Eosinophil-mediated lung inflammation associated with elevated natural killer T cell response in COVID-19 patients

Background/Aims@#Coronavirus disease 2019 (COVID-19) is associated with acute respiratory syndrome. The mechanisms underlying the different degrees of pneumonia severity in patients with COVID-19 remain elusive. This study provides evidence that COVID-19 is associated with eosinophil-mediated inflammation. @*Methods@#We performed a retrospective case series of three patients with laboratory and radiologically confirmed COVID-19 pneumonia admitted to Chosun University Hospital. Demographic and clinical data on inflammatory cell lung infiltration and cytokine levels in patients with COVID-19 were collected. @*Results@#Cytological analysis of sputum, tracheal aspirates, and bronchoalveolar lavage fluid (BALF) samples from all three patients revealed massive infiltration of polymorphonuclear cells (PMNs), such as eosinophils and neutrophils. All sputum and BALF specimens contained high levels of eosinophil cationic proteins. The infiltration of PMNs into the lungs, together with elevated levels of natural killer T (NKT) cells in BALF and peripheral blood samples from patients with severe pneumonia in the acute phase was confirmed by flow cytometry. @*Conclusions@#These results suggest that the lungs of COVID-19 patients can exhibit eosinophil-mediated inflammation, together with an elevated NKT cell response, which is associated with COVID-19 pneumonia.

Assessment of the Synergistic Effect of Seven Antimicrobial Combinations on Extensively DrugResistant Acinetobacter baumannii Strains / 대한임상미생물학회지

Background@#To solve the difficulty in determining the appropriate treatment regimen for patients infected with extensively drug-resistant Acinetobacter baumannii (XDRAB), it is necessary to develop various strategies to increase the therapeutic effect of antimicrobial agents. The purpose of this study was to select the treatment combination showing the greatest antimicrobial effect among seven candidate antimicrobial substances. @*Methods@#Seven strains of XDRAB were used in this study. The composition of the treatment consisted of colistin as the base and one of the seven antimicrobial substances, doripenem, minocycline, tigecycline, linezolid, fusidic acid, vancomycin, or alyteserin E4K peptide. The interaction between the drugs in each combination was evaluated by measuring the synergy rates using time-kill analysis. @*Results@#The synergy rates of the seven combinations tested in the time-kill assay in this study were as follows, in descending order from the combination with the highest synergy rate: colistin + minocycline (57.1%), colistin + alyteserin E4K (50.0%), colistin + tigecycline (42.9%), colistin + vancomycin (28.6%), colistin + doripenem (14.3%), colistin + fusidic acid (14.3%), and colistin + linzolid (0%). None of the combinations showed antagonism. The three combinations showing bactericidal activity and the rates of their bactericidal activity were colistin + alyteserin E4K combination (33.3%), colistin + minocycline (14.3%), and colistin + vancomycin (14.3%). @*Conclusion@#The colistin + minocycline and colistin + alyteserin E4K treatment combinations, which showed high synergy rates, can be considered as promising candidates for future in vivo experiments evaluating combination therapies.

A Case of African Tick-Bite Fever in a Returning Traveler from Southern Africa

African tick-bite fever (ATBF), caused by Rickettsia africae, is the second most frequent cause of fever after malaria in travelers returning from Southern Africa. As the Korean outbound travelers are increasing every year, tick-borne rickettsial diseases as a cause of febrile illness are likely to increase. We describe a febrile Korean returning traveler who showed two eschars after visiting the rural field in Manzini, Swaziland. We performed nested polymerase chain reaction using the eschar and diagnosed the patient with ATBF. He was treated with oral doxycycline for 7 days, and recovered without any complications. We believe that the present case is the first ATBF case diagnosed in a Korean traveler.

The Effect of Long-lasting Permethrin Impregnated Socks on Tick Bite in Korea

Background@#The risk of tick-borne diseases is decreased by increasing awareness and knowledge through prevention education. The aim of the present study was to evaluate the effect of long-lasting permethrin impregnated (LLPI) socks for tick bites. @*Methods@#A randomized open label study was conducted to determine the effectiveness of LLPI socks for prevention of tick bites among 367 adults living in a rural area. Participants completed questionnaires at the start of follow-up (July 2014) and at the end of follow-up (December 2014), and tick bites were reported. @*Results@#A total of 332 subjects completed the follow-up survey. The tick bite rate of the two groups was not significantly different (3.6% vs. 3.1%). But the tick bite rate of lower extremities of subjects wearing LLPI socks was significantly lower compared to that of subjects wearing general socks. @*Conclusion@#The tick bite rate was not different between the two groups, but the tick bite rate of lower extremities of LLPI was significantly lower than general groups. Further study is needed to investigate the effect of LLPI clothes with larger populations.

