Transcriptomic analysis of Vibrio alginolyticus challenged by Rhizoma coptidis reveals mechanisms of virulence genes.

Rhizoma coptidis, a Chinese herbal medicine widely used to treat various bacterial infections, has the potential to develop antibiotic substitutes to overcome the drug resistance of Vibrio alginolyticus. To study the inhibitory effect of R. coptidis on V. alginolyticus, we sequenced the transcriptomes of three groups of samples of wild-type V. alginolyticus (CK) and V. alginolyticus, which were stressed by 5 mg/mL R. coptidis for 2 h (RC_2 h) and 4 h (RC_4 h). CK was compared with RC_2 h and RC_4 h, respectively, and a total of 1565 differentially expressed genes (DEGs) (988 up-regulated and 577 down-regulated) and 1737 DEGs (1152 up-regulated and 585 down-regulated) were identified. Comparing RC_2 h with RC_4 h, 156 DEGs (114 up-regulated and 42 down-regulated) were identified. The ability of biofilm formation and motility of V. alginolyticus altered upon with different concentrations of R. coptidis. Interestingly, relative expression patterns of virulence genes appeared statistically significantly varied, upon different concentrations of R. coptidis extract. DEGs were annotated to the Gene Ontology (GO) database for function enrichment analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the results showed that the main enriched pathways, was those related to the virulence of V. alginolyticus. This study provides a new perspective for understanding the complex pathogenic mechanism of V. alginolyticus. R. coptidis could potnetially be used as alternative or complimnetary to antibiotics to treat infections after further research.

Clinical and Epidemiologic Characteristics and Therapeutic Management of Patients with Vibrio Infections, Bay of Biscay, France, 2001-2019.

Noncholera vibriosis is a rare, opportunistic bacterial infection caused by Vibrio spp. other than V. cholerae O1/O139 and diagnosed mainly during the hot summer months in patients after seaside activities. Detailed knowledge of circulating pathogenic strains and heterogeneities in infection outcomes and disease dynamics may help in patient management. We conducted a multicenter case-series study documenting Vibrio infections in 67 patients from 8 hospitals in the Bay of Biscay, France, over a 19-year period. Infections were mainly caused by V. alginolyticus (34%), V. parahaemolyticus (30%), non-O1/O139 V. cholerae (15%), and V. vulnificus (10%). Drug-susceptibility testing revealed intermediate and resistant strains to penicillins and first-generation cephalosporins. The acute infections (e.g., those involving digestive disorder, cellulitis, osteitis, pneumonia, and endocarditis) led to a life-threatening event (septic shock), amputation, or death in 36% of patients. Physicians may need to add vibriosis to their list of infections to assess in patients with associated risk factors.

Treatment Trial of Nile Tilapia (Oreochromis niloticus) Experimentally Infected with Vibrio alginolyticus Isolated from Sea bass (Dicentrarchus labrax).

BACKGROUND AND OBJECTIVE: In Egypt, Nile tilapia represents the main cultured type due to its economical price, palatability and easy culturing. This study was aimed to elucidate the pathogenicity of V. alginolyticus isolated from diseased sea bass and experimentally infected healthy Nile tilapia fish. MATERIALS AND METHODS: Healthy Nile tilapia fish were injected I/P with V. alginolyticus isolated from diseased sea bass. Symptoms and mortality rates of infected Nile tilapia fish were recorded during the experimental period. Re-isolation of V. alginolyticus was done from infected tilapia fish by bacteriological methods. For confirmation the pathogenicity of Vibrio isolated either from marine fish or tilapia fish, PCR test was done using tdh and bla gens. Liver and kidney function tests with histopathological examinations of some organs were performed. Treatment trial was done according to the antibiotic sensitivity test. RESULTS: The isolated Vibrio is highly pathogenic to Nile tilapia fish causing deterioration in all parameters which finished by severe mortalities. Treatment with florfenicol, enrofloxacin, or oxytetracycline reduced the mortality rate and improved liver and kidney function parameters of infected Nile tilapia fish. CONCLUSION: V. alginolyticus can infect both marine and fresh water fish inducing a high mortality rate. Treatment of infected fish with florfenicol, enrofloxacin, or oxytetracycline reduces the mortality rate.

Bactericidal effect on skin mucosa of dietary guava (Psidium guajava L.) leaves in hybrid tilapia (Oreochromis niloticus × O. mossambicus).

