Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Microbiological, immunological, and histological changes in the gut of Salmonella Enteritidis-challenged rats fed goat cheese containing Lactobacillus rhamnosus EM1107.

Cheeses are able to serve as suitable matrices for supplying probiotics to consumers, enabling appropriate conditions for bacteria to survive gastric transit and reach the gut, where they are assumed to promote beneficial processes. The present study aimed to evaluate the microbiological, immunological, and histological changes in the gut of Salmonella Enteritidis-challenged rats fed goat cheese supplemented with the probiotic strain Lactobacillus rhamnosus EM1107. Thirty male albino Wistar rats were randomly distributed into 5 experimental groups with 6 animals each: negative (NC) and positive (PtC) control groups, control goat cheese (CCh), goat cheese added with L. rhamnosus EM1107 (LrCh), and L. rhamnosus EM1107 only (EM1107). All animals, except NC group were challenged with Salmonella Enteritidis (109 cfu in 1 mL of saline through oral gavage). Microbial composition was assessed with high-throughput 16S rRNA sequencing by means of Illumina MiSeq (Illumina, San Diego, CA). Nuclear factor kappa B (NF-κB) from the animal cecum tissue was determined by real-time PCR and interleukins (TNF-α, IL-1ß, IL-10, and IFN-γ) by means of ELISA. Myeloperoxidase and malondialdehyde levels were determined biochemically. The administration of the L. rhamnosus EM1107 probiotic strain, either as a pure culture or added to a cheese matrix, was able to reduce Salmonella colonization in the intestinal lumen and lessen tissue damage compared with rats from PtC group. In addition, the use of cheese for the probiotic strain delivery (LrCh) was associated with a marked shift in the gut microbiota composition toward the increase of beneficial organisms such as Blautia and Lactobacillus and a reduction in NF-κB expression. These findings support our hypothesis that cheeses might be explored as functional matrices for the efficacious delivery of probiotic strains to consumers.

Iron uptake mechanisms as key virulence factors in bacterial fish pathogens.

This review summarizes the current knowledge about iron uptake systems in bacterial fish pathogens and their involvement in the infective process. Like most animal pathogens, fish pathogens have evolved sophisticated iron uptake mechanisms some of which are key virulence factors for colonization of the host. Among these systems, siderophore production and heme uptake systems are the best studied in fish pathogenic bacteria. Siderophores like anguibactin or piscibactin, have been described in Vibrio and Photobacterium pathogens as key virulence factors to cause disease in fish. In many other bacterial fish pathogens production of siderophores was demonstrated but the compounds were not yet chemically characterized and their role in virulence was not determined. The role of heme uptake in virulence was not yet clearly elucidated in fish pathogens although there exist evidence that these systems are expressed in fish tissues during infection. The relationship of other systems, like Fe(II) transporters or the use of citrate as iron carrier, with virulence is also unclear. Future trends of research on all these iron uptake mechanisms in bacterial fish pathogens are also discussed.

Virulence properties of three new Photobacterium species affecting cultured fish.

AIMS: Several virulence factors of three new Photobacterium species: Photobacterium toruni, Photobacterium malacitanum and Photobacterium andalusiense associated with diseases of cultured redbanded seabream (Pagrus auriga) were studied. The exoenzymatic activities, adherence and cytotoxic capabilities, and iron-uptake mechanisms were determined both in bacterial extracellular products (ECP) and whole bacterial cells. The histopathology damages provoked on redbanded seabream by the ECP was also studied. METHODS AND RESULTS: The highest exoenzymatic activities of the ECP were alkaline- and acid-phosphatase, phosphohydrolase and lipase. The ECP were strongly lethal for fish at 4-96 h post-inoculation (p.i). Histological changes were evident at 96 hpi of ECP, affecting head kidney, splenic parenchyma and heart. Cytotoxicity assays, on three fish lines and one human cell line, were conducted using whole bacterial cells and their ECP. The new species tested were cytotoxic only for fish cell lines using whole bacterial cells. Bacterial adherence showed an adherence index moderate on CHSE-214 cell line. All strains showed variable haemolytic activity, and were able to grow under iron-limiting conditions, although the CAS reactivitiy was very low. However, all strains produced high amounts of extracelullar citrate that could be used as iron carrier, and use haem as iron source, except the P. toruni strains because a deletion in the genomic region encoding this ability in all Vibrionaceae members. CONCLUSIONS: The toxic activity of the bacterial ECPs was thermolabile, and not associated with their thermoresistant lipopolysaccharide content. The virulence of the strains tested could not be related to the haemolytic activity. Iron uptake could be based on the use of endogenous citrate as iron carrier and P. toruni lacks the ability to use haem as iron source. SIGNIFICANCE AND IMPACT OF THE STUDY: The study analyses for the first time the virulence properties of three new species of Photobacterium pathogenic for fish.