The Effect of Long-lasting Permethrin Impregnated Socks on Tick Bite in Korea

Background@#The risk of tick-borne diseases is decreased by increasing awareness and knowledge through prevention education. The aim of the present study was to evaluate the effect of long-lasting permethrin impregnated (LLPI) socks for tick bites. @*Methods@#A randomized open label study was conducted to determine the effectiveness of LLPI socks for prevention of tick bites among 367 adults living in a rural area. Participants completed questionnaires at the start of follow-up (July 2014) and at the end of follow-up (December 2014), and tick bites were reported. @*Results@#A total of 332 subjects completed the follow-up survey. The tick bite rate of the two groups was not significantly different (3.6% vs. 3.1%). But the tick bite rate of lower extremities of subjects wearing LLPI socks was significantly lower compared to that of subjects wearing general socks. @*Conclusion@#The tick bite rate was not different between the two groups, but the tick bite rate of lower extremities of LLPI was significantly lower than general groups. Further study is needed to investigate the effect of LLPI clothes with larger populations.

Usefulness of Nested Polymerase Chain Reaction with Clinical Specimens for Diagnosis of Leptospirosis: a Case Series and a Review of Literature

A culture of the Leptospira species and the microscopic agglutination test (MAT) are considered as the reference standard for the diagnosis of leptospirosis, but both tests are imperfect for early diagnosis. We describe 4 patients diagnosed with leptospirosis using nested polymerase chain reaction (N-PCR) that targeted the 16S rRNA gene and the passive hemagglutination assay (PHA). In our 4 cases, Leptospira DNA in the urine, plasma, or cerebrospinal fluid (CSF), was detected by N-PCR in the early phase of leptospirosis, except in the sample from the buffy coat. Especially, case 3 showed that N-PCR with the urine and CSF was positive 8 days after symptom onset, but not for the plasma or buffy coat. We report 4 cases of leptospirosis that were diagnosed by N-PCR that targeted the 16S rRNA gene with urine, plasma, or CSF, but not the buffy coat. Three were cured by doxycycline but the case 4 was fatal. Detection of Leptospira DNA by PCR from the urine and CSF, in addition to plasma, may be helpful to confirm the diagnosis.

Evaluation of Synergistic Effect of Combined Treatment with Linalool and Colistin on Multidrug-Resistant Acinetobacter baumannii to Expand Candidate for Therapeutic Option / 대한임상미생물학회지

BACKGROUND: Acinetobacter baumannii infection is a significant health problem worldwide due to increased drug resistance. The limited antimicrobial alternatives for the treatment of severe infections by multidrug-resistant A. baumannii (MDRAB) make the search for other therapeutic options more urgent. Linalool, the major oil compound in Coriandrum sativum, was recently found to have high antibacterial activity against A. baumannii. The purpose of this study was to investigate the synergistic effect of linalool and colistin combinations against MDRAB and extensively drug-resistant A. baumannii (XDRAB).METHODS: A total of 51 strains of A. baumannii clinical isolates, consisting of 10 MDRAB and 41 XDRAB were tested. We determined the minimum inhibitory concentration (MIC) of linalool for the test strains using the broth microdilution method and searched for interactions using the time-kill assay.RESULTS: The time-kill assay showed that the linalool and colistin combination displayed a high rate of synergy (92.1%) (by synergy criteria 2), low rate of indifference (7.8%), and a high rate of bactericidal activity (74.5%) in the 51 clinical isolates of A. baumannii. The synergy rates for the linalool and colistin combination against MDRAB and XDRAB were 96% and 92.1%, respectively. No antagonism was observed for the linalool and colistin combination.CONCLUSION: The combination of linalool and colistin showed a high synergy rate, which may be beneficial for controlling MDRAB infections. Therefore, this combination is a good candidate for in vivo studies to assess its efficacy in the treatment of MDRAB infections.

