Protective Effects of Chicken Egg Yolk Immunoglobulins (IgYs) against Vibrio vulnificus Infections.

Vibrio (V.) vulnificus infection is a rare disease whose death rates exceed 50% despite aggressive antibiotic treatment and surgical debridement. The aim of this study was to assess the ability of specific anti-V. vulnificus immunoglobulins Y (IgYs) for preventing and treating V. vulnificus infections. IgYs were produced by immunizing egg laying hens with inactivated whole cell bacteria. Peritoneal cytokines, blood's bacterial load, and survival curves were obtained from both prophylactic and therapeutic mouse models. The results showed that the specific IgYs (i) inhibited the growth of V. vulnificus in vitro, (ii) dramatically reduced the inflammatory response and blood's bacterial load, and (iii) improved the survival rate of V. vulnificus-infected mice. These results prove that anti-V. vulnificus IgYs can be markedly effective means for the prophylaxis and the therapy of V. vulnificus infections.

Comprehensive identification of Vibrio vulnificus genes required for growth in human serum.

Vibrio vulnificus can be a highly invasive pathogen capable of spreading from an infection site to the bloodstream, causing sepsis and death. To survive and proliferate in blood, the pathogen requires mechanisms to overcome the innate immune defenses and metabolic limitations of this host niche. We created a high-density transposon mutant library in YJ016, a strain representative of the most virulent V. vulnificus lineage (or phylogroup) and used transposon insertion sequencing (TIS) screens to identify loci that enable the pathogen to survive and proliferate in human serum. Initially, genes underrepresented for insertions were used to estimate the V. vulnificus essential gene set; comparisons of these genes with similar TIS-based classification of underrepresented genes in other vibrios enabled the compilation of a common Vibrio essential gene set. Analysis of the relative abundance of insertion mutants in the library after exposure to serum suggested that genes involved in capsule biogenesis are critical for YJ016 complement resistance. Notably, homologues of two genes required for YJ016 serum-resistance and capsule biogenesis were not previously linked to capsule biogenesis and are largely absent from other V. vulnificus strains. The relative abundance of mutants after exposure to heat inactivated serum was compared with the findings from the serum screen. These comparisons suggest that in both conditions the pathogen relies on its Na+ transporting NADH-ubiquinone reductase (NQR) complex and type II secretion system to survive/proliferate within the metabolic constraints of serum. Collectively, our findings reveal the potency of comparative TIS screens to provide knowledge of how a pathogen overcomes the diverse limitations to growth imposed by serum.

Clinical Usefulness of Real-Time Polymerase Chain Reaction for the Diagnosis of Vibrio vulnificus Infection Using Skin and Soft Tissues.

Vibrio vulnificus is a halophilic gram-negative bacillus isolated in seawater, fish, and shellfish. Infection by V. vulnificus is the most severe food-borne infection reported in the United States of America. Here, we aimed to examine the clinical usefulness of polymerase chain reaction (PCR) using tissue specimens other than blood samples as a diagnostic tool for V. vulnificus infection. A retrospective study was conducted with patients who underwent real-time PCR of toxR in both blood and skin tissues, including serum, bullae, swab, and operation room specimens, between 2006 and 2009. The median V. vulnificus DNA load of 14 patients in real-time PCR analysis of serum at the time of admission was 638.5 copies/mL blood, which was within the interquartile range (IQR: 37-3,225). In contrast, the median value by real-time PCR using the first tissue specimen at the time of admission was 16,650 copies/mL tissue fluid (IQR: 4,419-832,500). This difference was statistically significant (P = 0.022). DNA copy numbers in tissues were less affected by short-term antibiotic administration than that in blood samples, and antibiotic administration increased the DNA copy number in some patients. We found, for the first time, that DNA copy numbers in tissues of patients infected by V. vulnificus were higher than those in blood samples. Additionally, skin lesions were more useful than blood samples as specimens for PCR analysis in patients administered antibiotics for V. vulnificus infection before admission.

An invertebrate-specific miRNA targeted the ancient cholinergic neuroendocrine system of oyster.

