Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Vibrio parahaemolyticus serovar O3:K6 gastroenteritis in northeast Brazil ˆ .C.T. de A. Figueiroa2, V.V.F. Nunes3, I.S. Miralles4 N.C. Leal1, S.C. da Silva1, V.O. Cavalcanti2, A 3 and E. Hofer 1 2 3 4
Centro de Pesquisas Aggeu Magalha˜es ⁄ Fiocruz, Cidade Universita´ria, Pernambuco, Brasil Laborato´rio Central Milton Sobral, Secretaria de Sau´de do estado do Pernambuco, Pernambuco, Brasil Instituto Oswaldo Cruz ⁄ Fiocruz, Avenida Brasil, Rio de Janeiro, Brasil Laborato´rio Central de Sau´de Pu´blica, Secretaria da Sau´de do estado do Ceara´ Avenida Bara˜o de Studart, Ceara´, Brasil
Keywords multiplex PCR, random-amplified polymorphic DNA, ribotyping PCR, Vibrio parahaemolyticus, virulence genes. Correspondence Nilma Cintra Leal, Centro de Pesquisas Aggeu Magalha˜es ⁄ Fiocruz, Avenida Moraes Rego s ⁄ n, Campus da UFPE, Cidade Universita´ria, 50670-420 Recife, Pernambuco, Brasil. E-mail:
[email protected] or
[email protected]
2007 ⁄ 1984: received 7 December 2007, revised 31 January 2008 and accepted 3 February 2008 doi:10.1111/j.1365-2672.2008.03782.x
Abstract Aims: To examine the virulence factors and the genetic relationship isolates of the serogroup O3 of Vibrio parahaemolyticus in outbreaks of diarrhoea in the northeast region of Brazil. Methods and Results: Eighteen samples of the O3:K6 and O3:KUT serotypes of V. parahaemolyticus were analysed by multiplex polymerase chain reaction (m-PCR) for detection of the tl, tdh and trh genes, by random-amplified polymorphic DNA (RAPD) using two primers, and by amplification of the rDNA 16S–23S region. The gene tl was amplified in all the samples, tdh in 16 while trh in none; amplification of rDNA 16S–23S generated only one profile; each RAPD primer produced two amplification patterns allowing grouping two tdh– Kanagawa-negative isolates. Conclusions: V. parahaemolyticus with characteristics of the pandemic clone appears to be widely disseminated in the studied region. Because of the genetic uniformity of the isolates, elucidation of outbreaks or tracking the source of contamination by the present molecular techniques seems useless. Significance and Impact of the Study: Detection of V. parahaemolyticus with virulence potential of pandemic clone from two outbreaks and from several isolated gastroenteritis cases points out the need for inclusion of this microorganism in the Brazilian routine monitoring of the diarrhoeas for elucidation of their aetiology.
Introduction Vibrio parahaemolyticus, a halophylic bacterium, autocthon of marine and estuary ecosystems, is responsible for isolated cases or outbreaks of gastroenteritis in humans. Diarrhoea caused by this vibrio is mostly related to eating raw or insufficiently cooked seafood (Blake et al. 1980). Its pathogenic action is dependent on the production of thermostable direct haemolysin (TDH) encoded by the gene tdh and evidenced by total haemolysis in Wagatsuma medium, known as the Kanagawa-positive phenomenon (Wagatsuma 1968). Another factor involved in the mechanism of pathogenicity is TDH-related haemolysin (TRH), which is not demonstrable in vitro and is encoded
by the gene trh, which in fact shows 68% homology with the gene tdh (Zhang and Austin 2005). Still another related factor is thermolabile haemolysin (TL), encoded by the gene tl, incapable of causing haemolysis in Wagatsuma agar, which is why it is not considered a virulence factor, although the gene is specific for V. paraheamolyticus (Su and Liu 2007). The genes of virulence factors are detected in the serogroups of V. parahaemolyticus isolated from human enteric processes and exceptionally in strains originating from marine ecosystems, identified as Kanagawa-negative (Blake et al. 1980). Various serogroups of V. parahaemolyticus are present in the environment as well as in enteric infections.