Role of Efflux Pump Gene adeIJK to Multidrug Resistance in Acinetobacter baumannii Clinical Isolates / 대한임상미생물학회지

BACKGROUND: The emergence of multidrug-resistant Acinetobacter baumannii as a nosocomial pathogen is one of the major public health problems. The aim of this study was to evaluate the role of an efflux pump gene adeJ for the multidrug resistance of A. baumannii clinical isolates.METHODS: Two groups (MDRAB and SAB) of A. baumannii clinical isolates were studied. The SAB group consisted of strains that did not meet the criteria of MDRAB and were susceptible to more categories of antibiotics than MDRAB. Antimicrobial susceptibility results obtained by VITEKII system were used in data analysis and bacterial group allocation. We performed real-time reverse transcription PCR to determine relative expression of adeJ. We compared relative expression of adeJ in comparison groups by considering two viewpoints: i) MDRAB and SAB groups and ii) susceptible and non-susceptible groups for each antibiotic used in this study.RESULTS: The mean value of relative expression of adeJ of MDRAB and SAB groups was 1.4 and 0.92, respectively, and showed significant difference (P=0.002). The mean values of relative expression of adeJ of susceptible and non-susceptible groups to the antibiotics cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem, tigecycline, piperacillin/tazobactam, ticarcillin/clavulanic acid, trimethoprim/sulfamethoxazole, piperacillin, and gentamicin showed statistically significant differences.CONCLUSION: The overexpression of adeIJK might contribute to the multi-drug resistance in A. baumannii clinical isolates. Further, the overexpression of adeIJK might be one of the factors contributing to the resistance to numerous antibiotics.

Distribution of adeG, adeB, adeE, adeY, abeM, and adeJ Efflux Pump Genes in Clinical Isolates of Acinetobacter Species from Korea

BACKGROUND: The aim of the present study was to determine the frequency of six efflux pump genes in Acinetobacter clinical isolates collected from South Korean hospitals. METHODS: In this study, we used a total of 339 Acinetobacter strains, comprising 279 Acinetobacter calcoaceticus–Acinetobacter baumannii (ACB) complex and 60 non-ACB complex strains. We performed specific PCR assays to detect adeG, adeB, adeE, adeY, abeM, and adeJ, transporter genes of the multidrug efflux pumps AdeFGH, AdeABC, AdeDE, AdeXYZ, AbeM, and AdeIJK, respectively. RESULTS: Frequencies of six efflux pump genes varied according to the species of Acinetobacter. Frequencies of adeE, abeM, and adeJ between A. baumannii group and A. nosocomialis group were found to be significantly different. Significant differences were found in the frequencies of adeB, adeE, adeY, and adeJ among the susceptible A. baumannii (SAB), multidrug-resistant A. baumannii (MDRAB), and extensively drug-resistant A. baumannii (XDRAB) groups within the 154 strains of A. baumannii. The frequencies of efflux pump genes in imipenem-susceptible and imipenem-nonsusceptible groups were significantly different for adeB, adeY, and adeJ. The frequencies of efflux pump genes in ciprofloxacin-susceptible and ciprofloxacin-nonsusceptible groups were significantly different for adeB and adeY. No significant difference was found in the frequency of efflux pump genes among groups sampled from different regions of Korea, across 86 strains of A. baumannii collected in 2012. CONCLUSIONS: The frequencies of six efflux pump genes obtained in this study demonstrate the fundamental epidemiological feature of efflux pump genes in Korean Acinetobacter clinical isolates.

Comparison of the Utility of dnaJ and 16S rDNA Sequences for Identification of Clinical Isolates of Vibrio Species

BACKGROUND: Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis. METHODS: Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus-specific sequence, gyrB V. navarrensis-specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test. RESULTS: The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement. CONCLUSIONS: Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.