The ABC-transporter hutCD genes of Photobacterium damselae subsp. piscicida are essential for haem utilization as iron source and are expressed during infection in fish.

The marine fish pathogen Photobacterium damselae subsp. piscicida utilizes haem compounds as the sole iron source. In a previous work, we characterized a gene cluster with ten potential haem uptake and utilization genes. Two of these genes, hutC and hutD, which are iron-regulated, conform a putative inner membrane haem ABC transporter. In this study, we constructed an insertional mutant, leading to the inactivation of hutCD genes. Reverse transcriptase-PCR analyses demonstrated that an insertion between the hutB and hutC genes abolished transcription of the downstream hutC and hutD genes. The hutCD mutant was unable to utilize haem as the sole iron source, demonstrating that the putative ABC-transporter proteins HutC and HutD are essential for haem utilization as an iron source in P. damselae subsp. piscicida. In addition, reverse transcriptase-PCR assays conducted with RNA samples isolated from experimentally infected fish revealed the presence of hutCD transcripts. The results demonstrate for the first time that haem uptake genes of a fish pathogen are expressed during the infective process in fish.

Survival outcome and cost-effectiveness with docetaxel and paclitaxel in patients with metastatic breast cancer: a population-based evaluation.

BACKGROUND: A randomized controlled trial showed longer overall survival (OS) with docetaxel compared with paclitaxel in metastatic breast cancer patients with prior exposure to anthracycline. We report a similar comparison using population-based data. METHODS: Data on patients treated with single-agent paclitaxel or docetaxel were retrospectively reviewed. OS was compared using a two-tailed log-rank test and expressed as Kaplan-Meier plots. A cost-effectiveness analysis was carried out using cost/patient and OS. RESULTS: Four hundred and thirty-five patients met eligibility criteria. Prognostic factors were balanced between docetaxel and paclitaxel groups. Median OS was significantly longer for docetaxel versus paclitaxel [10.9 versus 8.3 months; hazard ratio 0.76; 95% confidence interval (CI), 0.62-0.92; P = 0.006]. The median number of cycles administered were four (docetaxel) and three (paclitaxel). The incremental cost-effectiveness ratio was $2434/per month of median survival gained. In the sensitivity analysis, the results were robust except that paclitaxel dominated when the low end of the 95% CI of survival for docetaxel was compared with the high end for paclitaxel. CONCLUSION: This population-based study corroborated the randomized trial's conclusion that for patients with metastatic breast cancer, docetaxel provided superior survival compared with paclitaxel. Each additional month of survival had an incremental cost of $2434.

Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities.

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100 degrees C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.

Effects of soy phytoestrogens genistein and daidzein on breast cancer growth.

OBJECTIVE: To determine whether genistein and daidzein, the major phytoestrogens in soy, can stimulate breast cancer growth. DATA SYNTHESIS: Systematic search through primary English-language literature on MEDLINE (1966-January 2001), EMBASE (1982-January 2001) and Current Contents (1998-January 2001). DATA SOURCES: Genistein and daidzein at low concentrations were found to stimulate breast tumor growth in in vitro and in vivo animal studies, and antagonize the antitumor effect of tamoxifen in vitro. At high concentrations, genistein inhibited tumor growth and enhanced the effect of tamoxifen in vitro. CONCLUSIONS: Genistein and daidzein may stimulate existing breast tumor growth and antagonize the effects of tamoxifen. Women with current or past breast cancer should be aware of the risks of potential tumor growth when taking soy products.

