Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes Vibrio cholerae bile resistance, virulence factor expression, and intestinal colonization

The transmembrane transcriptional activators ToxR and TcpP modulate expression of Vibrio cholerae virulence factors by exerting control over toxT, which encodes the cytoplasmic transcriptional activator of the ctx, tcp, and acf virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in V. cholerae pathogenesis, the ToxR-activated ompU promoter was used to drive ompT transcription in a strain lacking OmpU. Likewise, the ToxR-repressed ompT promoter was used to drive ompU transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a toxR+ strain that expresses OmpT in place of OmpU, and a toxR− strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the toxR+ V. cholerae expressing OmpT in place of OmpU, but increased in toxR− V. cholerae expressing OmpU in place of OmpT. Additionally, the toxR+ V. cholerae expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced toxT transcription in this strain. Finally, the toxR+ V. cholerae expressing OmpT in place of OmpU was ≈100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for V. cholerae bile resistance, virulence factor expression, and intestinal colonization.

Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains.

Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139.

The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.

Observaciones preliminares del crecimiento de Paralichthys adspersus y presencia de infecciones por Vibrio spp. durante el ensayo de cultivo de Tumbes

La presente investigación es para evaluar la posibilidad de cultivo de Paralichthys adspersus (lenguado común) bajo las condiciones fisicoquímicas del agua del mar de la región de Tumbes. Luego de aclimatar 100 ejemplares de P. adspersus a la temperatura de 23,4 °C y 32 ‰ de salinidad, se distribuyó en dos tanques iguales de 1000 L a una densidad inicial de 50 ind./m3, realizando recambios diarios de 50 %, alimentados a saciedad durante todo el día con alimento balanceado de 42 % de proteínas (consumo total de alimento: 413 g). Mensualmente se muestreo el 100 % de los peces, determinando su tasa de crecimiento especifico, supervivencia y presencia de infecciones por Vibrio spp. La tasa de crecimiento específico durante esta experiencia fue de 0,41 %/día, la supervivencia luego de la aclimatación fue de 100 % hasta los 42 días de cultivo que alcanzó el 0 %, debido a la infestación por protozoarios parásitos del genero Amyloodinium en las branquias, descartando en todo momento la presencia de infecciones por Vibrio spp. La presente investigación permite precisar que P. adspersus se adapta a las condiciones fisicoquímicas de la región de Tumbes

Joh. Andreae Fischeri ... Consilia medica : quae in usum practicum & forensem pro scopo curandi & renunciandi adornata sunt : accessit eiusdem autoris consiliarius metallicus.

Mode of access: Internet.

Joh. Andreae Fischeri ... Consilia medica continuata : quae in usum practicum & forensem pro scopo curandi & renunciandi adornata sunt : Accessit eiusdem autoris Mantissa medicamentorum singularium.

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Joh. Andreae Fischeri ... Consilia medica iterum continuata : quae in usum practicum & forensem pro scopo curandi & renunciandi adornata atque singularibus experimentis, inter arcana domestica hactenus reservatis, illustra sunt /

Mode of access: Internet.

Joh. Andreae Fischeri ... Responsa practica et forensia selecta : ab anno MDCCVI. usque ad annum MDCCXIIX. ; quibus una cum indice generali loco mantissae ; accessit crügnerus redivivus, sive tr. De polychresta materia perlata ...

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Laboratory methods for the diagnosis of Vibrio cholerae.

Prepared by the National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC) in cooperation with the Pan American Health Organization (PAHO).

Intestinal ameliorative effects of traditional Ogi-tutu, Vernonia amygdalina and Psidium guajava in mice infected with Vibrio cholera.

Background: Cholera, a severe acute watery diarrhea caused by Vibrio cholerae is endemic in Nigeria with most cases occurring in the rural areas. In South West Nigeria, some individuals resort to alternative treatments such as Ogi-tutu, Psidium guajava and Vernonia amygdalina during infections. The effectiveness of these alternatives in the prevention and treatment of V. cholerae infection requires experimental investigation. Objective: This study was designed to investigate the ameliorative effects of Ogi-tutu, Vernonia amygdalina and Psidium guajava on intestinal histopathology of experimental mice infected with V. cholerae. Methods: Preliminary investigation of in vitro vibriocidal activities of these alternatives were carried out using agar cup diffusion assay. For ameliorative effects, adult mice were inoculated with 100 μl (106 cells) of Vibrio cholerae and dosed at 0 h (immediate prevention) and 4 h (treatment of infection) and their intestines were histopathologically evaluated. Results: The histopathological changes were the same irrespective of the treated groups, but the lesions varied in extent and severity. The ameliorative effects in decreasing order were V. amygdalina > P. guajava > Ogi-tutu. Conclusion: V. amygdalina gave the best ameliorative effects in the prevention and treatment of V. cholerae infection.

Identificación de las proteínas implicadas en la respuesta al estrés en Escherichia coli y Vibrio harveyi.

187 p.

Anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum (Linn)

Purpose: To evaluate the anti-vibrio potentials of acetone and aqueous leaf extracts of Ocimum gratissimum and determine its relevance in the treatment of vibrios infection. Methods: The agar-well diffusion method was used for screening the extracts for their anti-vibrio activity. Broth micro-dilution assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extracts. Time-kill assay was used to assess bactericidal and/or bacteriostatic activity. Results: The acetone extract showed activity against 47.5 % (19/40) of the test bacteria, while the aqueous extract had activity against 30 % (12/40). MIC and MBC values range for the acetone extract were 0.625 – 5.0 mg/mL and 2.5 – 10 mg/mL respectively. The range of MIC exhibited by the antibiotic (gentamicin) against the vibrios is 0.002 mg/mL and >0.256 mg/mL. Significant reduction in the bacterial density was at 2 × MIC after a 4 h interaction period, while bacterial density after 6 and 8 h interactions with extract was highly bactericidal. Growth inhibition and efficacy of the crude acetone extract were observed to be both concentration- and time-dependent. Conclusion: The bacteriostatic and bactericidal activities observed for Ocimum gratissimum leaf suggest that the plant is a potential source of bioactive components that may be effective in the treatment of vibrios infections.

Determination of Extracellular Molecules Produced by Vibrio Harveyi Using MS/MS

Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules produced by V. harveyi, and assessment of their relationship to QS. A total of 11 molecules were characterized, from which three could be related to QS. These findings provide a glimpse of the nature of novel secondary metabolites produced by V. harveyi and provide the groundwork for further research.