Assessment of Efflux Activity Using H33342 Accumulation in Tigecycline-Resistant Acinetobacter baumannii Clinical Isolates / 대한임상미생물학회지

BACKGROUND: Tigecycline resistance has emerged recently and has shown diverse mechanisms. The aim of this study was to assess the role of efflux activity in tigecycline resistance in 120 clinical isolates of A. baumannii using two methods: the H33342 accumulation assay and adeB real-time reverse transcriptase polymerase chain reaction. In addition, we analyzed the correlation between the expression level of adeB and H33342 accumulation level. METHODS: A. baumannii clinical isolates was divided into tigecycline-resistant (49 strains), intermediate (40 strains), and susceptible (31 strains) groups. The H33342 accumulation was measured in the absence or presence of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Real-time RT-PCR was performed to determine the relative expression of the adeB gene in A. baumannii clinical isolates. RESULTS: The level of H33342 accumulation in the resistant group was relatively lower than those in the other groups. The addition of CCCP caused a significantly increased fold change in H33342 accumulation in the tigecycline-resistant group. Significant difference in the fold change level in H33342 accumulation was found between tigecycline-susceptible and resistant isolates. Those findings support the role of efflux pumps of which substrates are H33342 in the resistance of tigecycline. Significant differences in the relative expression levels of adeB were shown between tigecycline-susceptible and resistant groups also. CONCLUSION: The results showed that several efflux pumps of which substrates were H33342 can contribute to tigecycline resistance. The adeB overexpression can also contribute to tigecycline resistance. It is possible that efflux pumps other than adeB efflux pumps contribute to tigecycline resistance because there was no correlation between fold change level in H33342 accumulation and adeB expression level.

In Vitro Interactions of Antibiotic Combinations of Colistin, Tigecycline, and Doripenem Against Extensively Drug-Resistant and Multidrug-Resistant Acinetobacter baumannii

BACKGROUND: Acinetobacter baumannii infections are difficult to treat owing to the emergence of various antibiotic resistant isolates. Because treatment options are limited for multidrug-resistant (MDR) A. baumannii infection, the discovery of new therapies, including combination therapy, is required. We evaluated the synergistic activity of colistin, doripenem, and tigecycline combinations against extensively drug-resistant (XDR) A. baumannii and MDR A. baumannii. METHODS: Time-kill assays were performed for 41 XDR and 28 MDR clinical isolates of A. baumannii by using colistin, doripenem, and tigecycline combinations. Concentrations representative of clinically achievable levels (colistin 2 microg/mL, doripenem 8 microg/mL) and achievable tissue levels (tigecycline 2 microg/mL) for each antibiotic were used in this study. RESULTS: The colistin-doripenem combination displayed the highest rate of synergy (53.6%) and bactericidal activity (75.4%) in 69 clinical isolates of A. baumannii. Among them, thedoripenem-tigecycline combination showed the lowest rate of synergy (14.5%) and bacteri-cidal activity (24.6%). The doripenem-tigecycline combination showed a higher antagonistic interaction (5.8%) compared with the colistin-tigecycline (1.4%) combination. No antagonism was observed for the colistin-doripenem combination. CONCLUSIONS: The colistin-doripenem combination is supported in vitro by the high rate of synergy and bactericidal activity and lack of antagonistic reaction in XDR and MDR A. baumannii. It seems to be necessary to perform synergy tests to determine the appropri-ate combination therapy considering the antagonistic reaction found in several isolates against the doripenem-tigecycline and colistin-tigecycline combinations. These findings should be further examined in clinical studies.

SmcR, the Quorum-sensing Master Regulator, Is Partially Involved in Temperature/Salinity-mediated Changes in Metalloprotease vvpE Expression in Vibrio vulnificus

Vibrio vulnificus, a gram-negative halophilic marine bacterium and opportunistic human pathogen, must withstand various environmental changes, especially simultaneous changes in temperature and salinity, from 25degrees C/2.5% to 37degrees C/0.9% (SCTS) upon entering the human body. In our previous study, SCTS stimulated vvpE expression even in the background of a mutation in luxS encoding LuxS enzyme for the biosynthesis of quorum-sensing (QS) signal molecule autoinducer-2 (AI-2), suggesting that the A1-2-mediated QS system is partially involved in the SCTS-mediated change of vvpE expression. In this study, we examined the effects of the QS master regulator SmcR on SCTS-mediated changes in vvpE expression and extracellular VvpE production. SCTS stimulated V. vulnificus growth, but with no increase in maximal growth levels. The SCTS-mediated prolongation of the stationary growth phase resulted in a significant increase in growth phase-dependent smcR and vvpE expressions. A mutation in smcR seriously repressed vvpE expression, but had no significant effect on V. vulnificus growth. However, the smcR mutation only partially attenuates SCTS-mediated changes in vvpE expression. These results indicate that SCTS stimulates the expressions of smcR and vvpE by stimulating V. vulnificus growth, and that SmcR is only partially involved in SCTS-mediated changes in vvpE expression.