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the intensification practices in global aquaculture, fish are often confined in small volumes, which can results in outbreak diseases. In this context, the use of antibiotics is very usual. Thus, looking for natural substance able to reduce the use of the antibiotics is imperative. Among them, there is a great interest at present in the study of medicinal plants such as guava (Psidium guajava L.). These plants could help to develop a more sustainable aquaculture all over the world. The application of guava in traditional medicine dates for centuries and it is widely used in tropical countries for the treatment of diseases in human and animals. AIM OF THE STUDY: The purpose of this work was to study the effects of the dietary administration of dried leaves of Psidium guajava on the skin mucosal immunity of hybrid tilapia (Oreochromis niloticus × O. mossambicus). Furthermore, the ability of this plant to inhibit the bacterial load in different tissues after an experimental infection with Vibrio harveyi was studied. MATERIALS AND METHODS: P. guajava leaves collection and the experimentation was carried out in Dominican Republic. Fish were fed with a commercial diet supplemented with guava leaf at different concentrations (0%, 1.5% and 3%) for 21 days before being intraperitoneally injected with V. harveyi (1 × 104 cells mL-1). Thereafter, several immune activities were measured in fish skin mucus and after 48 h of injection, the skin, spleen and liver were collected to analyse the bactericidal activity of guava leaf and the gene expression of some immune related genes. RESULTS: The administration of P. guajava leaves significantly modulated some immune-related enzymes (protease, antiprotease and peroxidase) in the skin mucus of hybrid tilapia. In addition, the bacterial load after V. harveyi infection in skin, spleen and liver significantly reduced in fish supplemented with guava leaves. Finally, the expression profile of hepcidin gene in skin and liver was modulated in fish feed with control diet after V. harveyi infection. CONCLUSION: These results demonstrate that the dietary intake of guava leaves increases the skin mucosal barrier defences of hybrid tilapia and confers protection against V. harveyi colonization.

In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis.

The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

Screening, partial purification of antivibriosis metabolite sterol-glycosides from Rhodococcus sp. against aquaculture associated pathogens.

The present study probed the antimicrobial potential of a rare mangrove associated actinomycetes against an array of aquatic bacterial pathogens causing disease outbreak in fin and shellfish. Antibacterial activity results implied that the mangrove associated actinomycetes RAS7 exhibited striking inhibitory activity against the tested aquatic bacterial pathogens. Identification of strain RAS7 through polyphasic and 16S rRNA sequencing affirmed that the strain belongs to Rhodococcus sp. Optimization of culture conditions for antibacterial activity by Rhodococcus sp. inferred that it grew well and exerted notable antagonistic activity in medium supplied with 1% galactose and peptone as carbon and nitrogen sources. Similarly, the strain grown in 0.1% tyrosine, 1% NaCl, pH 7.5 and temperature 35 °C recorded maximum bioactivity against the test pathogens. The crude ethyl acetate extract of Rhodococcus sp. at 200  µg/ml recorded markedly pronounced growth inhibitory activity ranged between 14 and 29 mm. The cytotoxic effect of crude extract against brine shrimp Artemia salina nauplii registered LC50 value of 134.294 µg/ml after 24 h of exposure. The secondary metabolite was separated using Ethyl acetate: Methanol (7:3) as solvent system through TLC. The TLC autobiogram mapped the active spot in TLC with Rf value of 0.84. Analysis of chemical constituents and FT-IR spectral analysis substantiated that the active principle in bioassay guided fraction was sterol-glycosides.

Outcomes of Third-Generation Cephalosporin Plus Ciprofloxacin or Doxycycline Therapy in Patients with Vibrio vulnificus Septicemia: A Propensity Score-Matched Analysis.

