Bacteriemia por Vibrio cholerae no-O1/no-O139 que porta una región homóloga a la isla de patogenicidad VpaI-7 / Bacteremia caused by Vibrio cholerae non-O1/non-O139 carrying a region homologous to pathogenicity island VpaI-7

Resumen Presentamos un caso de bacteriemia por Vibrio cholerae no-O1/ no-O139 en una mujer de 81 años con un cuadro de dolor abdominal, fiebre, vómitos, diarrea, coluria e ictericia, mientras visitaba una zona rural sin acceso a agua potable. La identificación se realizó por la técnica de espectrometría de masa MALDI-TOF, confirmándose una cepa no toxigénica no-O1/no-139. La caracterización molecular del aislado demostró la ausencia del gen de la toxina del cólera (CTX), y pilus TCP; sin embargo, presentó cinco de los seis genes de virulencia presentes en la isla de patogenicidad homóloga denominada VPaI-7 del V. parahaemolyticus (vcs N2+, vcs C2+, vcs V2+,toxR-, vspD+, T vopF+). Además, el aislado presentó los genes de virulencia hylA y rtxA. Este es el primer caso reportado en Chile de una cepa clínica de V. cholerae no-O1, no-O139 aislada de hemocultivos portador de un segmento homólogo de la isla de patogenicidad denominada VPaI-7 de V. parahaemolyticus, el cual codifica para un sistema de secreción tipo III (TTSS), que probablemente contribuye a su virulencia.

We report a case of V. cholerae non-O1 / non-O139 bacteremia in an 81-year-old woman with abdominal pain, fever, vomiting, liquid stools, choluria and jaundice, while visiting a rural area without access to potable water. The identification was made by the MALDI-TOF mass spectrometry technique and subsequently the non-toxigenic non-O1 / non-139 strain was confirmed in the national reference laboratory. The molecular characterization demonstrated the absence of the cholera toxin gene (CTX), and the TCP pilus, however, presented 5 of 6 virulence genes present in an island of homologous pathogenicity named VPaI-7 of V. parahaemolyticus (vcs N2 +, vcs C2 +, vcs V2 +, toxR-, vspD +, T vopF +) and in addition it was positive for hylAy rtxA virulence genes recognized outside the island. This is the first case reported in Chile of a clinical strain of V. cholerae non-O1, non-O139 isolated from blood culture that carries in its genome a homologous segment of the pathogenicity island named VPaI-7 of V. parahaemolyticus, which codifies for a type III secretion system (TTSS) that probably contributes to his virulence.

Molecular mechanism of acquisition of the cholera toxin genes.

One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTX. CTX makes use of the chromosome dimer resolution system of V. cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process.

Cholera toxin – A foe & a friend.

After De’s pivotal demonstration in 1959 of a diarrhoeogenic exo-enterotoxin in cell-free culture filtrates from Vibrio cholerae (of classical biotype), much insight has been gained about cholera toxin (CT), which is arguably now the best known of all microbial toxins. The subunit structure and function of CT, its receptor (the GM1 ganglioside), and its effects on the cyclic AMP system and on intestinal secretion were defined in the 1970s, and the essential aspects of the genetic organization in the 1980s. Recent findings have generated additional perspectives. The 3D-crystal structure of CT has been established, the CT-encoding operon has been shown to be carried by a non-lytic bacteriophage, and in depth knowledge has been gained on how the bacterium controls CT gene expression in response to cell density and various environmental signals. The mode of entry into target cells and the intracellular transport of CT are becoming clearer. CT has become the prototype enterotoxin and a widely used tool for elucidating important aspects of cell biology and physiology, e.g., cell membrane receptors, the cyclic AMP system, G proteins, as well as normal and pathological ion transport mechanisms. In immunology, CT has emerged as a potent, widely used experimental adjuvant, and the strong oral-mucosal immunogenicity of the non-toxic B-subunit (CTB) has led to the use of CTB as a protective antigen together with killed vibrios in a widely licensed oral cholera vaccine. CTB has also been shown to promote immunological tolerance against certain types of mucosally co-administered antigens, preferably tissue antigens linked to the CTB molecule; this has stimulated research and development to use CTB in this context for treatment of autoimmune and allergic diseases. In summary, in the 50 years after De’s discovery of CT, this molecule has emerged from being the cholera patient’s “foe” to also becoming a highly useful scientist’s “friend”.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore β-barrel oligomer to a functional diffusion channel.