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In fact, among those serotypes occurring in enteric infections, the type O3:K6 is one of the most frequent throughout the world including Latin America (Cabanillas-Beltran et al. 2006; Gil et al. 2007; Su and Liu 2007). In relation to Brazil, there are few references to V. parahaemolyticus in intestinal infections (Hofer 1983; Magalha˜es et al. 1991a,b; 1992) and in the colonization of cutaneous wounds (Rodrigues et al. 2001). There have been a number of investigations on V. parahaemolyticus in marine ecosystems of estuaries and in seafood in diverse areas of the country, as per the initial observations by Leita˜o and Arina (1975) and Gelli et al. (1975). However, only Hofer and Silva (1984, 1986) and Pereira et al. (2007) carried out the serological characterization of strains isolated from seafood. Based on these previous considerations, the present investigation examined the virulence factors and the possible genetic relation of isolates of serogroup O3 of V. parahaemolyticus involved in outbreaks of diarrhoea in Pernambuco (2001) and Ceara´ (2002) and in isolated cases in Alagoas (2002). Materials and methods Sample collection A total of 18 clinical strains of V. parahaemolyticus were analysed, which consisted of fecal isolates from patients with watery diarrhoea, where four (one adult and three children) were from an outbreak that occurred in a public school in Recife, Pernambuco in 2001; seven strains from adult patients were from an episode of food poisoning in Fortaleza, Ceara´ in 2002, and seven from isolated cases, all from adult individuals with diarrhoea, one from Pernambuco (2001) and six from Alagoas (2002). All organisms were recovered from patient’s stool in the Central Laboratories of Public Health of states Alagoas, Ceara´ and Pernambuco and reconfirmed by routine test carried out in the National Reference Laboratory for Cholera, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil. Bacteriological examination For recovering Vibrio spp., patient’s stool samples were routinely enriched in alkaline peptone water (pH 8Æ6) with 1% (w ⁄ v) NaCl and incubated overnight at 35C. All tubes showing growth in enrichment medium were subcultured on to thiosulphate-citrate-bile salts-sucrose agar (TCBS; Difco, Sparks, MD) which was then incubated at 35C for 24 h. Both sucrose-negative (green or blue) and sucrosepositive (yellow) colonies were confirmed as oxidase-positive and inoculated into the following screening media: 692
Kliger iron agar, lysine iron agar and motility agar. When results were compatible of representation, the pathogens were further identified by standard biochemical procedures, including Kanagawa test and antigenic characterization based on the protocols of Guidelines of Brazilian Health Ministry (MS 1992) and Hofer and Silva (1984, 1986). All strains were tested for slide agglutination with polyvalent and monovalent O and K V. parahemolyticus antisera (Denka Seiken Co. Ltd., Tokyo, Japan). Preceding the molecular analysis, all strains were subcultured on to TCBS agar plates, and two or three typical green colonies (sucrose-negative) were inoculated into alkaline peptone water, pH 8Æ6, and incubated for 24 h at 37C, followed by DNA extraction. The strain ATCC 17802 was used as control for the gene trh in polymerase chain reaction (PCR). Extraction of DNA The protocol described by Ausubel et al. (1987) was followed, except for the incubation step at 60C in which lysozyme was not added to retard cell lysis, to avoid DNA breakdown. Briefly, 1 ml of the culture was centrifuged, and the pellet homogenized in 500 ll of TE (Tris-HCl 10 mmol l)1, EDTA 1 mmol l)1, pH 8Æ0) and 10 ll of proteinase K (5 mg ml)1) and incubated at 60C for 20 min. Next, 100 ll of STE (sodium dodecyl sulfate, SDS 2Æ5%, Tris-HCl 10 mmol l)1, pH 8Æ0, EDTA 0Æ25 mol l)1) were added and the suspension incubated for 15 min at 60C, 5 min at room temperature and 5 min on ice, followed by the addition of 130 ll of ammonium acetate (7Æ5 mol l)1). After 15 min on ice, the material was centrifuged and the supernatant was transferred