Role of the virulence plasmid pR99 and the metalloprotease Vvp in resistance of Vibrio vulnificus serovar E to eel innate immunity.

Vibrio vulnificus biotype 2 serovar E (VSE) is a bacterial pathogen that produces a haemorrhagic septicaemia called vibriosis in eels. Its ability to grow in blood is conferred by a recently described virulence plasmid [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.]. In this study, we analyzed the role of this plasmid together with the role played by the metalloprotease (Vvp) in the interaction between bacteria and eel innate immunity. To this end, we compared and statistically analyzed the differences in resistance to serum and mucus factors (complement, selected antimicrobial peptides, transferrin and lysozyme) and also to phagocytosis/opsonophagocytosis between one VSE strain and its derivatives: a plasmid-cured strain and a vvp-deficient mutant. The wild-type and the metalloprotease-deficient strains were resistant to both the bactericidal action of fresh serum and the phagocytosis and opsonophagocytosis by eel phagocytes, confirming that Vvp is not involved in resistance to eel innate immunity. In contrast, the cured strain was sensitive to both the bactericidal action of eel serum activated by the alternative pathway and phagocytosis/opsonophagocytosis. Since no plasmid-encoded ORF, with homology to known genes, is related to the resistance to innate immunity [Lee CT, Amaro C, Wu KM, Valiente E, Chang YF, Tsai SF, et al. A common virulence plasmid in biotype 2 Vibrio vulnificus and its dissemination aided by a conjugal plasmid. Journal of Bacteriology, submitted for publication.], this function could be codified by one or more new genes. Further studies are underway to characterize the plasmid-encoded system responsible for V. vulnificus resistance to the innate immune system of eels.

Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin.

We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange. The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties. The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.

Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue.

Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificus virulence in mice.

Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA.

We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in E. coli DH5alpha reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E. coli or V. vulnificus. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of V. vulnificus mice.

Behavioural and serological human immunodeficiency virus risk factors among female commercial sex workers in Cambodia.

BACKGROUND: The spread of human immunodeficiency virus (HIV) in Cambodia is mainly caused by sexual transmission and the high-risk group in this country are female commercial sex workers (CSW). There are two types of CSW, direct CSW (DCSW) and indirect CSW (IDCSW), who are different from each other in sexual activities. This study was conducted in order to describe the risk factors on HIV for each type of CSW, and to establish effective preventive strategies against the HIV epidemic among CSW. METHODS: The participants, 143 DCSW and 94 IDCSW, were interviewed using a questionnaire to determine their demographic characteristics and behaviour. Blood samples were taken for serological tests on HIV, Chlamydia trachomatis and syphilis. The association between their behavioural pattern and their serological results was analysed. RESULTS: The questionnaire study showed that IDCSW had a riskier behavioural pattern than DCSW. The HIV seroprevalence rates of the DCSW and the IDCSW were 52.4% and 22.3%, respectively. Univariate logistic analyses showed a significant association between HIV antibody (HIV-Ab) and current age, age at commencement of commercial sex work, duration of commercial sex work, and the seropositivity of Chlamydia trachomatis-IgG antibody (CT-IgG-Ab) among the DCSW. The analyses also showed a significant relationship between HIV-Ab and CT-IgG-Ab among the IDCSW. CONCLUSIONS: Improving condom use rate is very important in order to prevent an HIV epidemic among the two types of CSW. This study also suggests it is important to prevent sexually transmitted disease (STD) such as Chlamydia trachomatis infection. The STD control programme could be efficient for HIV prevention, especially among DCSW.

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.

Cross-sectional study on risk factors of HIV among female commercial sex workers in Cambodia.

To describe epidemiological features on HIV prevalence among female commercial sex workers (CSWs), a cross-sectional study on sexual behaviour and serological prevalence was carried out in Cambodia. The CSWs were interviewed on their demographic characters and behaviour and their blood samples were taken for testing on sexually transmitted diseases, including HIV, Chlamydia trachomatis, syphilis, and hepatitis B. Associations between risk factors and HIV seropositivity were analysed. High seroprevalence of HIV and Chlamydia trachomatis IgG antibody (CT-IgG-Ab) was shown among the CSWs (54 and 81.7%, respectively). Univariate logistic regression analyses showed an association between HIV seropositivity and age, duration of prostitution, the number of clients per day and CT-IgG-Ab. Especially, high-titre chlamydial seropositivity showed a strong significant association with HIV prevalence. In multiple logistic regression analyses, CT-IgG-Ab with higher titre was significantly independently related to HIV infection. These suggest that existence of Chlamydia trachomatis is highly related to HIV prevalence.

Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection.

Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level. The effect of a high iron level on the virulence of V. vulnificus was therefore investigated in mice treated with iron dextran. The mice loaded with iron became highly susceptible to V. vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum. However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or D-galactosamine. Mice with or without iron-overloading died when the bacterial concentration in the blood reached 10(5) cfu/ml or above. Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron-overloaded mouse. V. vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration. Iron overload stimulated the production of tumor necrosis factor alpha (TNF-alpha), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF-alpha, appeared to be less active. Taken together, the major virulence factor of V. vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron-overloading. These two effects manifest other virulence factors, the host's as well as bacterial. Such factors, other than TNF-alpha stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.

Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan.

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.

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.

Cloning and nucleotide sequencing of the protease gene of Vibrio vulnificus.

The gene (vvp) encoding a thermolabile protease of Vibrio vulnificus was cloned and sequenced. The transcription start point was also determined by primer extension. The product of this gene is very likely the secretory neutral metalloprotease that has been purified and characterized previously.

Ribotyping of clinical Vibrio vulnificus isolates.

Restriction fragment length polymorphism analysis of rRNA genes (ribotyping) was used to differentiate Vibrio vulnificus isolates. Among the 10 restriction enzymes tested, HindIII was shown to provide the most discriminatory patterns. Stul was used for further analysis of strains that were indistinguishable with HindIII. Thirteen clinical V. vulnificus strains were analyzed for their ribotypes with HindIII, as well as Stul when necessary. Four of the clinical strains were isolated from different samples collected from the same patient, and were shown to have identical ribotypes. All the others gave unique ribotypes, indicating the large genetic divergence in V. vulnificus clinical isolates. The ribotype of V. vulnificus by HindIII remained unchanged after successive in vitro and in vivo passages. HindIII gave rise to five bands which were shown in every V. vulnificus strain but not in other vibrio species tested, suggesting that ribotyping with this restriction enzyme may be useful for confirming the identification of this bacterium.

Isolation and Characterization of Vibrio vulnificus Inhabiting the Marine Environment of the Southwestern Area of Taiwan.

Vibrio vulnificus, a marine bacterium, is of concern in Taiwan because it causes wound infections and sepsis with a high mortality rate every year. To examine for V. vulnificus, 13 samples of seawater or oysters were collected from nine sites in Yunlin, Chiayi, and Tainan. Seventy-seven strains of V. vulnificus were isolated from 11 samples. Among these environmental isolates, 72 (91%) were indole-positive, a characteristic of biotype 1. The remaining five strains although indole-negative, a characteristic previously found exclusively in biotype 2 strains, were all ornithine decarboxylase- and mannitol-positive, which has never been reported for biotype 2 strains. Based on the overall biochemical reactions obtained using a commercial identification system, these indole-negative strains appeared to be more like biotype 1. Fifty-seven ribotypes were identified among these isolates, indicating the great genetic divergence in this species. Of the 30 environmental isolates tested, 17 (56.7%) exhibited virulence comparable to the clinical isolates in the mouse, implying that a high proportion of the V. vulnificus strains in the marine environments might be pathogenic to humans. Copyright 1995 S. Karger AG, Basel

Differentiation of Vibrio vulnificus strains by an arbitrarily primed polymerase chain reaction.

A synthetic 17 mer oligonucleotide (5'-GTTGGGTAACGCCAGGG-3') was used as a primer for the arbitrarily primed polymerase chain reaction (AP-PCR) to differentiate various strains of Vibrio vulnificus. A total of 37 genomic DNAs that were extracted from the clinical and environmental strains were successfully differentiated. Among them, 32 profiles of the 37 strains were characterized. None of the environmental and clinical strains had the same amplification profile, suggesting the highly heterogeneous population existed in the strains of V. vulnificus. The size of the amplified sequences ranged from 0.3 to 2.0 Kb and the DNAs were separated to 12 to 20 bands by the 1.2% agarose gel. The clinical isolates from two independent episodes of V. vulnificus infections in a patient were shown to have the same profile, indicating that the second episode was due to recurrence rather than reinfection. The profiles of amplification were reproducible with different preparations of genomic DNA. Arbitrarily primed polymerase chain reaction can therefore be a useful tool for epidemiological study of V. vulnificus infection.

Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter.

The maltose transport system of Escherichia coli is a well-characterized member of the ATP binding cassette transporter superfamily. Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket. It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another. Periplasmic binding protein-dependent transport systems, like the maltose transport system of E.coli, possess a water-soluble ligand binding protein that is essential for transport activity. In addition to delivering ligand to the membrane-bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity. Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase. Genetic analysis indicates that, in general, two mutations are required for binding protein-independent transport and constitutive ATPase. The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG. Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex. Genetic alteration of this signalling pathway suggests that other agents might have similar effects. These could be potentially useful for modulating the activities of ABC transporters such as P-glycoprotein or CFTR, that are implicated in disease.

