SciELO - Scientific Electronic Library Online

 
vol.35 número3-4Grape seed extract proanthocyanidins downregulate HIV- 1 entry coreceptors, CCR2b, CCR3 and CCR5 gene expression by normal peripheral blood mononuclear cellsuvsZ1 mutation shows epistatic relations with uvsD153 and uvsJ1 mutations without any involvement with checkpoint control in Aspergillus nidulans índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados

Revista

Articulo

Indicadores

Links relacionados

Compartir


Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.35 n.3-4 Santiago  2002

http://dx.doi.org/10.4067/S0716-97602002000300017 

Biol Res 35: 433-440, 2002

 

Characterization by PCR of Vibrio parahaemolyticus
isolates collected during the 1997-1998 Chilean outbreak

JOSÉ LUIS CÓRDOVA1, JOSEFA ASTORGA2, WALLY SILVA3 AND CARLOS
RIQUELME
4

1. Fundación Ciencia para la Vida and Millennium Institute for Fundamental and Applied Biology. Av.
Marathón 1943, Ñuñoa, Santiago, Chile.
2. Sección de Microbiología de Alimentos, Instituto de Salud Pública, Av, Marathón 1000, Ñuñoa,
Santiago, Chile.
3. Sección de Bacteriología Clínica, Instituto de Salud Pública, Av, Marathón 1000, Ñuñoa, Santiago,
Chile.
4. Departamento de Oceanografía, Universidad de Antofagasta, Chile.

ABSTRACT

Between November 1997 and April 1998, several human gastroenteritis cases were reported in Antofagasta, a city in the north of Chile. This outbreak was associated with the consumption of shellfish, and the etiologic agent responsible was identified as Vibrio parahaemolyticus. This was the first report of this bacterium causing an epidemic in Chile. V. parahaemolyticus was the only pathogenic bacterium isolated from patient stools and from shellfish samples. These isolates were analyzed by polymerase chain reaction (PCR) amplification of the pR72H gene, a species-specific sequence. Based on the pR72H gene amplification pattern, at least three different isolates of V. parahaemolyticus were found. Two isolates (named amplicons A and C) generated PCR products of approximately 400 bp and 340 bp respectively, while another type of isolate designated B, did not generate a PCR product, regardless of which method of DNA purification was used. Sequence analysis of the amplicons A and C shows that they have an 80 bp and 183 bp conserved region at the 5'end of the gene. However, both isolates have different sequences at their 3' terminus and are also different from the pR72H sequence originally reported. Using this PCR assay we demonstrated that these three isolates were found in clinical samples as well as in shellfish. The warm seawater caused by the climatological phenomena "El Niño" perhaps favored the geographic dispersion of the bacterium (bacterial bloom) occurring in Antofagasta that occurred during that time of year.

Key terms: Vibrio parahaemolyticus, bacterial bloom, bacterial geographical dispersion, PCR amplification and
molecular typification.

INTRODUCTION

An acute colitis outbreak affected northern Chile between November of 1997 and April 1998. Clinical signs appeared after shellfish consumption. The bacterium Vibrio parahaemolyticus was isolated from patient stools and shellfish samples from areas where clinical cases were reported.

V. parahaemolyticus is a Gram negative bacterium that has been associated with human diarrhetic episodes affecting hundreds of people in extensive geographic areas and is associated with shellfish consumption (Janda et al., 1988). It has also been reported that the characteristics of the outbreak depend on the species of shellfish, the season, the location and the level of fecal pollution (Walkins and Cabelli, 1985).

V. parahaemolyticus pathogenicity is mediated by different mechanisms such as expressions of adhesins (Iijima et al., 1981), detection of an enterotoxin by ileal-loop assay (Twedt and Brown, 1974) in Kanagawa-positive strains, and production of a cell-associated Shiga-like cytotoxin (O'Brien et al., 1984). Thus, a specific assay was necessary for early detection. The assay was based on the PCR amplification of a species-specific gene sequence named pR72H was developed by Lee et al., (1995). Here we use this procedure to analyze bacterial isolates from patients and shellfish from the 1997-1998 Chilean outbreak. We identified at least three different types of isolates of V. parahaemolyticus occurring during this outbreak. Two bacterial isolates generated products when the pR72H gene was amplified by PCR, while one isolate did not. Furthermore, when the amplified DNA sequences were compared to that pR72H, differences were detected specially at the 3' termini.