to a new tube and purified with phenol : chloroform : isoamyl alcohol (25 : 24 : 1). DNA in the upper phase was precipitated with an equal volume of isopropanol. The sediment was dissolved in 10 ll of a solution of RNAse (10 mg ml)1) and stored at –20C. Multiplex PCR amplifications The genes tl, tdh and trh were determined by multiplex PCR performed in a Biometra thermocycler, model TRIOthermoblock, in 0Æ5-ml microtubes. The reactions were prepared containing 20 ng of purified DNA, 20 pmol l)1 of each primer (Bej et al. 1999), 2Æ5 ll of 10X PCR buffer, 1Æ5 mmol l)1 of MgCl2, 200 lmol l)1 of each dNTP, 2 U of Taq polymerase (Amersham Bioscience of Brazil Ltda, Sa˜o Paulo, Brazil) and water to a final volume of 25 ll. The thermocycler conditions were 30 cycles of 1 min at 94C, 1 min at 55C and 1 min at 72C, with a final extension at 72C for 7 min. The amplified DNA was separated on 1Æ5% agarose gel in Tris-borate-EDTA (TBE) buffer and then
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V. parahaemolyticus O3:K6 in Brazil
stained with ethidium bromide and photographed under ultraviolet (UV) light. Random-amplified polymorphic DNA (RAPD) RAPD analysis was carried out in a total volume of 25 ll containing 2Æ5 ll of 10X reaction buffer, 3 mmol l)1 of MgCl2, 200 lmol l)1 of each dNTP, 20 pmol l)1 of each primer, 2 U of Taq polymerase (Invitrogen), 20 ng of purified DNA and water to the final volume. Two primers were used in individual reactions: 785-CCGCAGCCAA and 791-GAGGACAAAG. The experiments included negative controls which contained the components of the reaction minus DNA, and each reaction was performed in triplicate. The reactions were carried out in a Biometra thermocycler, model TRIO-thermoblock, programmed for 30 cycles of 94C for 1 min, 36C for 1 min and 72C for 1 min and 30 s, followed by 72C for 10 min. The amplification product (8–10 ll) was submitted to electrophoresis in a 1Æ5% agarose gel, stained with ethidium bromide and photographed under UV light. Ribotyping PCR A pair of primers, 5¢-TTTCTGCAGYGGNTGGATCACCTCCTT-3¢ and 5¢-ACGAATTCTGACTGCCMRGGCAT-
CCA-3¢ (Chun et al. 1999), were utilized to amplify the spacer regions between the genes 16S and 23S. The reactions were prepared in a volume of 25 ll containing 2Æ5 ll of 10X PCR buffer (Tris-HCl 200 mmol l)1 pH 8Æ4, KCl 500 mmol l)1), 1Æ5 mmol l)1 of MgCl2, 200 lmol l)1 of each dNTP, 20 pmol l)1 of each primer, 20 ng of purified DNA, 2 U of Taq polymerase (Invitrogen) and water to the final volume. DNA was amplified under the same conditions used for the multiplex reaction, along with a negative control without DNA. Results Phenotypic analysis showed that 16 strains were Kanagawa-positive, where the two negative ones were from Pernambuco (2002) and Alagoas (2002) originating from isolated cases. However, in the serological characterization, all belonged to the serogroup O:3 including 10 as serotype O3:K6 (seven from food poisoning in Ceara´ and three from isolated cases with two from Alagoas and one from Pernambuco). In strains from the school outbreak in Pernambuco, it was not possible to identify the K antigen in four cultures (Table 1). From a genetic point of view, all the isolates amplified the gene tl, confirming the identity of the species, and only two strains did not show the gene tdh (Alagoas
Table 1 Phenotypic and genotypic characteristics of isolates of Vibrio parahaemolyticus from northeast of Brazil Multiplex PCR target gene
RAPD primer
Isolates
Location and year
Serogroup
tdh
trh
tl§
Kanagawa
785
791
IOC* 17375 17377 17378 17485 17384 17470 17483 17533 17535 17536 17537 17538 17539 17540 17541 17542 17543 17549
PE – 2001 outbreak PE – 2001 outbreak PE – 2001 outbreak AL – 2002 PE – 2001 outbreak AL – 2002 AL – 2002 AL – 2002 AL – 2002 PE – 2002 CE – 2002 outbreak CE – 2002 outbreak CE – 2002 outbreak CE – 2002 outbreak CE – 2002 outbreak CE – 2002 outbreak CE – 2002 outbreak AL – 2002
O3:KUT O3:KUT O3:KUT O3:KUT O3:KUT O3:KUT O3:KUT O3:K6 O3:K6 O3:K6 O3:K6 O3:K6 O3:K6 O3:K6 O3:K6 O3:K6 O3:K6 03:K6
+ + + + + + ) + + ) + + + + + + + +
) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )
+ + + + + + + + + + + + + + + + + +
+ + + + + + ) + + ) + + + + + + + +
A1 A1 A1 A1 A1 A1 A2 A1 A1 A2 A1 A1 A1 A1 A1 A1 A1 A1
B1 B1 B1 B1 B1 B1 B2 B1 B1 B2 B1 B1 B1 B1 B1 B1 B1 B1
*Oswaldo Cruz Institute. Thermostable direct haemolysin. Thermostable-related haemolysin. §Thermolabile haemolysin. PCR, polymerase chain reaction; RAPD, random-amplified polymorphic DNA; UT, untypable; PE, Pernambuco; AL, Alagoas; CE, Ceara´. ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 691–697
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M 1
2
3
4
5
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6
7
8
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
9 10 11 12 13
(a)
800 bp 800 bp
(b) Figure 2 Ribotype patterns of Vibrio parahaemolyticus strains obtained with spacers 16S–23S polymerase chain reaction (PCR) assay. Lanes: M, molecular size markers (100 pb); 1, Vp 17375; 2, Vp 17377; 3, Vp 17378; 4, Vp 17485; 5, Vp 17384; 6, Vp 17470; 7, Vp 17483 (Kanagawa-negative); 8, Vp 17533; 9, Vp 17535; 10, Vp 17536 (Kanagawa-negative); 11, Vp 17537; 12, Vp 17538; 13, Vp 17539; 14, Vp 17540; 15, Vp 17541; 16, Vp 17542; 17, Vp 17543; 18, Vp 175498.
800 bp
Figure 1 Agarose gel (1Æ0%) electrophoresis showing the results of polymerase chain reaction (PCR) amplification of tl (thermolabile haemolysin), tdh (thermostable direct haemolysin) and trh (thermostable-related haemolysin) genes. (a) Lanes: M, molecular size markers (100 pb); 1, strain 17375; 2, strain 17377; 3, strain 17378; 4, strain 17485; 5, strain 17384; 6, strain 17470; 7, strain 17483 (Kanagawanegative); 8, strain 17533; 9, strain 17535; 10, strain 17536 (Kanagawa-negative); 11, strain 17537; 12, strain 17538; 13, strain 17539. (b) Lanes: 1, strain 17540; 2, strain 17541; 3, strain 17542; 4, strain 17543; 5, strain 17549; 6, positive control tl (strain 17375); 7, positive control tdh (strain 17375); 8, positive control trh (strain 17802).
O3:KUT and Pernambuco O3:K6). The gene trh was not amplified in any of the samples (Table 1 and Fig. 1). The amplification of the 16S–23S spacer region of rDNA displayed a single profile for all the isolates (Fig. 2), showing five bands between 250 and 750 bp, where the size was estimated by comparison with the molecular marker of 100 bp. The isolates were also analysed with two random primers by the RAPD-PCR technique. Two profiles were distinguished with each primer. One grouped all the strains that were tdh-positive and another profile differed from the first by having only one band with the primer 791 and two bands with the primer 785, grouping the two isolates that were tdh– and Kanagawa-negative (Fig. 3). Discussion In this work, we attempted to identify molecular markers that would allow the identification of the source of 694
V. parahaemolyticus, responsible for two episodes of food poisoning distinct in time and location, where they occurred in two different cities in northeast Brazil. Using conventional bacteriological techniques, V. parahaemolyticus of the serogroup O3:KUT was identified in the outbreak of Pernambuco and O3:K6 in that which occurred in Ceara´. Other samples of V. parahaemolyticus, isolated in the same period and regions but not related epidemiologically, were studied as those of the isolated cases (out group). The same serogroups were present, pointing out that the serotype O3:K6 had not been reported in the northeast region and that type O3:KUT was cited on one occasion as the causative agent of gastroenteritis by Magalha˜es et al. (1991a,b, 1992), although no reference was made in these latter investigations to the predominance of one particular serogroup ⁄ serotype. The limited capacity of phenotypic techniques to differentiation strains in epidemiological analyses showed the necessity to use a molecular approach for the characterization of the virulence factors and the clonal relation among the isolates. Fundamental molecular techniques in the amplification-specific or random regions of DNA, as well as PCR of virulence genes can supply more precise information for the epidemiological tracing of outbreaks. One of these would be amplification of the 16S–23S spacer region, a variable structure capable of recognizing individual
