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Revista argentina de microbiología

versión On-line ISSN 1851-7617

Rev. argent. microbiol. v.36 n.4 Ciudad Autónoma de Buenos Aires oct./dic. 2004

 

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina

C. Bidinost1, H.A. Saka1, O. Aliendro1, C. Sola1, G. Panzetta-Duttari1, P. Carranza2, J. Echenique1, E. Patrito1, J.L. Bocco1*

1Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000, Córdoba, Argentina
2 Facultad de Ciencias Agropecuarias, Universidad Católica de Córdoba, Cno. Alta Gracia, Km 8, 5000, Córdoba, Argentina.
*Correspondencia. E-mail: jbocco@fcq.unc.edu.ar

SUMMARY
V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

Key words: non-O1 non-O139 V. cholerae, enterotoxin, cytotoxin, hemolysin, multiplex PCR

RESUMEN
Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina. En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.

Palabras clave:V. cholerae no-O1 no-O139, enterotoxina, citotoxina, hemolisina, multiplex PCR

Introduction

Cholera disease, caused by toxigenic Vibrio cholerae, is a major public health problem in developing countries. Epidemiological surveillance and comparative molecular analysis of isolates have demonstrated clonal diversity among epidemic strains and a continual emergence of new clones of toxigenic V. cholerae (13,20). The non-O1 non-O139 serogroups are associated to the emergence of new pathogenic variants of V. cholerae (21) and are potential receptors of virulence factors from toxigenic V. cholerae O1 (6).
Non-O1 non-O139 V. cholerae has been isolated from patients with acute secretory diarrhea worldwide (23). Nevertheless, the factors involved in the pathogenesis of non-O1 non-O139 serogroups are not well understood. It has been reported that these strains encode some putative virulence factors such as NAG-ST enterotoxin (2), hemolysins (24), and proteases (22). The study of these factors in clinical as well as environmental strains of non-O1 non-O139 V. cholerae isolated around the world will be of great value to elucidate the pathogenesis of non toxigenic cholera disease.
The aim of the present study was to investigate and compare the virulence determinants in fifteen non-O1 non-O139 V. cholerae strains isolated in Córdoba, Argentina. The presence of several virulence genes, and the ability to produce hemolysin, cytotoxin, protease and fluid accumulation, were analyzed. Also, the clonal relationship among the strains was determined by pulsed field gel electrophoresis (PFGE).  

Materials and methods

Bacteriology and serogrouping
Thirteen V. cholerae isolates were obtained from environmental water sources of Córdoba during the period 1991-1996: strains 1383, 208 and 286 from Suquía River; 140, 150, 183, 289 from La Cañada creek; 210 and 287 from Northern Main Canal, 211 and 288 from Southern Main Canal; 217 from Dolores River and 218 from Calabalunga River. During the same period, under a cholera surveillance program carried out at the “Centro de Enterobacterias-División Laboratorio Central de Salud Pública de la Provincia de Cordoba”, two V. cholerae clinical isolates were obtained from hospitalized patients who suffered an acute diarrhea syndrome indistinguishable from cholera, in Córdoba city (strains175 and 588).All the V. cholerae isolates included in this study did not agglutinate with either O1 or O139 antiserum.

Bacterial growth conditions and supernatant preparation
V. cholerae was grown on dialyzed Brain Heart Infusion Broth (BHI, Oxoid, England) (15) under shaking conditions at 37 ºC for 16 h. Cultures were centrifuged at 10.000 g and 4 ºC for 30 min and the supernatants were used as “centrifuged supernatants” (containing about 1 x 105– 1 x 106cfu ml-1) or were sterilized by filtration through a 0.22 μm low-binding protein filter (Millipore Corp.). Aliquots of the supernatants were heat-treated by incubation at 65 ºC during 15 min. Trypsin treatment was done as follow: culture supernatants were precipitated with 50% ammonium sulfate at 4 ºC, centrifuged, resuspended in 1/10 volume of phosphate-buffered saline (PBS) and dialyzed against Hepes 50 mM at 4 ºC for 1 h. Aliquots of the concentrated supernatants were incubated with 0.2 volume of trypsin 25 µg ml-1(Serva, Boehringer Ingelheim) at 37ºC during 3 hours. The reaction was stopped with fetal bovine serum (Gibco-BRL Laboratories, N.Y.) in a final dilution of 1/100. Concentrated supernatants without trypsin-treatment were used as controls.

