Role of Iron in Human Serum Resistance of the Clinical and Environmental Vibrio vulnificus Genotypes

Abstract

We recently reported a simple PCR procedure that targets a sequence variation of the virulencecorrelated gene locus vcg. It was found that 90% of all clinical isolates possessed the vcgC sequence variant, while 93% of all environmental isolates possessed the vcgE sequence variant. Here we report that the clinical genotype of Vibrio vulnificus is significantly better able to survive in human serum than is the environmental genotype. The presence of a siderophore-encoding gene, viuB, influenced serum survivability among all isolates of V. vulnificus tested. Those strains positive for viuB (all C-type strains but very few E-type strains) showed greater serum survivability than those lacking viuB (most E-type strains). The addition of iron (in the form of ferric ammonium citrate) to human serum restored the survival of E-type strains lacking viuB to levels not significantly different from those of C-type and E-type strains that possess viuB. These findings suggest that viuB may dictate serum survival in both C-and E-type strains of V. vulnificus and may explain why some strains (C-and E-type strains) are pathogenic and others (predominately E-type strains) are not. Additionally, C-type strains exhibited a cross-protective response against human serum, not exhibited by E-type strains, after incubation under nutrient and osmotic downshift conditions that mimicked estuarine waters. This suggests that the nutrient/osmotic environment may influence the survival of V. vulnificus following entry into the human body, leading to selection of the C genotype over the E genotype.Vibrio vulnificus is a gram-negative estuarine bacterium capable of causing fatal septicemia after ingestion of raw or undercooked seafood and infection of wounds following exposure to water containing this pathogen (14, 15). Primary septicemias result in mortality rates exceeding 50%, increasing to more than 90% for patients in shock, despite aggressive treatment (8, 9). Most patients suffering from septicemia have predisposing factors which result in elevated serum iron levels (liver dysfunction, alcohol-induced cirrhosis, hemochromatosis, and thalassemia major) or immunodeficiency (1,2,4,8,14,15,22,23). The FDA estimates that between 12 and 30 million Americans are susceptible to infection, although only ca. 30 cases are reported yearly (14,15).While most clinical and environmental isolates of V. vulnificus possess several virulence factors, few environmental isolates appear capable of human pathogenesis. Virulent isolates of V. vulnificus are generally characterized as encapsulated, resistant to human serum, and able to utilize iron-saturated transferrin. Conversely, avirulent isolates lack one or more of these attributes. However, previous serum studies have indicated that all encapsulated strains, whether of clinical or of environmental origin, were to some extent resistant to human serum, while virulence varied drastically with 50% lethal dose (LD 50 ) values ranging from 1.5 ϫ 10 0 to 7.0 ϫ 10 5 cells (10). Even so, of those strains with LD 50 v...

Distribution of Virulence Markers among Vibrio vulnificus Isolates of Clinical and Environmental Origin and Regional Characteristics in Japan

“…In addition, multiplex polymerase chain reaction (PCR) studies by Panicker et al [28] showed that clinical isolates were more likely to be positive for the siderophore-encoding gene viuB than environmental isolates. Regarding the prevalence of viuB , Bogard et al [29] found that serum survivability of the viuB- positive isolate was greater than that of the viuB -lacking ones. Bacterial genotyping methods focusing on differences between clinical and non-clinical types have been performed for individual or two to four combinations of genes; however, genotypic analysis of a combination of five genes participating in V. vulnificus infection has not been reported.…”

Implications of Chitin Attachment for the Environmental Persistence and Clinical Nature of the Human Pathogen Vibrio vulnificus

“…V. vulnificus is widely recognized as a human pathogen, although genetic analyses have revealed that strains of this species are not equally pathogenic. Genotyping has revealed differences not only in pathogenic potential but also in the ecological niches in which these strains reside (11,12,53). Genetic dimorphisms among V. vulnificus strains have been documented on a genome-wide scale; thus, it has been proposed that C-and E-genotype strains may reflect distinct ecotypes (10,13).…”

“…The use of multilocus sequence typing and phylogenetic analyses of sequenced genomes has further delineated C and E genotypes into two distinct evolutionary lineages (9,10). Indeed, previous studies have shown that C-and E-genotype strains display different ecologies, in which E-genotype strains seem to have a distinctive advantage in inhabiting oysters, whereas C-genotype strains are more successful in infecting the human host (7,(11)(12)(13). Furthermore, genome comparisons have allowed the identification of several putative virulence factors (such as the genomic XII region) that could potentially aid C-genotype strains in disease progression (10,14,15).…”

“…Additionally, due to its ability to resist digestion by acid, chitin has been shown to provide V. cholerae with protection from lethal acid stress, offering an effective means for gastric transit inside the human host (34,35). V. vulnificus C-genotype strains have been shown to resist the bactericidal effects of human serum compared to their E-genotype counterparts and also to exhibit a more robust cross-protective response in the presence of multiple stressors (12,13). To our knowledge, the effect of V. vulnificus-chitin interactions on stress resistance has not yet been investigated.…”

Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin

“…Previous studies have provided further distinctions between these genotypes. Specifically, we have found that C-genotype strains demonstrate superior survival in human serum relative to E-genotype strains (12)(13)(14), and genome comparisons have allowed the identification of several putative virulence factors that could potentially aid this bacterium in disease progression (15,16).…”