BACKGROUND: Combination therapy with a third-generation cephalosporin (TGC) and a tetracycline analogue is recommended for Vibrio vulnificus infection. The combination of a TGC and ciprofloxacin has synergistic in vitro bactericidal activity against V. vulnificus. No clinical study has compared the standard regimen with TGC plus ciprofloxacin therapy for V. vulnificus infection. METHODS: Patients with a confirmed V. vulnificus infection at two medical centers in Korea from 1991 to 2016 were enrolled in this study. The patients were grouped according to the type of antibiotic administered. A retrospective propensity-score-matched case-control study of patients treated with TGC plus doxycycline or TGC plus ciprofloxacin was performed. The clinical characteristics and outcomes of the patients were analyzed. RESULTS: A total of 218 patients were confirmed to have V. vulnificus septicemia during the study, and the 30-day survival rate was 39% (85/218). The patients were classified into the following six treatment groups: TGC monotherapy (n = 82), TGC plus doxycycline therapy (n = 42), TGC plus ciprofloxacin therapy (n = 39), ciprofloxacin monotherapy (n = 14), other ß-lactam monotherapy (n = 10), and other (n = 31). The survival rates of these groups were as follows: TGC monotherapy (35%), TGC plus doxycycline (38%), TGC plus ciprofloxacin (54%), ciprofloxacin monotherapy (29%), other ß-lactam (20%), and other (39%). The 30-day survival rate showed no significant difference between the TGC plus doxycycline and TGC plus ciprofloxacin groups (log-rank test, P = 0.18). Among the 81 patients treated with TGC plus doxycycline or TGC plus ciprofloxacin, 12 per treatment group were selected by propensity-score matching. There was no significant difference in the baseline characteristics or the frequency of fasciotomy between the two groups. The 30-day survival rate showed no significant difference between the TGC plus doxycycline (50%) and TGC plus ciprofloxacin (67%) groups (log-rank test, P = 0.46). CONCLUSION: Our data suggest that the outcome of TGC plus ciprofloxacin therapy was comparable to that of TGC plus doxycycline therapy in patients with V. vulnificus septicemia.

Efficacy assessment of commercially available natural products and antibiotics, commonly used for mitigation of pathogenic Vibrio outbreaks in Ecuadorian Penaeus (Litopenaeus) vannamei hatcheries.

Bacterial diseases cause high mortality in Penaeus (Litopenaeus) vannamei postlarvae. Therefore, appropriate application of efficient therapeutic products is of vital importance for disease control. This study evaluated through in vitro analyses the antimicrobial effectiveness of commercial therapeutic products used for P. vannamei bacterial diseases and antibiotics against pathogenic Vibrio strains circulating in Ecuadorian hatcheries. Twenty strains were isolated from 31 larvae samples with high bacterial counts from 10 hatcheries collected during mortality events. The strains virulence was verified through challenge tests with Artemia franciscana nauplii and P. vannamei postlarvae. Through 16S rRNA sequence analysis, strains showed a great similarity to the Vibrio sequences reported as pathogens, with 95% belonging to the Harveyi clade. Through antibiograms and minimal inhibitory concentration (MIC) in vitro tests we found that furazolidone, ciprofloxacin, chloramphenicol, norfloxacin, nalidixic acid, florfenicol, fosfomycin and enrofloxacin inhibited the growth of all or most of the strains. Less efficient antibiotics were penicillin, oxytetracycline and tetracycline. A multiple antibiotic resistance (MAR) index of 0.23 showed some level of resistance to antibiotics, with two MAR prevalent patterns (Penicillin-Oxytetracycline and Penicillin-Oxytetracycline-Tetracycline). From a total of 16 natural products (five probiotics, nine organic acids and two essential oils), only three (one probiotic, one organic acid and one essential oil) were effective to control most of the strains. Shrimp producers can apply relatively simple in vitro analyses, such as those employed in this study, to help take adequate management decisions to reduce the impact of bacterial diseases and increase profit.

In vitro and in vivo antibacterial activity of tigecycline against Vibrio vulnificus.

BACKGROUND/PURPOSE: The aim of this study is to investigate the role of tigecycline in Vibrio vulnificus infection. METHODS: Eight randomly selected clinical V. vulnificus isolates were studied to obtain the minimal inhibitory concentrations (MICs) of minocycline, cefotaxime, and tigecycline, and the time-kill curves of tigecycline alone or in combination with other drugs. A peritonitis mouse model was used for the evaluation of the therapeutic efficacy of tigecycline alone or cefotaxime in combination with minocycline or tigecycline. RESULTS: The MIC of minocycline, cefotaxime, and tigecycline for eight clinical V. vulnificus isolates was 0.06-0.12 µg/mL, 0.03-0.06 µg/mL, and 0.03-0.06 µg/mL, respectively. In time-killing studies, at the concentration of 1 × MIC, the inhibitory effect of tigecycline persisted for 24 hours in five of eight isolates. With 2 × MIC and trough level, the inhibitory effect was noted in all isolates for 24 hours. With the combination of minocycline plus cefotaxime and tigecycline plus cefotaxime at 1/2 × MIC, the bactericidal effect was noted in 25% and 62.5% of eight isolates and synergism in 50% and 75% of isolates. With a low (1.25 × 105 CFU/mL) inoculum, all infected mice survived with tigecycline alone, tigecycline plus cefotaxime, or minocycline plus cefotaxime on the 14th day. At the inoculum of 1.25 × 106 CFU, the survival rate was 33.3% on the 14th day in the tigecycline plus cefotaxime-treated group, but none of the mice treated by tigecycline alone or minocycline plus cefotaxime survived (33.3% vs. 0%, p = 0.01 by Fisher's exact test). CONCLUSION: Our in vitro combination and animal studies indicate that tigecycline could be an option for the treatment of invasive V. vulnificus infections.