Antimicrobial susceptibility test
Bacterial strains were examined by the disk diffusion method according to National Committee for Clinical Laboratory Standards (NCCLS) (17) to determine the susceptibility to the following antibiotics (Oxoid, England): chloramphenicol (30 μg), ampicillin (10 μg), cephalothin (30 μg), trimethoprim /sulfame-thoxazole (1.25/23.75 μg), ciprofloxacin (5 μg), gentamicin (10 μg), kanamycin (30 μg), nalidixic acid (30 μg), norfloxacin (10 μg), streptomycin (10 μg), and tetracycline (30 μg). When no interpretive criteria for V. cholerae were available based on the NCCLS guidelines, breakpoints for Enterobacteriaceae were applied. ATCC strains E. coli 25922, S. aureus 25923, P. aeruginosa 27853 and E. faecalis 29212 were used as controls.

Polymerase chain reaction assay (PCR)
Three sets of multiplex and one simplex PCR were used to determine the presence of the genes: ctxA (encoding the enzymatic subunit of cholera toxin) and tcpA (encoding the major structural subunit of toxin co-regulated pilus, El Tor variant) (10); zot (encoding zonula occludens toxin) and ace (encoding accessory cholera enterotoxin) (21); toxR (encoding the transcriptional activator ToxR) (10) and hlyA (encoding the El Tor hemolysin) (1); and stn (encoding the termostable toxin NAG) (18). The primers used are shown in Table 1.

The following were added to obtain each 25 μl of PCR mixture: 2.5 μl of 10X PCR amplification buffer (500 mM KCl; 100 mM Tris-HCl pH 9.0; 1% Triton X-100, Promega Corporation), 2.5 μl of MgCl250 mM, 0.25 μl dNTPs (25 mM each), 0.4 μl (20 pmol) of each of the primers, 0.25 μl (1.25 U) of Taq DNA polymerase (Promega Corporation) and 1 μl of DNA template. DNA samples were prepared as follow: bacterial colonies were resuspended in 500 μl of water, centrifuged, washed twice with water and boiled for 10 minutes. The PCR conditions for all the reactions included a first step of 10 min at 94 ºC and a final step of 10 min at 72 ºC. The middle step consisted of 30 cycles of: a) ctxA - tcpA: 1.5 min at 94 ºC, 1.5 min at 60 ºC and 1.5 min at 72 ºC; b) zot - ace: 1.5 min at 94 ºC, 1.5 min at 55 ºC and 1.5 min at 72 ºC; c) toxR – hlyA: 1.0 min at 94 ºC, 1.0 min at 62 ºC and 4.0 min at 72 ºC; d) stn: 1.5 min at 94 ºC, 1.5 min at 53 ºC and 1.5 min at 72 ºC. V. cholerae O1 biotype El Tor and non-O1 non-O139 V. choleraestn+were used as positive controls.

Determination of protease and hemolytic activities
The protease activity was estimated inoculating approximately 103 cfu of each strain with a Steers multireplicator onto LB agar plates supplemented with 3% of casein. To achieve the desired density of the bacterial suspensions, 0.5 Mc Farland turbidity inoculum obtained from overnight colonies emulsified in sterile saline (0.85% NaCl) were properly diluted. Regular colony counts were performed to verify the final inoculum density in terms of cfu ml-1. The plates were incubated 18 h at 37 ºC and triplicates of the diameter of the clear halo around colonies were measured. Protease activity was classified in low (+), intermediate (++) and high (+++) when the halo ranged from 7-9 mm, 10-12 mm and 13-15 mm, respectively.
The hemolytic activity was determined by the production of a clear halo around bacterial colonies grown on LB-agar plates supplemented with 5% (v/v) sheep red blood cells, overnight at 37 ºC. To quantify the hemolytic activity, sterile culture supernatants were diluted 1/10 in PBS pH 7.3 and 0.8 ml of dilution was mixed with 0.2 ml of a 5% (v/v) sheep red blood cells suspension equilibrated in PBS. The mixture was incubated at 37º C during 30 min, centrifuged at 2,000 rpm during 5 min and the absorbance was measured spectrophotometrically at 540 nm. A volume of 0.2 ml of 5% (v/v) red blood cells suspension was mixed with 0.8 ml of PBS or 0.8 ml of water and used as negative and 100% of hemolysis controls, respectively. Values were expressed as the percentage of hemolysis.