Acetylcholine (ACh) is the main neurotransmitter in the cholinergic neuroendocrine system and plays an indispensable role in modulating diverse immune responses. As important transporters in choline uptake, choline transporter-like proteins (CTLs) can control ACh synthesis and release indirectly in multiple organisms. In this study, cgi-miR-2d, an invertebrate-specific miRNA in oyster Crassostrea gigas, is proved to repress the synthesis/release of ACh by targeting CgCTL1 and choline uptake in haemocytes during the early stage of pathogen infection. In short, an opposite expression pattern between CgCTL1 and cgi-miR-2d is observed during Vibrio splendidus infection, accompanied by changes in haemolymph ACh. In addition, the expression level of CgCTL1 is found to be significantly repressed after cgi-miR-2d overexpression in vivo, while both haemocyte choline and haemolymph ACh are also decreased simultaneously, similar to the finding in CgCTL1 knock-down assay. As a result, the expression of two tumour necrosis factor-like proteins and the bacteriostatic activity of oyster haemocytes are found to be altered significantly by either gain-of-function cgi-miR-2d or knock-down of CgCTL1. To our knowledge, this is the first miRNA identified in invertebrates that can target the ancient cholinergic system and augment immune response during infection.

Correlations between Clinical Features and Mortality in Patients with Vibrio vulnificus Infection.

Vibrio vulnificus is a common gram-negative bacterium, which might cause morbidity and mortality in patients following consumption of seafood or exposure to seawater in Southeast China. We retrospectively analyzed clinical data of patients with laboratory confirmed V. vulnificus infection. Twenty one patients were divided into a survival group and a non-surviving (or death) group according to their clinical outcome. Clinical data and measurements were statistically analyzed. Four patients (19.05%) died and five patients gave positive cultures from bile fluid, and 16 other patients gave positive culture from blood or blisters. Ten patients (47.62%) had an underlying liver disease and marine-related events were found in sixteen patients (76.2%). Patients with heavy drinking habits might be at increased mortality (p = 0.028). Clinical manifestations of cellulitis (47.6%), septic shock (42.9%) and multiple organ failure (28.6%) were statistically significant when comparing survivors and non-survivors (p = 0.035, p = 0.021 and p = 0.003, respectively). The laboratory results, including hemoglobin < 9.0 g/L (p = 0.012), platelets < 2.0 × 109 /L, prothrombin time activity (PTA) <20%, decreased serum creatinine and increased urea nitrogen were statistically significant (p = 0.012, p = 0.003, p = 0.028 and p = 0.028, respectively). Patients may be at a higher risk of mortality under situations where they have a history of habitual heavy alcoholic drink consumption (p = 0.028, OR = 22.5, 95%CI 1.5-335.3), accompanied with cellulitis, shock, multiple organ failure, and laboratory examinations that are complicated by decreased platelets, hemoglobin and significantly prolonged prothrombin time (PT).

Viable but Nonculturable and Persister Cells Coexist Stochastically and Are Induced by Human Serum.

Dormancy holds a vital role in the ecological dynamics of microorganisms. Specifically, entry into dormancy allows cells to withstand times of stress while maintaining the potential for reentry into an active existence. The viable but nonculturable (VBNC) state and antibiotic persistence are two well-recognized conditions of dormancy demonstrated to contribute to bacterial stress tolerance and, as a consequence, yield populations that are tolerant to high-dose antibiotics. Aside from this commonality, more evidence is being presented that indicates the relatedness of these two states. Here, we demonstrate that VBNC cells are present during persister isolation experiments, further indicating that these cells coexist and are induced by the same conditions. Interestingly, we reveal that VBNC cells can exist stochastically in unstressed growing cultures, a finding that is characteristic of persisters. Furthermore, human serum induces the formation of both VBNC cells and persisters, a finding not previously described for either dormancy state. Lastly, we describe the role of toxin-antitoxin systems (TAS) in the induction of the VBNC state and report that these TAS, which are classically implicated in persister cell formation, are also induced during incubation in human serum. This study provides evidence for the recently proposed "dormancy continuum hypothesis" and substantiates the physical and molecular relatedness of VBNC and persister cells in a standardized model organism. Notably, these results provide new evidence for the clinical significance of VBNC and persister cells.

Host-like carbohydrates promote bloodstream survival of Vibrio vulnificus in vivo.

