Starks Angela M, Bourdage Keri L, Thiaville Patrick C, Gulig Paul A
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida College of Medicine, Gainesville, FL, USA.
Mol Microbiol. 2006 Jul;61(2):310-23. doi: 10.1111/j.1365-2958.2006.05227.x.
Vibrio vulnificus is Gram-negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran-treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony-forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran-treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid-containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12-15 h infection period were not significantly different in iron dextran-treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non-iron dextran-treated mice with clinical strains demonstrated that the greatest effect of iron dextran-treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran-treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15-28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram-negative bacteria.
创伤弧菌是一种革兰氏阴性菌，可污染牡蛎，食用生牡蛎后会引发高致死性败血症以及伤口感染。我们之前描述过两组创伤弧菌菌株，在皮下接种右旋糖酐铁处理的小鼠中具有不同程度的毒力。强毒临床菌株和减毒环境菌株都能从皮肤损伤处和肝脏中大量回收；然而，减毒环境菌株在接种物中需要显著更多的菌落形成单位（cfu）才能产生致死性感染。使用其中一些菌株以及另一临床菌株，我们现在探究临床菌株和环境菌株之间不同的感染能力是否归因于小鼠宿主体内细菌生长或死亡速率的差异。因此，我们构建了一个标记质粒pGTR902，其仅在存在阿拉伯糖时作为复制子起作用，而阿拉伯糖在动物组织中含量不高。将含有pGTR902的创伤弧菌菌株接种到右旋糖酐铁处理和未处理的小鼠中。测量从小鼠中回收的维持了标记质粒的细菌比例，使我们能够监测体内分裂次数，从而监测生长速率；而测量回收的含标记质粒的细菌数量则能够测量小鼠体内弧菌的死亡情况。在12 - 15小时的感染期内，所有菌株在右旋糖酐铁处理的小鼠体内的细菌分裂次数没有显著差异；然而，一种环境菌株的死亡速率与临床菌株相比显著更高。用临床菌株感染未用右旋糖酐铁处理的小鼠表明，右旋糖酐铁处理的最大影响是提高了生长速率，而一种临床菌株在未处理的小鼠中也出现了死亡增加的情况。接种到右旋糖酐铁处理小鼠体内的创伤弧菌在感染的最初4小时内复制极快，倍增时间约为15 - 28分钟。相比之下，一种环境菌株表现出早期生长速率降低。这些结果表明，自然存在的创伤弧菌毒力差异可以通过在宿主体内快速复制或抵抗宿主防御的不同能力来解释。标记质粒pGTR902对于从区分动物宿主体内细菌生长与死亡的角度研究细菌毒力应该对大多数革兰氏阴性菌有用。