Background & objectives: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results: Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Cluster of cases of clinical cholera due to Vibrio cholerae 010 in east Delhi.

A total of 514 samples of acute diarrhoeal stools received over a period of four months yielded 315 isolates morphologically and biochemically resembling V. cholerae. Out of 315 isolates, 223 (70.8%) were identified as V. cholerae 01, 20 (6.4%) as 0139 and 42 (13.3%) as 010. Thirty (9.5%) isolates did not agglutinate with any of the available antisera. All V. cholerae 010 isolates showed complete homogeneity in their biochemical and physiological properties. This strain appears to be closely related to El Tor biotype of V. cholerae 01, since it was positive for some of the tests used for identification of El Tor. The ability of strain 010 to grow in the presence of 6 per cent salt provides it the status of an important environmental pathogen. Acquisition of some virulence genes from El Tor vibrios by this strain 010 appears to be one of the mechanisms involved in the emergence of this serogroup.

Susceptibilidad de vibrio cholerae 01 a los antibióticos y quimioterápicos / Vibrio cholerae susceptibility to antibiotics and drug therapy

Se estudió la susceptibilidad antimicrobiana de 200 cepas de vibrio cholerae 01, biotipo El Tor, aisladas en pacientes con diarrea en el Centro Médico Naval, durante los primeros trimestre de 1991-1992. El serotipo prevalente en 1991 fue el lnaba (94,7 por ciento) y en 1992 el Ogawa (92 por ciento). 137 cepas proceden de pacientes masculinos y 63 del sexo femenio. El antibiograma realizado por serotipos mediante el método de kirby y bauer, dio como resultado 100 por ciento de sensibilidad en ambos serotipos para las quinolonas, pequeñas variaciones frente a sulfametoxazol-trimetropim, cloranfenicol e imipenem y mayor sensibilidad del serotipo Ogawa para ceftazidime, ampilicina y tetraciclinas, ambos serotipos a la ampicilina, la susceptibilidad no depende del serotipo (p < 0.05). 47 por ciento de las cepas eran resistentes a ampicilina, 2 por ciento a oxitetraciclina y 1.5 por ciento a doxiciclina, 2 cepas fueron resistentes a los tres antibióticos. La concentración mínima inhibitoria se determinó por el método de dilución en Agar con inóculo múltiple. 80 por ciento de las cepas resistentes a la ampicilina producen la enzima betalactamasa detectado con el método lodométrico.

Recuperación e identificación de vibrio choloerae en mariscos provenientes de diferentes mercados de Lima Metropolitana y Callao / Identification and recuperation of vibrio cholerae en sellfish from Lima and Callao market

A fines de 1991, se inició en el Perú una epidemia de cólera de gran inagnitud, en cuanto a morbi-mortalidad y extensión geográfica, que invadió a 19 países produciendo 600,000 casos y más de 6000 muertes. Esta enfermedad ha sido relacionada con el consumo de numerosos productos de pesca, entre ellos los mariscos como vehículos de transmisión del vibrio cholerae. El método utilizado fue el reconocimiento por el ICMSF modificado. Se analizó 104 muestras de mariscos, obteniéndose 0.96 por ciento de positividad correspondiente a la muestra de marisco tipo almeja, comprobándose mediantes pruebas serológicas, pertenecia al serotipo inaba. Asimismo, se comprobó la localización del microorganismo en las valvas del marisco y no en el músculo. También se puso de manifiesto la efectividad del método mediante un segundo enriquecimiento en APA pH=8,6 que proporcionó una mayor selectividad.