Sialic acids are found on all vertebrate cell surfaces and are part of a larger class of molecules known as nonulosonic acids. Many bacterial pathogens synthesize related nine-carbon backbone sugars; however, the role(s) of these non-sialic acid molecules in host-pathogen interactions is poorly understood. Vibrio vulnificus is the leading cause of seafood-related death in the United States due to its ability to quickly access the host bloodstream, which it can accomplish through gastrointestinal or wound infection. However, little is known about how this organism persists systemically. Here we demonstrate that sialic acid-like molecules are present on the lipopolysaccharide of V. vulnificus, are required for full motility and biofilm formation, and also contribute to the organism's natural resistance to polymyxin B. Further experiments in a murine model of intravenous V. vulnificus infection demonstrated that expression of nonulosonic acids had a striking benefit for bacterial survival during bloodstream infection and dissemination to other tissues in vivo. In fact, levels of bacterial persistence in the blood corresponded to the overall levels of these molecules expressed by V. vulnificus isolates. Taken together, these results suggest that molecules similar to sialic acids evolved to facilitate the aquatic lifestyle of V. vulnificus but that their emergence also resulted in a gain of function with life-threatening potential in the human host.

Immune responses evoked by infection with Vibrio anguillarum in zebrafish bath-vaccinated with a live attenuated strain.

Live attenuated vaccines are a promising application to control bacterial fish diseases. A live attenuated Vibrio anguillarum vaccine candidate was established in our laboratory to protect fish against vibriosis. To elucidate the mechanism of immunoprotection, it is necessary to compare the different immune responses to infection between vaccinated and non-vaccinated fish. In this study, the expression levels of pathogen-specific antibodies and immune-related genes upon challenge at 28 days post-vaccination were compared between vaccinated and non-vaccinated zebrafish. In the results, the specific antibody levels against virulent V. anguillarum in the vaccinated group did not rise significantly following infection, which suggested that high-affinity antibodies were induced by the vaccine. In the non-vaccinated group, the specific IgM response was triggered at 3 days post-infection and showed a delayed antibody response. Meanwhile, the transcription levels of the genes encoding the pro-inflammatory cytokine IL-1ß and the chemokine IL-8 were more highly up-regulated in non-vaccinated fish than in vaccinated fish. This suggests that the overwhelming inflammatory response trigged by infection in non-vaccinated zebrafish was controlled in vaccinated zebrafish. Interestingly, the expression levels of adaptive immune-related genes were increased in vaccinated fish after challenge, compared to the non-vaccinated fish. These results suggest that inoculation with the live attenuated vaccine triggered protection by curbing inflammation and strengthening the adaptive immune response.

Tumor necrosis factor-α and mortality in patients infected with Vibrio vulnificus.

Serum tumor necrosis factor-α (TNF-α) was evaluated in Vibrio vulnificus-infected patients at admission. The median TNF-α concentration in the non-survivor group was determined to be 261.0 pg/mL, in contrast to 69.5 pg/mL in the survivor group (P = 0.001). Hence, serum TNF-α concentration may potentially be an early predictor of the mortality in patients with Vibrio septicemia.

Comparison of necrotizing fasciitis and sepsis caused by Vibrio vulnificus and Staphylococcus aureus.