Presence of high-affinity iron uptake systems in fish-isolated and environmental strains of Vibrio anguillarum serotype O3.

This work describes the presence of high-affinity iron uptake systems in Vibrio anguillarum serotype O3 strains (subgroups A and B), isolated from diseased fish and environmental samples, as well as the presumptive effect of iron on their virulence. All strains demonstrated an ability to grow under iron-limiting conditions, production of catechol-type siderophores and synthesis of iron-regulated outer membrane proteins. However, clear differences were found depending on the isolation source, suggesting a more efficient iron uptake system in fish-isolated strains. Comparing the iron-regulated outer membrane protein profiles with those described for O1 and O2 serotypes, we found a protein which is specific for serotype O3, and another one that allows the differentiation of serotype O3 fish isolates from environmental strains. Moreover, only the strains showing this protein increased their virulence when iron was added to the inoculum in pathogenicity assays.

A new ciprofloxacin stepdown program in the treatment of high-risk febrile neutropenia: a clinical and economic analysis.

STUDY OBJECTIVE: To determine treatment outcomes and economic impact of a ciprofloxacin stepdown program for high-risk febrile neutropenic adults from the hospital's perspective. DESIGN: Unblinded, two-phase, single-center study. SETTING: Adult leukemia and stem cell transplant unit. PATIENTS: High-risk adults with febrile neutropenia. INTERVENTION: Two conditions were analyzed: a multidisciplinary ciprofloxacin stepdown program involving a reduction in parenteral ciprofloxacin dose from 400 to 200 mg (i.v.-i.v.) and conversion to oral ciprofloxacin (i.v.-p.o.) when criteria were met; and no i.v.-i.v. stepdown program. MEASUREMENTS AND MAIN RESULTS: Forty-six sequential treatment courses were compared with 42 treatment course from 6-month periods in preintervention (P1) and postintervention (P2) phases. Assessed parameters were clinical and microbiologic outcomes, adverse drug reactions (ADRs), and direct medical resource use and costs (1998 $Canadian) for the episode of febrile neutropenia. A decision analytic model was used to map probabilities and costs and to conduct sensitivity analyses. To supplement standard statistical testing, 1,000 bootstrap samples were created, and the mean cost difference was calculated between phases for each sample. Patient demographics, percentage i.v.-p.o. stepdown, and duration of therapy were similar between phases. Clinical success (83% P1, 81% P2), microbiologic eradication (15% P1, 24% P2), and possible ADRs (6% P1, 9% P2) did not differ. Intravenous-to-intravenous dose stepdown occurred in 33% of P2 and no P1 treatment courses (p<0.001). Resource use and costs were similar between phases, although a reduction was seen in the drug's mean total cost/day ($58 P1, $52 P2, p=0.04). There was also a trend toward a decrease in mean total treatment costs ($4,843 P1, $3,493 P2, p=0.08). In 1,000 bootstrap samples, 99.8% showed a cost advantage for P2. The model was robust to sensitivity analyses. CONCLUSION: This intervention influenced administration of ciprofloxacin without apparent compromise of patient outcomes and resulted in a reduction in total costs of treating febrile neutropenia.

Ferric-reductase activities in Vibrio vulnificus biotypes 1 and 2.

In this paper, the ferric-reductase activities of Vibrio vulnificus were investigated. This species comprises two biotypes pathogenic for humans and eels that are able to express different mechanisms for iron acquisition. All strains of both biotypes used in this study were able to reduce ferric citrate, irrespective of the iron levels in the growth medium. Some variation in the degree of reduction was observed among the strains, with the highest values corresponding to one acapsulated environmental strain of biotype 1. When cell fractions were tested, only those from periplasm and cytoplasm showed reductase activity whereas no activity was detected in membranes. Low temperatures inhibited these activities in both whole cells and cell fractions. At least six bands with ferric-reductase activity were identified in all strains using native polyacrylamide gels. These data demonstrate that the two biotypes of V. vulnificus produce similar ferric-reductases mainly located in the periplasm and cytoplasm and these could be involved in iron acquisition.

Isolation of a hemin and hemoglobin binding outer membrane protein of Vibrio vulnificus biotype 2 (serogroup E).

