary growth phase, decreasing water activity of the treatment media (0.98, 0.96, and 0.94), and treatment in food products (apple and orange juices, chicken and vegetable soups, and rehydrated powdered milk) resulted in up to a 1.6-, 3.9-, and 2.5-fold maximum change in D_MS values, respectively. Whereas an exponential relationship between the amplitude of ultrasonic waves and D_MS values was found, the relationship between static pressure and D_MS values was better described by a quadratic equation. The energy transferred into the medium determined the lethality of the ultrasonic waves regardless of the combination of pressure (0, 50, 100, 200 and 300 kPa) and amplitude (34, 62, 90, 117 and 145 μm) applied. There was an exponential relationship between D_MS values and the power input: an increase of 134 W increased the inactivation rate ten times regardless of the treatment medium. No C. sakazakii cells with sublethally injured cytoplasmic membrane or with sublethal oxidative damage occurred after MS treatments, but the results indicated that damage to the outer membrane preceded microbial death.     

婴儿配方乳粉中阪崎克罗诺杆菌解旋酶恒温基因扩增检测方法的建立

目的：建立一种检测婴儿配方乳粉中阪崎克罗诺杆菌的解旋酶恒温基因扩增方法。方法：根据阪崎克罗诺杆菌ITS基因设计特异性引物,优化解旋酶恒温基因扩增法反应条件UvrD helicase、T4 gp32的浓度,人工添加阪崎克罗诺杆菌确定检出限,多种致病菌在建立的解旋酶恒温基因扩增体系中扩增验证特异性,电泳检测扩增产物。结果：解旋酶恒温基因扩增法检测婴儿配方乳粉中阪崎克罗诺杆菌得到与设计序列长度一致的100 bp基因片段,检出限为10 CFU/g,优化反应条件UvrD helicase、T4 gp32的终浓度分别为0.1、5.0 μg。结论：解旋酶恒温基因扩增法用于检测婴儿配方乳粉中阪崎克罗诺杆菌的特异性强、灵敏度高、耗时短,为婴儿配方乳粉中阪崎克罗诺杆菌的快速检测提供了新的方法。     

基质辅助激光解吸电离飞行时间质谱技术对阪崎克罗诺杆菌的鉴定及溯源

目的研究不同样品前处理方法对VITEK~?基质辅助激光解吸电离飞行时间质谱(VITEK~?MALDI-TOF MS,以下简称VITEK MS)鉴定阪崎克罗诺杆菌的影响及该方法对阪崎克罗诺杆菌的分型溯源能力,研究VITEK MS与MALDI 7090~(TM)MALDI-TOF/TOF MS(以下简称MALDI 7090)所得图谱的异同。方法选取3种不同样品前处理方法处理18株阪崎克罗诺杆菌野生菌株、2株阪崎克罗诺杆菌标准菌株和2株非阪崎克罗诺杆菌标准菌株,比较VITEK MS鉴定结果与传统鉴定结果的一致性;通过VITEK MS的鉴定结果和图谱的比对,研究不同样品前处理方法对阪崎克罗诺杆菌鉴定的影响;利用VITEK MS质谱图特征峰的聚类分析,对20株阪崎克罗诺杆菌进行溯源研究;同时比较VITEK MS与MALDI 7090所得图谱,研究两者的异同。结果 20株试验菌株的VITEK MS鉴定结果均为阪崎克罗诺杆菌,与传统的鉴定结果一致,并与2株非阪崎克罗诺杆菌标准菌株能明显地区分;3种不同样品前处理方法得到的鉴定谱图与标准谱图比对获得的鉴定结果可信度之间差异无统计学意义(P0.05),但质谱图中质谱峰数量和相对峰强度有明显差异,其中甲酸提取法在2 000~15 000 m/z范围内,相对峰强度5 m V的质谱峰数量达15个以上。在相似水平为80%的条件下,VITEK MS的聚类分析将甲酸提取法的20株阪崎克罗诺杆菌分为5个群,通过聚类分析及菌株来源能够推测阪崎克罗诺杆菌传播途径。VITEK MS和MALDI 7090得到的谱图,两者特征峰的出峰位置与相对峰强度无明显差异。结论 MALDI-TOF MS技术可以准确鉴定阪崎克罗诺杆菌;甲酸提取法更适用于阪崎克罗诺杆菌的MALDI-TOF MS鉴定;MALDI-TOF MS技术对阪崎克罗诺杆菌的溯源研究具有重要价值;VITEK MS与MALDI 7090所得谱图无明显差异。     

Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil

Staphylococcus aureus produces exoproteins that contribute to its ability to colonize the mammary gland such as hemolysins, coagulase, slime, and protein A. This study characterized phenotypically and genotypically these virulence factors in 50 Staph. aureus isolates. These isolates were obtained from milk samples from subclinical mastitis cases identified in 15 dairy cattle farms located in the state of Rio de Janeiro, Brazil. All of the confirmed Staph. aureus samples were PCR positive for the coa gene, which displayed 3 different size polymorphisms. The amplification of the spaA X region yielded a single amplicon for each isolate with the prevalent amplicon sized 315 bp. The Staph. aureus isolates were 24 and 16% positive for the hla and hlb genes, respectively, and 22 and 20% positive for the icaA and icaD genes, respectively. Amplification of the agr gene RNAIII was positive in 74% of the strains. Twenty-seven different profiles were identified among the samples, indicating a great diversity of Staph. aureus involved in the etiology of mastitis cases in the analyzed region. These findings are valuable to the comprehension of the distribution of the profiles of Staph. aureus strains isolated from subclinical mastitis cases in the state of Rio de Janeiro.     

江苏地区肉鸡屠宰链弯曲菌分离菌株毒力基因分布及分子分型研究

目的了解江苏地区肉鸡屠宰生产链中弯曲菌分离菌株毒力基因分布及不同环节分离菌株遗传相关性。方法利用特异性引物对肉鸡屠宰过程不同环节分离所得的96株弯曲菌分离菌株的10种致病相关毒力基因进行聚合酶链式反应(polymera sechainre action,PCR)检测,应用多位点序列分型(multilocus sequence typing,MLST)方法对分离菌株进行分子分型研究,通过与弯曲菌MLST数据库比对获得各株菌等位基因值、序列型(sequence types,STs)及克隆复合体。结果96株分离菌株中,flaA、cadF和cdtB毒力基因携带率均为100%,其次为cdtC、iam和cdtA毒力基因,携带率较高,分别为96.9%、86.5%和84.4%,另外3个毒力基因flhA、virB11、ciaB携带率均较低,分别为25.0%、15.6%、11.5%;格林-巴利综合症相关毒力基因wlaN在所有菌株中均无检出。MLST分型结果显示,96株弯曲菌可分为10个STs,其中2个为新STs,形成1种优势克隆复合体CC828(56株)和4种未定义的克隆复合体,表现为较低的遗传多样性。结论肉鸡屠宰生产链中弯曲菌毒力基因分布广泛,不同环节弯曲菌分离菌株遗传多样性较低,表明弯曲菌在肉鸡屠宰生产链中存在交叉污染。     

Short communication: Global transcriptome analysis of Lactococcus lactis ssp. lactis in response to gradient freezing

Lactic acid bacteria are often preserved as starter cultures by freezing to extend shelf stability as well as maintain cell viability and acidification activity. Previous studies showed that the endocyte extracted from gradient-freezing pretreated cells could act as lyoprotectant in the lyophilization process of Lactococcus lactis ssp. lactis. In this study, the molecular mechanisms of L. lactis in response to gradient freezing exposure are described using high-throughput sequencing. Nineteen of 56 genes were upregulated after gradient freezing, whereas 37 genes were downregulated. Further validation results of quantitative real-time PCR experiments were consistent with the RNA sequencing. Gene Ontology (http://www.geneontology.org/) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) pathway were used to analyze the differentially expressed genes. Several pathways, such as glutathione metabolism, ATP-binding cassette transport, metabolism of cell wall and cell membrane components, and stress response-related pathways, were affected by gradient freezing. Six genes relevant to freezing stress response were selected for quantitative real-time PCR, including 3 upregulated genes (hisK, eutD, dukA) and 3 downregulated genes (als, yedF, pepN). The Gene Ontology enrichment and KEGG pathway analyses showed these genes may influence stress response-related pathways, improving the survival of the L. lactis under freezing stress. The identification of these genes deepened an understanding about their response under freezing stress, helping us find potential genes or pathways related to gradient freezing for further research on lyoprotectants.