Analysis of cytotoxic activity
Sterile culture supernatants were examined for their cytotoxic activity on Cos-7 cells monolayers. Eukaryotic cells were grown in Dulbecco modified Eagle’s medium (DMEM) (Sigma Chemical Co. Ltd., USA) supplemented with 5% (v/v) of fetal bovine serum (FBS) (Gibco-BRL Laboratories, N.Y.) at 37ºC in a humidified 5% CO2 atmosphere.
a) Microscopic examinations. Roughly 4 x 105 Cos-7 cells were seeded on 3-cm culture dishes and incubated during 24 h under standard conditions as described above. Then the culture media was replaced with DMEM supplemented with 1% (v/v) FBS and inoculated with 1/10 volume of sterile culture supernatant. As negative control, the Cos-7 cells were inoculated with BHI alone. Morphological changes of the cells were examined by phase-contrast microscopy in a Zeiss Axiovert 135IM microscope.
b) Quantification of cytotoxic activity. About 2 x 104 Cos-7 cells were added in each well of the 96-well plates and incubated 24 h under standard conditions. At this time the number of cells reached 4 x 104 cells/well. After fresh medium replacement with 1% (v/v) FBS, the cells were inoculated with 1/10 volume of culture supernatant and incubated for 2 h. The cell viability was measured using a CellViability AQ 96 system (Promega Corporation) according to the manufacturer conditions. Cos-7 cells treated with 1/10 volume of BHI were used as viability control. A mix of DMEM containing 1/10 volume of BHI was used as a blank. The absorbance at 490 nm was obtained with an ELISA Reader Plate (BioRad, Richmond Calif.). The values were expressed as the reciprocal of the absorbance at 490 nm in order to indicate cytotoxicity. Each determination was done by duplicate and the results are the media from two individual experiments.

Rabbit ileal loop assay
The ligated ileal loop test was performed as described by De and Chatterjee (5). Briefly, New Zeeland rabbits weighing 2.0 – 2.5 kg were starved for 48 h before the experiment. Rabbits were anesthetized by subcutaneous injection of ketamine (40.0 mg kg-1) and acepromazine (5.0 mg kg-1). The small intestine was withdrawn and ligated at a distance of 10 cm from the ileocaecal region. Ten to twelve intestinal loops of 6-8 cm, separated by uninoculated segments of 2 cm, were ligated in each animal. The loops were inoculated with 1 ml of bacterial suspension adjusted to 5 x 104- 5 x 105cfu ml-1(14). Negative control loops were injected with PBS alone or with E. coli DH5a, while positive control loops were injected with V. cholerae O1 biotype El Tor. After 18-20 h the animals were killed, the loops were excised and the fluid accumulation (FA) was expressed as the ratio between volume (ml) and length (cm) of each loop. Each strain was assayed at least in three rabbits.

Pulsed-field gel electrophoresis (PFGE)
The genomic DNA was prepared in agarose plugs as previously described (3) and digested twice with 14 U of Not I per 100 μl block. The chromosomal DNA fragments were analyzed by PFGE on a CHEF DR-II apparatus (BioRad, Richmond Calif.). The electrophoresis was carried out during 24 h at 200 V with pulse times of 0.1 to 35.0 sec. V. cholerae O1 biotype classical and El Tor, and V. cholerae O139 were used as controls.
 

Results

Identification of virulence genes by PCR
All the strains belonging to clinical or environmental sources yielded the toxR and hlyA specific amplicons, with the exception of strain 211 that was negative for hlyA (Table 2). As expected, none of the strains amplified the virulence genes ctxA, ace and zot, contained in the CTXF prophage, nor tcpA whose product is a colonization factor and acts as a receptor for the CTXF bacteriophage (Table 2) or stn, encoding the toxin NAG-ST (data not shown). The absence of the CTXF prophage was confirmed by Southern blot of genomic DNA using the ctxA PCR fragment as a probe (data not shown).

 3

Antimicrobial susceptibility and production of protease and hemolysin

Most of the non-O1 non-O139 V. cholerae isolates were susceptible to all antibiotics tested, but three environmental strains were resistant to ampicillin (strains 150, 183 and 211), and one showed intermediate resistance level to streptomycin (strain 140) (data not shown).
To characterize the secreted components potentially involved in epithelial cell damage, we investigated the production of proteases and hemolysins. All the strains were positive for protease activity in 3% casein plates. For easier comparison, this activity was classified in low, intermediate and high according to the size of the halo as described in Materials and Methods (Table 2). All the strains but strain 211 produced β-hemolysis when grown on sheep blood-agar plates. However, the quantification of the hemolytic activity in the sterile supernatants showed differences between isolates. Strains were considered markedly or weakly hemolytic according to activities higher than 60% (strains 140, 150, 175, 183, 218 and 588) or lower than 10% (strains 1383, 208, 210, 211, 217, 286, 287, 288 and 289), respectively (Table 2). All heat-inactivated supernatants lost their hemolytic activity (data not shown).

Cytotoxic and enterotoxic activities

The production of cytotoxic compounds was evaluated on eukaryotic Cos-7 cells. Most of the supernatants from non-O1 non-O139 V. cholerae produced cell rounding and decolling of the monolayer after 2-6 h of treatment (Fig 1). This effect was also seen using other cell lines, e.g. CHO cells (data not shown). However, after 2 h of incubation some strains induced a drastic reduction in cell viability (strains 1383, 150, 175, and 286) while the others have a slightly detectable activity (Table 2). All the supernatants showed more than five fold reductions in cytotoxicity when they were filter-sterilized. This effect was probably due to protein adsorption since it was reverted using albumin-saturated filters (data not shown). To study the putative polypeptide nature of the cytotoxic compound, supernatants were concentrated and treated with trypsin, which largely diminished the cytotoxic activity (Fig 2). Also, a heat treatment of supernatants (65ºC, 15 min) completely abolished this activity (Fig 2).