Coordinate Regulation of Vibrio vulnificus Heme Receptor HupA Expression by Cyclic AMP-receptor Protein and Ferric Uptake Regulator

Vibrio vulnificus causes rapid progressing fulminant infections in susceptible individuals, especially those with elevated serum iron levels. This ferrophilic bacterium can directly acquire iron from heme-containing proteins, such as, hemoglobin, via its heme receptor protein HupA. This study was undertaken to determine the roles of cyclic AMP-receptor protein (Crp) as an activator and of ferric uptake regulator (Fur) as a repressor in regulating hupA expression at various iron and glucose concentrations. Under severely iron-deficient conditions, hupA expression in the absence of Crp was induced albeit at low levels and repressed by the addition of iron. In contrast, hupA expression in the presence of Crp was increased by the addition of iron. Under moderately iron-deficient and iron-sufficient conditions, iron addition repressed hupA expression in the presence of Fur, but not in the absence of Fur. Glucose addition repressed hupA expression in the presence of Fur but not in the absence of Fur. Furthermore, a mutation in cyaA encoding adenylate cyclase required for cAMP synthesis hupA expression, and this repression was prevented by the exogenous addition of cAMP. These results indicate that hupA expression is under the coordinate control of cAMP or Crp, which responds to glucose availability, and of Fur, which responds to iron availability, and that Crp is not essential for the constitutional expression of hupA, but is required for the optimal expression of hupA, whereas Fur is essential for the prevention of hupA over-expression.

Vibrio vulnificus Hemolysin Is Easily Inactivated in Spite of Being Produced at High Levels in Cirrhotic Ascites by a fur Mutation

Vibrio vulnificus produces Hemolysin/cytolysin (VvhA), which is one of the most potent exotoxins capable of killing mice at submicrogram levels. However, V. vulnificus growth and vvhA expression are severely repressed and extracellular VvhA produced at low levels is easily inactivated in human body fluids. This study was conducted to obtain additional unequivocal evidence of the enigmatic characteristic of VvhA. V. vulnificus growth was stimulated, vvhA expression was de-repressed, and extracellular VvhA production was increased in cirrhotic ascites, a human ex vivo experimental system, by a mutation of fur encoding ferric uptake regulator, which acts as a transcriptional repressor. However, regardless of the presence or absence of the fur mutation, extracellular VvhA activity was not detected in cirrhotic ascites. These results indicate that VvhA is easily inactivated even when vvhA expression and extracellular VvhA production are maintained at high levels in cirrhotic ascites.

Change of Vibrio vulnificus Metalloprotease VvpE Production by Temperature and Salinity

Vibrio vulnificus, a gram-negative halophilic marine bacterium and opportunistic pathogen, must withstand various environmental changes, especially the simultaneous change of temperature and salinity (SCTS) from 25degrees C/2.5% to 37degrees C/0.9% upon entering the human body. Previous studies have suggested that temperature and salinity may affect the production of metalloprotease VvpE via the LuxS-mediated autoinducer-2 quorum sensing system (AI-2-QSS). However, this hypothesis remains to be verified through coherent experiments. In this study, SCTS stimulated V. vulnificus growth with no increase in total growth levels. The SCTS-mediated prolongation of the stationary growth phase resulted in a significant increase in growth phase-dependent luxS and vvpE transcriptions; however, SCTS did not affect luxS or vvpE transcription levels during the exponential growth phase. SCTS also advanced extracellular VvpE production, which was consistent with vvpE transcription and V. vulnificus growth. SCTS-mediated modulation of vvpE expression was slightly attenuated but still observed in the background of a luxS mutation which seriously repressed vvpE expression. These results indicate that SCTS stimulates luxS and vvpE expression by stimulating V. vulnificus growth; however, the LuxS-mediated AI-2-QSS plays only a minor role, if any, in the SCTS-mediated modulation of vvpE expression.

