Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 30, Frederiksberg C, Denmark
Int J Food Microbiol. 2012 Apr 2;155(1-2):60-8. doi: 10.1016/j.ijfoodmicro.2012.01.019. Epub 2012 Jan 27.
Campylobacter jejuni is the leading cause of bacterial diarrheal disease in humans, and contaminated poultry and poultry products are recognized as the main vehicle of infection. Despite the significance of C. jejuni as a foodborne pathogen, little is known about its response to stress, and, especially, how its virulence is modulated under such conditions. The aim of this study was to assess the effect of temperature shift in a broth model system on virulence expression and cell survival of three different Campylobacter jejuni strains: two clinical (TB1048 and NCTC11168) and one chicken isolate (DFVF1099). Firstly, cells were transferred from 42 to 4°C to investigate the effect of low temperature storage for short (30 min) and long (24 h) periods of time. A shift in temperature from 4 to 37°C for 30 min was performed to investigate the effect of a momentary increase in temperature. Virulence properties were evaluated by analyzing transcriptions of the virulence genes cdtB, ciaB, cadF and the stress associated genes clpP, htrB using reverse transcription quantitative PCR (RT-qPCR) and by the ability of the C. jejuni strains to adhere to and invade Caco-2 cells. Similar cell survival and no growth was seen for all strains at 4°C and after transfer to 37°C for 30 min. Interstrain variation was observed as transcription levels of cdtB, cadF and clpP were upregulated in NCTC11168 but not in DFVF1099 after 24h at 4°C. Bioinformatic analysis of invasion associated genes, showed differences in one gene, cipA between DFVF1099 and NCTC11168 resulting in a 14 amino acid deletion and 28 amino acid addition at the N and C terminal ends respectively of the CipA protein of DFVF1099. In contrast to DFVF1099, strains NCTC1168 and TB1048 were able to invade Caco-2 cells. Invasion ability was not affected by temperature shifts, as was also displayed by RT-qPCR analysis of another invasion associated gene, ciaB. The adhesion capacity was increased only for the TB1048 strain with incubation time. In conclusion, this study showed that low storage temperature is not enough to control the survival and virulence expression of C. jejuni. The clinical strains appeared to be more virulent than the chicken isolate as measured by the Caco-2 invasion assay which could be due to differences in CipA functionality. The RT-qPCR analysis and Caco-2 assay showed to be useful tools for differentiating virulence potentials of three C. jejuni strains under growth conditions where cell survival rates were similar.
空肠弯曲菌是人类细菌性腹泻病的主要病原体，受污染的家禽及其产品被认为是主要的感染源。尽管空肠弯曲菌作为食源性病原体具有重要意义，但人们对其应激反应知之甚少，特别是在这种情况下其毒力是如何调节的。本研究旨在评估在肉汤模型系统中温度变化对 3 株不同空肠弯曲菌（2 株临床分离株 TB1048 和 NCTC11168，1 株鸡分离株 DFVF1099）的毒力表达和细胞存活的影响。首先，将细胞从 42°C转移到 4°C，以研究短期（30 分钟）和长期（24 小时）低温储存对毒力的影响。将温度从 4°C升高到 37°C 30 分钟，以研究温度瞬时升高的影响。通过反转录定量 PCR（RT-qPCR）分析毒力基因 cdtB、ciaB、cadF 和应激相关基因 clpP、htrB 的转录，以及空肠弯曲菌菌株黏附和侵袭 Caco-2 细胞的能力来评估毒力特性。所有菌株在 4°C 时和转移到 37°C 30 分钟后，均出现类似的细胞存活和无生长现象。在 4°C 培养 24 小时后，NCTC11168 株的 cdtB、cadF 和 clpP 转录水平上调，但 DFVF1099 株没有上调，观察到种间差异。侵袭相关基因的生物信息学分析显示，DFVF1099 和 NCTC11168 之间的 cipA 基因存在差异，导致 DFVF1099 的 CipA 蛋白在 N 和 C 末端分别有 14 个氨基酸缺失和 28 个氨基酸添加。与 DFVF1099 相反，NCTC1168 和 TB1048 株能够侵袭 Caco-2 细胞。RT-qPCR 分析另一个侵袭相关基因 ciaB 显示，温度变化对侵袭能力没有影响。随着孵育时间的延长，只有 TB1048 株的黏附能力增加。综上所述，本研究表明，低温储存不足以控制空肠弯曲菌的存活和毒力表达。与鸡分离株相比，临床分离株的毒力似乎更强，这可以归因于 CipA 功能的差异。RT-qPCR 分析和 Caco-2 测定是区分 3 株空肠弯曲菌在细胞存活率相似的生长条件下的毒力潜力的有用工具。