The eel pathogen Vibrio vulnificus biotype 2 (serogroup E) is able to use hemin (Hm) or hemoglobin (Hb) as the sole iron source for growth in vitro and in vivo. The mechanism of heme-iron acquisition in this bacterium requires a direct interaction through binding sites on the bacterial surface (constitutive outer membrane proteins). Using affinity chromatography techniques, a unique protein of around 36.5 kDa was isolated from cell envelopes of E86 strain regardless of the affinity ligand used, hemoglobin or hemin. This protein was purified from both iron-enriched and iron-restricted grown cells. These results support the hypothesis that in this pathogen Hm- and Hb-iron acquisition is mediated by a common protein receptor which recognizes the heme prosthetic group of Hb.

Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.

Isolation of heme-binding proteins from Vibrio anguillarum using affinity chromatography.

Using affinity chromatography techniques, several hemin- and hemoglobin-binding proteins of 97, 56 and 39 kDa were isolated from cell envelopes of Vibrio anguillarum strain H775-3 (serotype O1) and of 56, 46 and 37 kDa from strain RV22 (serotype O2). All these proteins were isolated under iron-rich as well as iron-poor conditions. Proteins of 39 kDa in H775-3 and of 37 kDa in RV22 isolated by hemin affinity could also bind biotinylated hemoglobin after being transferred to nitrocellulose, which suggests that they could be the common receptors for the heme group in V. anguillarum.

Identification of heme-binding proteins in the cell membranes of Vibrio anguillarum.

Two strains of Vibrio anguillarum belonging to O1 and O2 serotypes were examined for their ability to bind hemin and hemoglobin. Whole cells as well as membrane extracts from both strains could clearly bind hemin and hemoglobin constitutively. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled hemoglobin and also by hemin, suggesting the existence of specific receptors for heme groups in the cell membranes. Several hemin-binding and hemoglobin-binding bands with similar molecular sizes were detected in polyacrylamide gels as well as in Western blots. Only two of these protein bands in both strains were iron-regulated while the others were independent of the cell iron status. We conclude that both serotypes of V. anguillarum possess heme-binding abilities by means of membrane proteins.

Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly preferred site of insertion of Tn7 into the chromosome of Vibrio anguillarum.

The possible usefulness of Tn7 as a tool for genetic studies in Vibrio anguillarum was examined. Using the plasmid pRK2073 as the transposon donor, Tn7 transposes at high frequency into the chromosome of V. anguillarum. However, hybridization analysis of the mutants DNA digested with different enzymes revealed that all isolates have the insertions in the same site. This indicates that like in many other gram-negative bacteria, Tn7 shows a specificity of transposition in the chromosome of V. anguillarum. Plasmid pRK2013 proved to be a very useful delivery vector for transposon mutagenesis in V. anguillarum.

Distribution of plasmid- and chromosome-mediated iron uptake systems in Vibrio anguillarum strains of different origins.

We investigated the incidence of plasmid-mediated and chromosome-mediated iron uptake systems in strains of Vibrio anguillarum that belong to serotypes O1 and O2 and were isolated from different fish species and in different geographic areas. All of the strains gave positive reactions in CAS agar medium and in the Arnow test, which indicated that catechol types of siderophores were produced. The majority of V. anguillarum serotype O1 strains harbored a 65-kb plasmid similar to plasmid pJM1 from strain 775, which encodes the siderophore anguibactin and its outer membrane receptor, protein OM2. All of the isolates harboring this plasmid promoted the growth of an anguibactin-deficient receptor-proficient mutant derived from strain 775, but none of these isolates promoted the growth of mutants lacking receptor OM2. Furthermore, under iron-limiting conditions all of these strains induced outer membrane proteins that were identical in size to protein OM2 of strain 775. In contrast, none of the serotype O2 strains contained a high-molecular-weight plasmid, but all of them induced the growth of mutants defective in the anguibactin-mediated system regardless of the presence or absence of receptor OM2. The serotype O2 strains, but not the plasmid-bearing serotype O1 strains, also induced the growth of Salmonella typhimurium enb-1 which utilizes only enterobactin as a siderophore.(ABSTRACT TRUNCATED AT 250 WORDS)