Antimicrobial peptide-loaded gold nanoparticle-DNA aptamer conjugates as highly effective antibacterial therapeutics against Vibrio vulnificus.

Vibrio vulnificus causes fatal infections in humans, and antibiotics are commonly used in treatment regimens against V. vulnificus infection. However, the therapeutic effects of antibiotics are limited by multidrug resistance. In this study, we demonstrated that an antimicrobial peptide (AMP), HPA3PHis, loaded onto a gold nanoparticle-DNA aptamer (AuNP-Apt) conjugate (AuNP-Apt-HPA3PHis) is an effective therapeutic tool against V. vulnificus infection in vivo in mice. HPA3PHis induced bacterial cell death through the disruption of membrane integrity of V. vulnificus. The introduction of AuNP-Apt-HPA3PHis into V. vulnificus-infected HeLa cells dramatically reduced intracellular V. vulnificus by 90%, leading to an increase in the viability of the infected cells. Moreover, when V. vulnificus-infected mice were intravenously injected with AuNP-Apt-HPA3PHis, a complete inhibition of V. vulnificus colonization was observed in the mouse organs, leading to a 100% survival rate among the treated mice, whereas all the control mice died within 40 hours of being infected. Therefore, this study demonstrated the potential of an AMP delivered by AuNP-Apt as an effective and rapid treatment option against infection caused by a major pathogen in humans and aquatic animals.

Efficacy of Ceftriaxone, Cefepime, Doxycycline, Ciprofloxacin, and Combination Therapy for Vibrio vulnificus Foodborne Septicemia.

Foodborne Vibrio vulnificus infections are associated with higher rates of sepsis and mortality than wound infections; however, antibiotic efficacy studies have not been performed in foodborne infection models. The efficacies of ceftriaxone, cefepime, doxycycline, ciprofloxacin, and combination therapy were assessed in V. vulnificus intestinal infection in mice in order to model foodborne infections. In accordance with prior studies of cefotaxime, cefepime was synergistic with doxycycline and ciprofloxacin in vitro; combination therapy significantly decreased bacterial growth, by ≥2 log10 units, from that with antibiotic monotherapy (P < 0.01). In vivo, survival rates in the ceftriaxone (50%), doxycycline (79%), and ciprofloxacin (80%) groups were significantly higher than those in the control group (0%) (P < 0.0001). Survival was significantly higher with ceftriaxone-doxycycline (91%) or ceftriaxone-ciprofloxacin (100%) therapy than with ceftriaxone (50%) (P ≤ 0.05). Survival with cefepime-doxycycline (96%) or cefepime-ciprofloxacin (90%) therapy was significantly higher than that with cefepime alone (20%) (P < 0.001). There was no difference in survival between the combination therapy groups. Thus, we conclude that combination therapy was the most effective treatment for foodborne V. vulnificus septicemia. In a septic patient with a recent ingestion of raw seafood, cefepime in combination with doxycycline or ciprofloxacin should be initiated for coverage of resistant Gram-negative organisms and V. vulnificus pending a microbiological diagnosis. Once a diagnosis of foodborne V. vulnificus septicemia is established, treatment can safely transition to ceftriaxone in combination with doxycycline or ciprofloxacin.