Most of the isolates showed enterotoxic activity although the FA values indicated variations between the strains (Table 2). The difference in FA ratios between highly and lower hemolytic strains was not statistically significant (p>0.05). Nevertheless, highly hemolytic strains produced higher FA ratios respect to the negative control (E. coli DH5a) and lower hemolytic strains produced lower FA ratios respect to the positive control (V. cholerae O1), both with statistically significant differences (p<0.05) according to a non-parametric test (ANOVA, Kruskal-Wallis).

Genetic relationship between non-O1 non-O139 V. cholerae isolates
Two pairs of indistinguishable genetic patterns were observed, one for the two clinical strains (175 and 588) and the other for two environmental strains isolated in Córdoba city (208 and 210). Ten different patterns were obtained for the others isolates (Fig. 3) and one was non-typeable (strain 140, not shown). All the patterns were different from that of toxigenic V. cholerae O1 biotype classical and El Tor, and O139 serogroup (data not shown).

DISCUSSION

In this study we analyzed fifteen strains of non-O1 non-O139 V. cholerae isolated in Córdoba, Argentina, during the period 1991-1996. All the isolates lacked the ctxA gene encoding the A subunit of the cholera toxin. Also, the genes ace, zot, tcpA and stn, associated to pathogenicity in V. cholerae, were absent in these isolates. On the contrary, all the strains contained the toxR gene, encoding the transcriptional regulator ToxR, present in most O1 and non-O1 non-O139 V. cholerae strains.
All the strains analyzed were proteolytic. It has been reported that V. cholerae proteases interfere with the intercellular tight-junctions of cultured epithelial cells (16). Although most strains analyzed in this work were cytotoxic, some did not affect the integrity of the Cos-7 cell monolayer. All the strains except for V. cholerae 211 harbored the hlyA gene encoding the El Tor hemolysin and showed hemolytic phenotype. Nevertheless, two groups of strains were determined, with hemolytic activities higher than 60% and lower than 10%, respectively. The two clinical isolates are in the former group, suggesting a putative role for the hemolytic activity in the pathogenesis of the cholera-like disease. Hemolysins from non-O1 non-O139 V. cholerae have been described to have cytotoxic and cell vacuolating activity on cultured HeLa and Vero cells (4, 8). The supernatants of some non-O1 non-O139 V. cholerae studied in this work produced dramatic cytotoxic effect on Cos-7 cell monolayers (Table 2) although they did not produce vacuolization on the same cell line. The results suggest that a proteic compound produce the cytotoxic activity. The molecular mechanisms responsible for this activity remain to be further characterized.
Most of the isolates, including environmental strains, showed enterotoxic activity. The highly hemolytic strains are also highly enterotoxic, suggesting a putative relation between the hemolytic activity and the enterotoxic phenotype as previously described (12). Both clinical isolates were highly enterotoxic and hemolytic although they showed a significant difference in cytotoxicity. At present, the pathogenesis of non-O1 non-O139 V. cholerae gastroenteritis is not well understood. It is possible that a concerted action of several secreted enzymes, as demonstrated for the accessory toxins in V. cholerae O1 (9), may be essential for the pathogenesis of non-O1 non-O139 V. cholerae to cause disease in the absence of classical cholera toxin. Also, as recently suggested, non-O1 non-O139 V. cholerae may produce previously undiscovered colonization factors that induce fluid accumulation by unknown mechanisms (7).
The DNA profiles of non-O1 non-O139 V. cholerae strains indicate that both clinical isolates are closely related, suggesting a probably common origin. The isolates from water sources showed a variety of genetic profiles coexisting in this environment.
Other authors recently described the genetic diversity of V. cholerae O1 in Argentina (19). This is the first study documenting the potential virulent factors, as determined from cell culture and “in vivo” studies, of non-O1 non-O139 V. cholerae isolates from clinical and environmental sources in Argentina.

Acknowledgements

We are grateful to Dr. L. Patrito for his encouragement and support. We thank Dr. A. Vicente for provided the V. cholerae stn positive strain and Drs. A. Vindel and M. Usera for technical assistance in PFGE. This work was supported by grants from CONICET, Agencia Córdoba Ciencia and SECyT Universidad Nacional de Córdoba Argentina. C.B. and A.S. were supported by CONICET fellowships. J.L.B. is a career investigator of CONICET.

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Recibido:7/1/2004
Aceptado:25/10/04