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M 1 2 3 4 5 6 7 8 9 101112131415161718 (a)
600 bp
M 1 2 3 4 5 6 7 8 9 101112131415161718 (b)
600 bp
Figure 3 (a, b) Results of random-amplified polymorphic DNA (RAPD)–polymerase chain reaction (PCR) agarose (1Æ5%) gel electrophoresis profiles of Vibrio parahaemolyticus strains produced using primers 785 and 791 and run on a 1Æ5% agarose gel. Lanes: M, molecular size markers (100 pb); 1, Vp 17375; 2, Vp 17377; 3, Vp 17378; 4, Vp 17485; 5, Vp 17384; 6, Vp 17470; 7, Vp 17483 (Kanagawa-negative); 8, Vp 17533; 9, Vp 17535; 10, Vp 17536 (Kanagawanegative); 11, Vp 17537; 12, Vp 17538; 13, Vp 17539; 14, Vp 17540; 15, Vp 17541; 16, Vp 17542; 17, Vp 17543; 18, Vp 175498.
characteristics of bacterial species and even between stains of the same species, identifying clones. In V. parahaemolyticus, this region is repeated nine times in the genome, and the spaces can vary in size and in the nature of the encoded rRNA. The variation in size in some species is already sufficient for associating highly related bacteria. Maeda et al. (2000) identified six types of spacer regions
V. parahaemolyticus O3:K6 in Brazil
in V. parahaemolyticus. In this investigation the same profile was determined for all the strains analysed. Another technique utilized is the amplification of random regions of DNA (RAPD), which grouped strains of the serogroup O3:K6 and related ones (O3:KUT) in a single profile, using diverse primers, characterizing them as one clone (Okuda et al. 1997; Matsumoto et al. 2000; Gil et al. 2007). In the 18 strains studied, two profiles were detected with each primer, curiously discriminating between the Kanagawa-positive and -negative isolates. These primers can be utilized for characterizing isolates with respect to the Kanagawa phenomenon, at least in strains of the serotype O3:K6, at the same time that a clonal relation is established. The presence of the genes tl and tdh and absence of the gene trh in all the related strains studied and the uniformity of the profiles obtained by the amplification of the 16S–23S spacer region and by two primers in the RAPD reaction led us to suppose that the pandemic strain O3:K6 and variants are involved (Matsumoto et al. 2000; Nair et al. 2007). The absence of the gene tdh in two samples nonrelated to the outbreaks (17483 and 17536), one from Pernambuco and the other from Alagoas, does not rule out the condition of a pandemic strain, as there is the possibility of spontaneous mutation in the gene tdh during storage (Nishibuchi et al. 1985). The great genetic uniformity of the isolates of V. parahaemolyticus responsible for gastroenteritis in some regions of northeast Brazil, in the years 2001 and 2002, with the techniques utilized, did not favour resorting to these molecular tools for the elucidation of outbreaks or in tracing the source of contamination. However, the presence of a clone with the characteristics of a pandemic clone in the two outbreaks and in various isolated cases of gastroenteritis, suggests that this is disseminated in the region. In fact, such situation finds support in the observations made with the serotype O3:K6 in various countries of the Far East (Okuda et al. 1997; Chiou et al. 2000; Matsumoto et al. 2000; Wong et al. 2000, 2007), which is why the clone has been considered pandemic. Based on this, it is suspected that the occurrence of V. parahaemolyticus in enteric processes in the population of northeast Brazil does not reflect the actuality, as the higher temperature of the sea water and its presence in seafood (oysters and mussels) are factors that are extremely favourable for the dissemination of the microorganism (Hofer and Silva 1986; Magalha˜es et al. 1991a; DePaola et al. 2003). In view of these considerations, particularly because of the virulence potential of the pandemic clone, routine monitoring in the recovery of V. parahaemolyticus in Brazil is recommended for the aetiologic elucidation of diarrhoea cases.
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