Virulence factors and antimicrobial resistance in environmental strains of Vibrio alginolyticus

Vibrio alginolyticus has acquired increasing importance because this microorganism may be pathogenic to aquatic animals and humans. It has been reported that some V. alginolyticus strains carry virulence genes derived from pathogenic V. cholerae and V. parahaemolyticus strains. In this work V. alginolyticus was isolated from oyster samples acquired from a food-market in Mexico City. Thirty isolates were identified as V. alginolitycus. Strains showed β-haemolysis and proteolytic activity and produced a capsule. Strains displayed swimming and swarming motility and 93.3% of them produced siderophores. Several genes encoding virulence factors were detected using PCR amplification. These included proA, wza, vopD, vopB, hcp, vasH and vgrG genes, which were present in all strains. Other genes had a variable representation: tdh (86.6%), lafA (96.6%), pvsA (62%) and pvuA (16%). The trh gene could not be amplified from any of the strains. The antimicrobial resistance profile revealed that more than 90% of the strains were resistant to beta-lactams antibiotics, 60% to cephalotin, 45% to amikacin, 16% to cephotaxime, and 10% to pefloxacin, while 100% were susceptible to ceftriaxone. The V. alginolyticus strains isolated from oysters showed multiple resistance to antibiotics and several virulence factors described in well-characterized pathogenic vibrios (AU)

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Inhibition of marine Vibrio sp. by pyoverdine from Pseudomonas aeruginosa PA1.

Siderophores are low-molecular-weight chemicals that are secreted by many microorganisms to chelate iron from the external environment in order to facilitate their growth and diverse metabolisms. In this study, a fluorescent siderophore, pyoverdine, secreted by Pseudomonas aeruginosa PA1 was purified by affinity chromatography using Cu-sepharose. Pyoverdine was determined to have a molecular mass of 1333.54 Da, as determined by MALDI-TOF/TOF, and belong to type I pyoverdine, as determined by PCR analysis of its corresponding outer membrane ferri-pyoverdine receptor. Pyoverdine showed different degrees of inhibitory effects on the growth of marine Vibrio sp. strains. It was also shown that the biofilm developed by Vibrio parahaemolyticus WzW1 and Wz2121 and Vibrio cyclitrophicus HS12 was significantly reduced, alone with the repressed growth in the presence of pyoverdine. Siderophore production was determined in the strains of Vibrio sp. in response to the pyoverdine-induced iron-limited conditions. The siderophore production of most Vibrio sp. was up-regulated, with the exception of the bacteria that produced little siderophore. Furthermore, Apostichopus japonicus cultured in pyoverdine pretreated seawater showed a relative percent of survival of 89% when they were challenged by Vibrio splendidus. Our results demonstrated that pyoverdine may be a promising agent that could be potentially applied to treat vibriosis.

In vivo efficacy of the combination of ciprofloxacin and cefotaxime against Vibrio vulnificus sepsis.

OBJECTIVES: The in vivo efficacy of a cefotaxime-ciprofloxacin combination against Vibrio vulnificus and the effects on rtxA1 expression of commonly used antibiotics are unknown. METHODS: In vitro time-kill studies were performed to evaluate synergism. Female BALB/c mice were injected subcutaneously with 1×10(7) or 1×10(8) cfu of V. vulnificus. Antibiotic therapy was initiated at 2 h after inoculation in the following four therapy groups: cefotaxime; ciprofloxacin; cefotaxime-plus-ciprofloxacin; and cefotaxime-plus-minocycline. The cytotoxicity of V. vulnificus for HeLa cells was measured using the lactate dehydrogenase assay; rtxA1 transcription was measured in a transcriptional reporter strain using a ß-galactosidase assay. RESULTS: In vitro time-kill assays exhibited synergism between cefotaxime and ciprofloxacin. In the animal experiments, the 96-h survival rate for the cefotaxime-plus-ciprofloxacin group (85%; 17/20) was significantly higher than that of the cefotaxime-plus-minocycline (35%; 7/20) and cefotaxime alone (0%; 0/20) groups (P<0.05 for both). Bacterial counts in the liver and spleen were significantly lower in the cefotaxime-plus-ciprofloxacin group 24 and 48 h after treatment, relative to the other groups. At sub-inhibitory concentrations, ciprofloxacin inhibited more effectively rtxA1 transcription and mammalian cell cytotoxicity than either minocycline or cefotaxime (P<0.05 for both). CONCLUSIONS: Ciprofloxacin is more effective at reducing rtxA1 transcription and subsequent cytotoxicity than either minocycline or cefotaxime, and the combination of ciprofloxacin and cefotaxime was more effective in clearing V. vulnificus in vivo than previously used regimens. These data suggest that the combination of ciprofloxacin and cefotaxime is an effective option for the treatment of V. vulnificus sepsis in humans.

Resveratrol--a potential inhibitor of biofilm formation in Vibrio cholerae.

Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae.

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.