BACKGROUND: Vibrio vulnificus can cause a rapidly progressive fatal soft-tissue infection. Staphylococcus aureus is the most common cause of skin and soft-tissue infections reported worldwide, and, in particular, methicillin-resistant Staphylococcus aureus has emerged as the most common isolate in emergency departments. The purposes of the present study were to compare the specific characteristics of Vibrio vulnificus and Staphylococcus aureus infections and to compare the clinical outcomes of Vibrio vulnificus, methicillin-resistant Staphylococcus aureus, and methicillin-sensitive Staphylococcus aureus necrotizing infections. METHODS: One hundred and fifteen patients with necrotizing fasciitis caused by Vibrio vulnificus (sixty patients) or Staphylococcus aureus (fifty-five patients) were retrospectively reviewed over a six-year period. Differences in mortality, patient characteristics, clinical presentations, laboratory data, and hospital course were compared between the Vibrio vulnificus and Staphylococcus aureus groups. RESULTS: Nineteen patients (including eleven in the Vibrio vulnificus group and eight in the Staphylococcus aureus group) died, resulting in a mortality rate of 16.5%. We found significant differences between the two groups with regard to hypotension, fever, the interval between contact and admission, the interval between the diagnosis of necrotizing fasciitis and the first operation, and admission to the intensive care unit. The patients in the Vibrio vulnificus group had significantly lower total white blood-cell counts, higher banded white blood-cell counts, and lower platelet counts as compared with those in the Staphylococcus aureus group. The proportion of patients who were hypotensive (as indicated by a systolic blood pressure of ≤ 90 mm Hg) was significantly greater in the methicillin-resistant Staphylococcus aureus subgroup than in the methicillin-sensitive Staphylococcus aureus subgroup. Patients with hepatic dysfunction were significantly more likely to have Vibrio vulnificus infection, and those with diabetes mellitus were significantly more likely to have Staphylococcus aureus infection. CONCLUSIONS: Necrotizing fasciitis caused by Vibrio vulnificus and Staphylococcus aureus is a surgical emergency. Vibrio vulnificus infection progresses more rapidly and the clinical characteristics are more fulminant than either methicillin-resistant Staphylococcus aureus or methicillin-sensitive Staphylococcus aureus infection.

Primary Shewanella algae bacteremia mimicking Vibrio septicemia.

Shewanella algae infections are rare in humans. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. We report a case of primary S. algae bacteremia occurring after the ingestion of raw seafood in a patient with liver cirrhosis that presented a fulminent course of necrotizing fasciitis.

A fatal case of Vibrio vulnificus meningoencephalitis.

The objective of this paper is to report a rare case of Vibrio vulnificus presenting as meningoencephalitis without a wound infection. Vibrio vulnificus is capable of causing severe and often fatal infections in susceptible individuals. It commonly causes necrotizing wound infections, primary septicemia, and gastroenteritis. A 69-year-old man had meningoencephalitis with lesion on the red nucleus, substantia nigra, basal ganglia, and dentate nucleus as the initial clinical manifestation of a V. vulnificus infection. This is the first case of V. vulnificus infection in which MRI demonstrated the involvement of deep nuclei of the brain.

The importance of serum creatine phosphokinase level in the early diagnosis, and as a prognostic factor, of Vibrio vulnificus infection.

BACKGROUND: Vibrio vulnificus infection causes rapidly progressive skin lesions and sepsis in compromised hosts with liver cirrhosis, and is often fatal. Early diagnosis and rapid treatment are important. OBJECTIVES: To clarify the characteristics of V. vulnificus infection that distinguish it from other cutaneous and soft-tissue bacterial infections and to confirm that serum creatine phosphokinase (CPK) levels are useful in early diagnosis, and are a prognostic factor for, V. vulnificus infection. METHODS: We analysed the clinical and laboratory findings (especially serum CPK levels) in eight patients with V. vulnificus infection who were treated at the Saga Medical School Hospital between January 1989 and December 1999. RESULTS: All eight patients had liver dysfunction and typical skin manifestations. Six had eaten raw seafood before onset. Seven patients had initial skin manifestations in their legs or feet and eventually died, despite prompt therapy in the intensive care unit. CPK levels of six of these seven patients were already elevated at their initial presentation. Only one patient, with skin manifestations solely on his left hand, showed and maintained a normal CPK level and survived. In 23 patients with cutaneous and soft-tissue infections (10 with necrotizing fasciitis, three with erysipelas, 10 with cellulitis), only three patients with necrotizing fasciitis and streptococcal toxic shock syndrome (STSS) showed CPK elevation. CONCLUSIONS: A high level of serum CPK in cutaneous or soft-tissue bacterial infection is considered useful for an early diagnosis of V. vulnificus infection and STSS. A history of eating raw seafood, underlying liver disease and multiple lesions suggest a diagnosis of V. vulnificus infection, rather than STSS.

A cytotoxin-producing strain of Vibrio cholerae non-O1, non-O139 as a cause of cholera and bacteremia after consumption of raw clams.

We report a case of a cholera-like gastroenteritis subsequent with bacteremia in a healthy man following consumption of raw clams. Although we failed to recover the organism from the patient's stool culture, his blood culture was positive for a non-cholera toxin-producing yet cytotoxin-producing non-O1 and non-O139 Vibrio cholerae.