MATERIAL AND METHODS

Bacterial isolates

Samples from patient stools and shellfish were cultured overnight at 35ºC using T1N1 medium (Difco). Total culture samples were plated into TCBS agar and incubated again at 20ºC overnight. Green colonies (sucrose+) were isolated and individually grown in T1N1 overnight before biochemical analysis. Clinical samples were processed as described by McLaughlin (1997), while shellfish samples were processed as described elsewhere (Elisa et al., 1995).

Biochemical characterization of V.
parahaemolyticus isolates

Biochemical assays included: a) differential growth in mediums such as LIA, MIO, TSI, TCBS, and Simmons citrate; b) sugars fermentation; c) amino acid requirement (Möller medium); d) salt tolerance; e) string test; f) enzymatic activity (oxidase); and g) pathogenicity (Kanagawa test).

Purification of V. parahaemolyticus DNA

The method was performed as described in Maniatis et al., (1982). Briefly, bacteria cultures before phenol extractions were concentrated by centrifugation at 4,000 x g. Bacterial pellets were resuspended and incubated at 37ºC for 2 h in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% sarcosyl and 50 mg/ml proteinase K). Bacterial genomic DNA was extracted with phenol-chloroform and precipitated with 2 volumes of ethanol in sodium acetate 0.3 M, pH 5.2 and the pellet was washed twice with 70% ethanol, resuspended in sterile distilled water and stored at 4ºC until use. Bacterial DNA was also extracted using a chelex 100 resin (Biorad Laboratories) as described by De Lambellerie et al., (1992).

PCR amplification

The primers reported by Lee et al., (1995) were used to amplify bacterial DNA by PCR: VP32 5' CGAATCCTTGAACATACGCAGC-3', and VP33 5'-TGCGAATTCGATAGGGTGTTAACC-3'. Primers, 1µM of each, were mixed with Supermix cocktail that contained 15 mM MgCl2; 500 mM KCl; 100 mM Tris-HCl buffer, pH 8.3 y 0.1% (w/v) of gelatin; 200 µM nucleotide mixture (dATP, dCTP, dGTP and dTTP); y 2.5 U of Taq DNA polimerase (GIBCO, Grand Island, NY). Then, 1 µg of ADN was added to 25 µl of PCR reaction mixture plus 50 µl of mineral oil. The thermocycler (M. J. Research Inc., Las Vegas, NV) was programmed as follows: 1 cycle at 95ºC for 5 min, 35 cycles of 1 min at 94ºC, 1 min at 55ºC and 1 min at 74ºC. Then , 1 cycle of 10 minutes at 72ºC (Mullis and Faloona 1987; Kellogg et al., 1994). The PCR products were analyzed electrophoretically in 1.5 % agarose-TBE buffer. Isolated DNA amplification fragments were cloned into TA cloning system (Invitrogen San Diego, CA) according to manufacturer recommendations and sequenced as described elsewhere (Sanger 1981).

RESULTS

Biochemical identification of isolates

All the isolates were subjected to biochemical characterization. The following profile was obtained for each.

Triple sugar iron (TSI, K/A -,-), Lysine iron agar (LIA, K/K -,-), Motility indol ornithine (MIO, M+O+); Simons citrate (+); Indol (+); ability to ferment sugars: arabinose (+/-), glucose (+), lactose (-), manitol (+), mannose (+) and sucrose (-); not visual growth at 0% and 10% of NaCl; amino acids: arginine (-), lysine (+), ornithine (+); esculin (-) and Voges Proskauer (-). These results indicate that they are identified as V. parahaemolyticus.