In vitro and in vivo bactericidal activity of Vitex negundo leaf extract against diverse multidrug resistant enteric bacterial pathogens.

OBJECTIVE: To investigate in vitro and in vivo antibacterial potentials of Vitex negundo (V. negundo) leaf extracts against diverse enteric pathogens. METHODS: Water and methanol extracts of V. negundo leaves were evaluated against enteric bacterial pathogens by using standard disc diffusion, viable bacterial cell count methods, determination of minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). RESULTS: Methanol extract of V. negundo leaves showed potent antibacterial activity (inhibition zone: 9.9-22.6 mm, MIC: 200-3200 µg/mL, MBC: 200-6400 µg/mL) against all the pathogenic enteric bacteria (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio mimicus, Echerichia coli, Shigella spps., and Aeromonas spps) tested. Methanol extract of V. negundo leaves showed potent bactericidal activity both in vitro laboratory conditions (MBC, 200-400 µg/mL) and in the intestinal environment (Dose, 1-2 mg/mL) of infant mice against pathogenic Vibrio cholerae, the major causative agent of cholera. Furthermore, assays using the mice cholera model showed that V. negundo methanol extract can protect mice from Vibrio cholerae infection and significantly decrease the mortality rate (P<0.0001). CONCLUSIONS: For the first time we showed that methanol extract of V. negundo leaves exhibited strong vibriocidal activity both in vitro and in vivo conditions. Therefore, it will be useful to identify and isolate the active compounds of this extract that could be a good alternative of antibiotics to treat cholera.

Quantitative PCR and in vivo efficacy of antibiotics in the treatment of Vibrio vulnificus infection in a mouse model.

The Centers for Disease Control and Prevention (CDC) recommend oral or intravenous doxycycline plus a third-generation cephalosporin or fluoroquinolone alone for the treatment of Vibrio vulnificus infections. Until now, no study has compared oral with parenteral administered doxycycline with respect to their in vivo efficacy. In the present work, ICR mice infected with a high dose of V. vulnificus were administered ciprofloxacin, ceftriaxone, and doxycycline. The bacterial DNA copy number in surviving and non-surviving mice was determined using quantitative polymerase chain reaction (qPCR). In this setting, ciprofloxacin was the most effective monotherapeutic drug, but a higher survival rate (50%) was achieved using the combination therapy of intraperitoneal doxycycline plus ceftriaxone. The blood of non-surviving mice at 12 h post-infection contained at least 10(4) DNA copies/µL, in contrast to 10(2) to 10(3) DNA copies/µL in surviving mice. Thus, in the treatment of V. vulnificus infections in humans, when the intravenous form of doxycycline is unavailable, ciprofloxacin might be a better option than oral doxycycline to lower mortality. In addition, our results demonstrate that qPCR can be a useful tool for identifying the V. vulnificus load in infected patients, with the DNA copy number providing a marker of either disease severity or mortality.

Kalopanax pictus as feed additive controls bacterial and parasitic infections in kelp grouper, Epinephelus bruneus.

Feeding kelp grouper, Epinephelus bruneus (26.1 ± 1.4), with 0%, 0.1%, 1.0%, and 2.0% Kalopanax pictus extract-supplementation diets, for 30 days reduced mortality on being challenged intraperitoneally 100 µl with bacterium Vibrio alginolyticus (2.1 × 10(7) cfu ml(-1)) and ciliate parasite Philasterides dicentrarchi (2.3 × 10(7) ciliates ml(-1)). The red blood cells (RBC), white blood cells (WBC), haemoglobin, haematocrit, lymphocytes, and monocytes levels significantly increased in kelp grouper fed with all doses of K. pictus-supplementation diets and challenged with bacterium and parasite when compared to control. However, the levels of mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), neutrophils, and thrombocytes did not significantly. The phagocytic activity, complement activity, and antiprotease activity did not significantly change in kelp grouper fed with 0.1% K. pictus-supplementation diets and challenged with bacterium and parasite. The respiratory activity, lysozyme activity, bactericidal activity, total protein level, and myeloperoxidase levels significantly increased in kelp grouper fed with all the doses of K. pictus-supplementation diet and challenged with bacterium and parasite. However, α2-macroglobulin level significantly increased with 1.0% diet, but not with 0.1% and 2.0% diets. Therefore this study suggests that 1.0% and 2.0% K. pictus-supplementation diets positively protected and enhanced the immune system in kelp grouper E. bruneus against V. alginolyticus and P. dicentrarchi infection.