Genetic analysis of periplasmic binding protein dependent transport in Escherichia coli. Each lobe of maltose-binding protein interacts with a different subunit of the MalFGK2 membrane transport complex.

Escherichia coli is able to accumulate maltose and maltodextrins by an ATP-binding cassette transporter known as the maltose transport system. This transport system is comprised of five proteins: the LamB protein in the outer membrane; the periplasmic maltose-binding protein (MBP); two integral inner membrane proteins, MalF and MalG; and MalK, which is associated with the cytoplasmic face of the inner membrane. It has been previously suggested that MBP interacts with MalF and MalG during sugar transport across the inner membrane. In two independent genetic studies, reported here, residue 210 of MBP has been identified as an important site for its interaction with MalF. In one study, allele-specific suppressors of a malF mutation, malF506, were isolated and yielded mutations which altered residue tyrosine 210 of MBP to aspartic acid. In the other study, dominant mutations in malE (the structural gene of MBP) were isolated; one of these altered the same tyrosine residue (210) to cysteine. It was shown that the Y210C MBP mutant is also an allele-specific suppressor malF506, and that of the suppressor MBP alleles also exhibited dominant-negative phenotypes. Previously it was shown that alterations at residues glycine 13 and aspartate 14 of MBP can result in suppression of a malG mutant. From these results and those described, it is possible to propose a simple model in which the amino-terminal lobe of MBP interacts with MalG and the carboxy-terminal lobe of MBP interacts with MalF. The locations of residues 13, 14 and 210 on the three-dimensional structure of MBP are in keeping with this model.

Investigation and elimination of epidemic methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit.

A recent outbreak of methicillin-resistant Staphylococcus aureus (MRSA) infection in neonatal intensive care unit (NICU) in a teaching hospital was investigated. A total of 25 MRSA isolates, 16 from the patients and nine from the staffs (carriers), was collected for the study. The possible relationship among the isolates was investigated by using antibiograms and restriction enzyme analysis of plasmid DNA. Control strategy of the MRSA nosocomial infection was proposed after the study of antibiograms and plasmid profile. There were four plasmid patterns (I-IV), each containing 7, 8, 2, and 8 isolates. Six of the 16 isolates from the patients had identical plasmid patterns as the carriers (I and II). Person to person transfer via hand contact by medical personnel was found to be the most frequent mode of transmission identified in the outbreak of MRSA nosocomial infection in the NICU. Our strategy for control of the outbreak and elimination of the MRSA from all patients and carriers was successful after intensive surveillance and control measures. These included (a) strict isolation and cohorting; (b) hand washing between patient contacts to prevent transmission; (c) treatment of the carrier state in health care workers and patients with safe and effective topical agents such as mupirocin.

Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli.

Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles. The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild-type MBP to these mutant vesicles produced unexpected responses. Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Noradrenergic feeding elicited via the paraventricular nucleus is dependent upon circulating corticosterone.

Feeding behavior elicited by injection of norepinephrine (NE) into the paraventricular nucleus (PVN) of satiated rats has been shown to be abolished by hypophysectomy. To determine the specific nature of this dependence of NE's action on pituitary hormones, the efficacy of PVN-injected NE was examined in rats subjected to hypophysectomy, as well as to adrenalectomy, thyroidectomy, and gonadectomy, and also in operated rats receiving hormone replacement therapy. The feeding response induced by NE was almost completely abolished in adrenalectomized as well as hypophysectomized animals, but remained unimpaired after thyroidectomy and gonadectomy. The NE response was significantly restored in hypophysectomized rats by daily subcutaneous injections of corticosterone, but not by thyroxine, testosterone, insulin, or the mineralocorticoid deoxycorticosterone. In adrenalectomized rats, subcutaneous corticosterone implants as well as daily corticosterone injections (as opposed to deoxycorticosterone injections) effectively restored the NE eating effect. Radioimmunoassay of plasma corticosterone indicated that the level of hormone was positively correlated with the strength of the animals' response to the NE injection. These findings demonstrate that the loss of response to NE subsequent to hypophysectomy is due to a disruption of the hypothalamo-pituitary-adrenal axis. The glucocorticoids of the adrenal gland appear to be the essential humoral factor interacting permissively with PVN-injected NE to elicit feeding.

[Typing of Bacteroides fragilis by bacteriocin production].

Sixty-four strains of Bacteroides fragilis isolated from clinical specimens were tested for their bacteriocin production. It showed that 65.6% of them produced bacteriocin(s) and gave different patterns of inhibition. Among them 9 with clear inhibition zones were chosen as the indicator strains. With this indicator s,et 75.2% of 109 clinically isolated B. fragilis strains could be typed, with type 482 as the predominant type. The colonial dissociants gave the same bacteriocin type, but small and opaque colonies tend to lose their bactericidal ability.