PCR amplification of DNA of V.
parahaemolyticus isolates

The VP32 y VP33 primers used to amplify the pR72H fragment described by Lee et al., (1995) were reported as species-specific because they identified all tested V. parahaemolyticus isolates and gave a negative PCR amplification when other related organisms were evaluated. Therefore, these primers were used to confirm the nature of isolates that were initially characterized biochemically. DNA was isolated from three V. parahaemolyticus isolates that were arbitrarily designated A, B and C as described in the Materials and Methods section.

Figure 1 (Lanes 3 and 5) shows the PCR product from isolates A and C respectively, while Lane 4 corresponds to isolate B, which did not generate PCR products.

Figure 1B (Lanes 6 and 7) illustrates the amount of DNA used in the PCR cocktail for isolates A and B respectively. Clearly, enough high molecular weight DNA was present in the sample (arrow) and very little degradation is observed. Furthermore, the gel shows that isolate B has two additional DNA fragments that could represent plasmids (Lane 7) and which are not detected in isolate A (Lane 6).

The size of the PCR products obtained from the amplification of DNA of isolates A and C is different than the expected 387 bp. The amplicons were approximately 400 bp for the product from isolate A and 340 bp for the product from isolate C, for the first time showing differences from the results described by Lee et al., (1995).

Furthermore, isolate B did not generate PCR amplification, which may indicate the presence of a new strain of V. parahaemolyticus clearly differing from those previously reported.

Figure 1. Molecular identification of V. parahaemolyticus isolates. After biochemical identification, the isolates were amplified by PCR using the reported primer species specific for V. parahaemolyticus. Based on PCR products, it was possible to identify the presence of at least 2 different isolates (Lanes 3 and 5). These isolates were arbitrarily designated as isolate A (Lane 3) with a larger PCR product than expected; isolate C, which generated a PCR product size as expected (Lane 5); and isolate B which did not generate a PCR product (Lane 4). Negative PCR amplification in isolated B (Fig. 2A, Lane 4) was not due to lack of DNA in the reaction tube, as there is enough DNA of high molecular weight (arrow) for isolates A and B, as shown in figure 2B, lanes 6 and 7, respectively. Lane 2 is PCR control reaction without DNA template. Molecular weight markers are as indicated (Lane 1).

To confirm that the 340 and 400 bp PCR products belong to V. parahaemolyticus and not to a non-specific amplification, despite the high stringency condition used, the PCR products were cloned and sequenced. The results are shown in Table I and compared to the 387 bp reported for pR72H. The PCR product from isolate A has 338 bp, 11 bp longer than pR72H. It also contains two well-defined areas: one of 187 bp in the 5' region that is 100% homologous to the reported pR72H sequence, with the exception of one bp missing at position 42 and one base change (G for A) at position 121. A second region of 200 bp at the 3' end, which is completely different than the reported pR72H sequence with the exception of that of the reverse primer sequence. These results are based on the sequence of three independent clones. On the other hand, sequence of the PCR product of the isolate C has a segment of 80 bp at the 5' end highly homologous to the reported pR72H sequence, except for 5 bp at positions 36, 40, 53, 55 and 75. The remaining segment of 258 bp is different than the reported pR72H. Thus, the A and C isolates have only 80 bp in common at the 5' position and are completely different at the 3' region. Therefore, these V. parahaemolyticus isolates appear to be significantly different from those previously reported (Lee et al., 1995).

TABLE I

V. parahaemolyticus DNA sequences. PCR products from isolates A and C were cloned,
sequenced and compared with the reported pR72H species-specific sequence. Primers are in
bold. "." means identical bp, and "Ø"means no base.

To evaluate whether these two V. parahaemolyticus isolates were present in shellfish as well as in clinical samples, 33 new shellfish isolates were evaluated by PCR. As shown in Figure 2 (lanes 2 and 3), both isolates are present in shellfish. Figure 2, (lanes 3, 4, 6, 7 and 8) shows that both isolates are also present in clinical samples, including isolate B as shown in Lane 5. Total PCR analysis of these 33 isolates are summarized in Table 2, 19 of them correspond to isolate C, 11 to isolate B and 3 to isolate A. Table II also indicates that all 3 isolates bloomed simultaneously, as they were recovered during the same time from samples from different cities.