Metalloprotease is not essential for Vibrio vulnificus virulence in mice.

Previous work suggested that a metalloprotease, Vvp, may be a virulence factor of Vibrio vulnificus, which causes severe wound infection and septicemia in humans. To determine the role of Vvp in pathogenesis, we isolated an isogenic protease-deficient (PD) mutant of Vibrio vulnificus by in vivo allelic exchange. This PD mutant was as virulent as its parental strain in mice infected intraperitoneally and was 10-fold more virulent in mice infected via the oral route. Furthermore, the PD mutant was indistinguishable from its parental strain in invasion from peritoneal cavity into blood stream, enhancement of vascular permeability, growth in murine blood, and utilization of hemoglobin and transferrin. These data suggest that Vvp is not essential for virulence in the mouse. However, the cytolysin activity in the culture supernatant of the PD mutant was found to be twofold higher than that of the wild-type strain and remained for a much longer period. The higher cytolysin activity of the PD mutant may be associated with the enhanced virulence in mice infected via the oral route.

Survival of Vibrio vulnificus in whole blood from patients with chronic liver diseases: association with phagocytosis by neutrophils and serum ferritin levels.

Vibrio vulnificus causes severe wound infections and sepsis, mostly in persons with chronic liver diseases. Survival of this organism in the whole blood collected from healthy volunteers and patients with chronic hepatitis, liver cirrhosis, and hepatoma was analyzed as an indication of susceptibility. The bacterial numbers in the blood after 5 h of incubation tended to increase with the severity of the liver disease and differed significantly between hepatoma patients and healthy volunteers (P<.05). Survival of V. vulnificus in the whole blood correlated positively with serum ferritin concentration (r=.266; P<.05) and percentage of transferrin iron saturation (r=. 200; P<.05) and correlated negatively with serum C4 concentration (r=-.198; P<.05) and phagocytosis by neutrophils (r=-.204; P<.05). Among these parameters, low phagocytosis activity (P<.01) and high ferritin level (P<.01) in the blood were the independent predictors.

Direct identification of Vibrio vulnificus in clinical specimens by nested PCR.

This study was performed to establish optimal nested PCR conditions and a high-yield DNA extraction method for the direct identification of Vibrio vulnificus in clinical specimens. We designed two sets of primers targeting the V. vulnificus hemolysin/cytolysin gene. The target of the first primer set (P1-P2; sense, 5'-GAC-TAT-CGC-ATC-AAC-AAC-CG-3', and antisense, 5'-AGG-TAG-CGA-GTA-TTA-CTG-CC-3', respectively) is a 704-bp DNA fragment. The second set (P3-P4; sense, 5'-GCT-ATT-TCA-CCG-CCG-CTC-AC-3', and antisense, 5'-CCG-CAG-AGC-CGT-AAA-CCG-AA-3', respectively) amplifies an internal 222-bp DNA fragment. We developed a direct DNA extraction method that involved boiling the specimen pellet in a 1 mM EDTA-0.5% Triton X-100 solution. The new DNA extraction method was more sensitive and reproducible than other conventional methods. The DNA extraction method guaranteed sensitivity as well, even when V. vulnificus cells were mixed with other bacteria such as Escherichia coli or Staphylococcus aureus. The nested PCR method could detect as little as 1 fg of chromosomal DNA and single CFU of V. vulnificus. We applied the nested PCR protocol to a total of 39 serum specimens and bulla aspirates from septicemic patients. Seventeen (94.4%) of the 18 V. vulnificus culture-positive specimens were positive by the nested PCR. Eight (42.1%) of the 19 culture-negative samples gave positive nested PCR results.

Vibrio vulnificus septicemia in a patient with severe aplastic anemia.

A 53-year-old man with severe aplastic anemia developed sporadic Vibrio vulnificus septicemia 1 day after eating raw fish and shellfish. Although V. vulnificus infection is potentially fatal, he was saved by immediate and sensitive antibiotic administration. Patients with chronic hematologic disease are susceptible to infection by this organism and are prone to developing septicemia when they eat raw seafood. It is necessary for a patient with this infection to be given effective antibiotics as quickly as possible.