Virulence Characteristics of Sucrose-fermenting Vibrio vulnificus Strains / 대한진단검사의학회지

We identified 6 sucrose-fermenting Vibrio vulnificus strains and examined their virulence characteristics. They were all encapsulated, motile, capable of producing toxins and utilizing transferrin-bound iron, cytotoxic to cultured cells, and virulent enough to kill mice. They could be definitely identified only by genetic identification methods such as PCR, and not by conventional culture-based identification methods such as API 20E (bioMerieux, France). These results indicate that it is essential to adopt genetic approaches as early as possible in order to avoid misdiagnosis of such strains, especially in clinical situations.

Effect of Iron-Chelator Deferiprone on the In Vitro Growth of Staphylococci

The standard iron-chelator deferoxamine is known to prevent the growth of coagulase-negative staphylococci (CoNS) which are major pathogens in iron-overloaded patients. However, we found that deferoxamine rather promotes the growth of coagulase-positive Staphylococcus aureus. Accordingly, we tested whether deferiprone, a new clinically-available iron-chelator, can prevent the growth of S. aureus strains as well as CoNS. Deferiprone did not at least promote the growth of all S. aureus strains (n=26) and CoNS (n=27) at relatively low doses; moreover, it could significantly inhibit the growth of all staphylococci on non-transferrin-bound-iron and the growth of all CoNS on transferrin-bound iron at relatively high doses. At the same doses, it did not at least promote the growth of all S. aureus strains on transferrin-bound-iron. These findings indicate that deferiprone can be useful to prevent staphylococcal infections, as well as to improve iron overload, in iron-overloaded patients.

Close Encounter of V. vulnificus with Host Cells is a Prerequisite to the Cytotoxicity of yet Unidentified New Virulence Factor

Live Vibrio vulnificus is highly cytotoxic to host cells in vivo and in vitro. The two most representative cytotoxins, cytolytic hemolysin and elastolytic protease, have been regarded to play major roles in the cytotoxicity of V. vulnificus. To further determine roles of the two cytotoxins in V. vulnificus pathogenesis, we constructed a double mutant of vvhA and vvpE genes, encoding a hemolysin and a protease, respectively. However, the cytotoxicity and the LD50 of a vvhA/vvpE double mutant showed no significant difference from those of the isogenic wild type strain. From these results, we came to speculate that yet unidentified, key cytotoxic factors should play a major role in the cytotoxic activity of V. vulnificus. The HeLa cells encountered with V. vulnificus became rounded, following detachment from the bottom of culture plate, and were killed eventually. However, the bacterial culture supernatant did not show any effect on the morphology and viability of HeLa cells. Also, no cytotoxicity could be noted when V. vulnificus was not allowed to contact with HeLa cells in the TranswellR system. Chloramphenicol, at lower concentration than minimum inhibitory concentration (MIC), decreased the cytotoxicity of a vvhA/vvpE double mutant to HeLa cells in a dose dependent manner. These results suggest that close encounter of V. vulnificus with host cells is a prerequisite to the cytotoxicity and that a yet unidentified virulence factor (s) should play an important role in the contact-dependent cytotoxicity.

Effect of Salinity, Temperature, and Glucose on the Production of Vibrio vulnificus Hemolysin

Among the exotoxins produced by V. vulnificus, hemolysin (HS) has been reported to be the most potent one. To investigate the factors up- or down-regulating HS production in the context of pathogenesis, we observed the effects of salinity or/and temperature shifting, glucose, and acidic pH on the production of HS by V. vulnificus C7184 strain in vitro. Significantly more HS was produced when V. vulnificus was cultured in 0.9% salinity and 37 degrees C than in 2.5% and 25 degrees C. When the culture condition reflecting natural habitat of V. vulnificus (2.5% salinity and 25degrees C) was changed into that reflecting human body (0.9% salinity and 37 degrees C), 2.5 fold or more HS was produced than in the V. vulnificus being cultured continuously in 0.9% NaCl at 37 degrees C. This result suggests that V. vulnificus somehow recognizes the shifting in salinity and temperature and stimulate HS production. Glucose addition in the culture medium resulted in a dose- dependent decrease in the HS production. Glucose itself and acidic pH resulting from its metabolism both appeared to inhibit the HS production. Glucose in itself had more dominant role in suppressing the HS production than the lowered pH accompanying the metabolism of glucose. This result suggests that HS production is down-regulated in the presence of glucose and under environmental acidic pH.