Figure 2. Identification of V. parahaemolyticus isolates in clinical and shellfish samples. DNA isolated from V. parahaemolyticus from different samples was subjected to PCR amplification. Isolates from shellfish generated PCR amplification bands presented in Lane 2 (isolate A) and Lane 3 (isolate C). Lanes 4, 5, 6, 7, and 8 are bands from the amplification of DNA of clinical isolates. Lane 6 shows a type A isolate, lanes 4, 7, and 8 show type C isolates, and Lane 5 shows no PCR amplification bands (isolate type B). Molecular weight markers are as indicated (Lane 1).

To determine progression in time and distance of the bacterial bloom, the clinical cases and shellfish isolates were arranged according to dates in Figure 3. These results indicate that the V. parahaemolyticus bloom began in Antofagasta in November 1997, and appeared again one month later in the Tarapacá Region. By January, which is summer in the Southern hemisphere, the bloom reached its maximum extension, contaminating Tarapacá, Antofagasta, Atacama and Coquimbo. Furthermore, the highest number of clinical cases was detected during this later period. The bloom remained until April 1998, after which the numbers of clinical cases dropped considerably.

Further molecular characterization of the three identified strains of V. parahaemolyticus including the sequencing of their 16S rRNA genes will be the subject of a subsequent disclosure.

·
·
   
·
27
O
5
O
0  

O

7
       
·
   

Figure 3. Geographic distribution of V. parahaemolyticus bloom. The outbreak began in November of 1997, after which shellfish and clinical samples were taken and bacterium was isolated and characterized. The maps shows how the bacterial bloom expanded over time. Light dots represent isolates from shellfish or seawater, and dark dots represent human clinical samples.

DISCUSSION

Two hundred and ninety eight people were affected during the colitis outbreak that affected the northern part of Chile from November 1997 until April 1998. By culturing patient stools and shellfish material, it was possible to identify V. parahaemolyticus, a Gram negative bacterium associated with human gastroenteritis in other countries (Walkins and Cabelli, 1985; Janda et al., 1988). This is the first time that Chile was affected by a V. parahaemolyticus outbreak (Fig. 3).

The facts that the diarrhea occurred after shellfish consumption and during the El Niño event with a higher-than-normal seawater temperature, plus the wide distribution of the bacterial bloom suggested that V. parahaemolyticus was a candidate bacterium responsible for this outbreak. V. parahaemolyticus identification from shellfish and human clinical cases were carried out via biochemical assays. However, the biochemical analysis did not demonstrate that V. parahaemolyticus isolated from shellfish was the same as that isolated from clinical cases. Thus, a confirmatory assay was necessary to demonstrate that the human cases were due to V. parahaemolyticus found in shellfish.

Using a previously-described PCR procedure, bacterial identities were confirmed as shown in Figure 1. However, the PCR products generated have different lengths than the 387 bp expected from the work of Lee et al. (1995)(Fig 1). Furthermore, isolate B did not produce a PCR product, which is the first report on negative PCR for these primers considered as specific for V. parahaemolyticus.

For further characterization, the PCR products were cloned, sequenced and compared with the previously reported pR72H sequence for V. parahaemolyticus (Table II). Sequences derived from isolates A and C differ from the pR72H reported sequence, with only 187 (48.6%) and 80 (23.6%) homology at the 5' region respectively, while the 3' end region sequences of both isolates were found to be completely different. These results indicate the existence of at least three different V. parahaemolyticus blooming during the outbreak. The same thee types of isolates were detected in stools from clinical cases as well as from shellfish samples (Figure 2), demonstrating the direct relationship between human disease and contaminated shellfish consumption.

The reason for the sequence diversity found among the A and C types of isolates could be due to the fact that these organisms might have originated in different geographical locations. Unfortunately, we have no means of tracking the origins of these bacteria.

The lack of a PCR product from isolate B suggests the existence of a new isolate that is not detected by the reported VP32-VP33 primers that amplified the pR72H DNA fragment. We have not pursued experiments to demonstrate whether the pR72H gene is present or not in isolate B. However, we must consider the possibility that these reported primers may fail as an early detection marker of a new outbreak if this is caused by isolate B whether in Chile or elsewhere.

