Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice

Abstract

SummaryVibrio 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.

Global Gene Expression as a Function of the Iron Status of the Bacterial Cell: Influence of Differentially Expressed Genes in the Virulence of the Human Pathogen Vibrio vulnificus

“…In a recent study, Adams et al (1) determined that parenteral iron inoculation in mice increases the iron content in the epidermis and dermis at a high iron dose (5 mg). We did not measure the iron concentration in the skin of the animals used in this study, but our results demonstrate that one of the components involved in the fast replication of V. vulnificus in that organ, previously observed by Starks et al (62,63), is the active transport of iron (which may be mainly from transferrin), as demonstrated by the involvement of the tonB genes, which is still crucial for a successful infection in this animal model. It is worth noticing that in iron-overloaded animals the wild-type strain can establish a rapid infection and that the LD 50 value is 4 to 5 log smaller than that for non-iron-treated animals.…”

“…Thus, the lethal concentration of bacteria can be reached faster in the iron-overloaded mouse model (19). Starks et al (62) showed that iron increased the replication of V. vulnificus in skin, but they also observed a reduction in susceptibility of some strains to being killed by animal defenses. There is also a correlation between utilization of iron from transferrin and virulence, in which the siderophore vulnibactin appears to play a role in infections in suckling mice (34,70) and in normal mice (23), where transferrin saturation levels are expected to be close to the normal range.…”

Hepcidin-Induced Hypoferremia Is a Critical Host Defense Mechanism against the Siderophilic Bacterium Vibrio vulnificus

“…To analyze the mode of action of minihepcidin PR73, we used V. vulnificus CMCP6 containing pGTR905, a marker plasmid that confers resistance to chloramphenicol, as described in Starks et al (Starks et al, 2006). The plasmid replicates only in the presence of arabinose, which is not found in mouse tissues.…”

Vibrio vulnificus: Disease and Pathogenesis

“…Blood taken from patients with chronic liver disease showed decreased neutrophil activity compared to the blood of healthy individuals, and survival of V. vulnificus in human blood is inversely correlated with phagocytosis by neutrophils (44). Based on these data and a study showing increased neutrophil concentrations in the peritoneal cavity upon infection with V. vulnificus, the response of neutrophils to V. vulnificus has been implicated as a disease determinant for this organism (44,56,144,154). One of these studies, however, did use unencapsulated strains (the significance of which is discussed later in this review), thus diminishing the potential role neutrophils play in controlling V. vulnificus infection (44,154).…”

“…Precisely how excess serum iron confers an advantage to V. vulnificus is still unclear; however, two theories have been put forth. One study evaluating growth of this bacterium in mice demonstrated that the presence of excess iron dramatically increased the growth rates of clinical strains, suggesting that excess iron enhances growth of this pathogen (144). Other researchers have found that excess iron resulted in decreased neutrophil activity and concluded that excess iron resulted in a compromised immune response (45).…”