The bacterial bloom began in November 1997 and lasted until April 1998. The greatest number of cases was reported in January 1998, affecting a large geographic area (Fig 3). We believe the climatological effects caused by El Niño, which warmed the sea water, have favored this bacterial bloom. The number of clinical cases dropped considerably during February, March, and April, despite the fact that the bloom remained. This may due to the adequate measures taken by the Chilean Health Ministry to mitigate the V. parahaemolyticus impacts, especially those related to human shellfish consumption.

ACKNOWLEDGEMENTS

We thank the P. Valenzuela & B. Méndez Foundation for its continuous support. We also thank Mr. Rodrigo Martinez for DNA sequencing.

We would also like to dedicate this paper to our co-author Josefa Astroga, who passed away recently.

REFERENCES

ELISA LE, KAYSNER CA, JACKSON L, TAMPLIN M L (1995) Vibrio cholerae, V. parahaemolyticus and V. vulnificus and another Vibrio sp. (Chapter 9).In: AOAC INTERACTION (eds) Bacteriological Analytical Manual, 8th ed. MD, USA: FDA/CFSAN         [ Links ]

KELLOGG DE, TYBALKIN L, CHEN S, MUKHAMEDOVA N, VLASIK T, SIEBERT PD, CHENCIK A (1994) TaqStart antibody: "Hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. Biotechniques 16:1134-1137         [ Links ]

DELAMBELLERIE X, ZANDOLF C, VIGNOLI C, BOLLET C, DEMICCO PP (1992) A one step microbial DNA extraction method using chellex 100 suitable for gene amplification. Res Microbial 143:785-790         [ Links ]

IIJIMA Y, TAMADA H, SHIMODA S(1981) Adherence of Vibrio parahaemolyticus and its relationship to pathogenicity. Can J Microbio. 27:1252-1259         [ Links ]

JANDA J M, POWERS C, G BRYANT R, ABBOTT SL (1988) Current perspectives on the epidemiology and pathogenesis of clinically significant Vibrio spp. Clin Microbiol Rev 1:2445-267         [ Links ]

LEE CY, PAN SF, CHEN CH (1995) Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR. Appl Environ Microbiol 61:1311-1317         [ Links ]

MCLAUGHLIN JC (1997) Vibrio (Chapter 35) In: MURRAY PR, BARON EJ, PEALLER MAF, TENOVER C, YOLKEN RH (eds) Manual of Clinical Microbiology 6th ed. WA, USA: ASM Press. pp: 465-476         [ Links ]

MANIATIS T, FRITSCH EF, SAMBROOK J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Pp: 280-281         [ Links ]

MULLIS K, FALOONA F (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335-350         [ Links ]

OBRIEN AD, CHEN ME, HOLMES RK, KAPER J, LEVINE MM (1984) Environmental and human isolates of Vibrio cholerae and Vibrio parahaemolyticus produce a Shigella dysenteriae 1 (shiga-loke cytotoxin). Lancet 1:77-78         [ Links ]

SANGER F (1981) Determining the nucleotides sequences in DNA. Sci 214:226-341         [ Links ]

TWEDT RM, BROWN DF(1974) Studies on the enteropathogenicity of Vibrio parahaemolyticus in the ligated rabbit ileum. In: SAKAZAKI R, TAKEDA Y (eds) International symposium on Vibrio parahaemolyticus. Tokyo: Saikon Publishing Co. pp: 211-217         [ Links ]

WALKINS WD, CABELLI VJ (1985) Effect of fecal pollution on Vibrio parahaemolyticus densities in an estuarine environment. Appl Environ Microbiol 49:1307-1313         [ Links ]

Corresponding author: José L. Córdova. Fundación Ciencia para La Vida. Av Marathón 1943, Ñuñoa, Santiago, Chile, South America. Telephone: (56-2)239-8969. FAX: (56-2)237-2259. e-mail: jcordova@bionova.cl

Received: May 6, 2002. In revised form: October 10 ,2002. Accepted: October 15, 2002

Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons