Journal of Applied Microbiology 2003, 95, 1277–1284
doi:10.1046/j.1365-2672.2003.02105.x
A biochemical protocol for the isolation and identification of current species of Vibrio in seafood D. Ottaviani, L. Masini and S. Bacchiocchi Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Cupa di Posatora, Ancona, Italy 2003/0197: received 7 March 2003, revised 30 June 2003 and accepted 1 July 2003
ABSTRACT D . O T T A V I A N I , L . M A S I N I A N D S . B A C C H I O C C H I . 2003.
Aims: We report a biochemical method for the isolation and identification of the current species of vibrios using just one operative protocol. Methods and Results: The method involves an enrichment phase with incubation at 30C for 8–24 h in alkaline peptone water and an isolation phase on thiosulphate-citrate-salt sucrose agar plates incubating at 30C for 24 h. Four biochemical tests and Alsina’s scheme were performed for genus and species identification, respectively. All biochemical tests were optimized as regards conditions of temperature, time of incubation and media composition. The whole standardized protocol was always able to give a correct identification when applied to 25 reference strains of Vibrio and 134 field isolates. Conclusions: The data demonstrated that the assay method allows an efficient recovery, isolation and identification of current species of Vibrio in seafood obtaining results within 2–7 days. Significance and Impact of the Study: This method based on biochemical tests could be applicable even in basic microbiology laboratories, and can be used simultaneously to isolate and discriminate all clinically relevant species of Vibrio. Keywords: Alsina’s scheme, biochemical identification, isolation, seafood, Vibrio spp.
INTRODUCTION In recent years researchers have focused their attention on the halophilic noncholerae vibrios. These organisms are not only natural inhabitants of aquatic environments, but are also more and more frequently involved in human gastroenteric episodes, because of consumption of raw or insufficiently cooked seafood, and in systemic pathologies by contact with sea water (Blake et al. 1980; Howard and Bennet 1993; Abbott et al. 1994; Bock et al. 1994; Matte et al. 1994; Shin et al. 1996; Dalsgaard et al. 1997; Kumamoto and Vukich 1998). Recent studies have shown that some species of vibrios considered as nonpathogenic were, instead, able to produce tetrodotoxin (TTX) and Correspondence to: Donatella Ottaviani, Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche via Cupa di Posatora 3, Ancona 06100, Italy (e-mail:
[email protected]).
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anhydrotetrodotoxin (AnhTTX) (Simidu et al. 1987, 1990; Matsumura 1995; Lee et al. 2000). Therefore, it is very important to define standardized methods for the isolation and identification of these microorganisms. To our knowledge no single method is able to isolate simultaneously Vibrio cholerae, V. parahaemolyticus and other halophilic vibrios. The methods are complex and specific for V. cholerae or V. parahaemolyticus (ISO 8914 1990; Elliot et al. 2001; Yukiko et al. 2001) and always involve the use of culture media containing antimicrobials, although specific vibrios show strain-specific susceptibility to some antimicrobials (Ottaviani et al. 2001). In Vibrio spp. nonselective, rather than selective, enrichment is more effective for the detection of injured bacteria, and among environmental stresses refrigeration storage is more injurious than frozen storage (Ray et al. 1978). Several studies of molecular biology investigated the genetic profile of Vibrio spp. and to date at least 37
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genospecies of Vibrio have been discovered and are currently well known. However, the correct identification of environmental isolates is still in discussion because of their biochemical variability (West et al. 1986; Pujalte et al. 1993; Ortigosa et al. 1994). Indeed, the common biochemical commercial kits are obsolete and unable to recognize Vibrio spp., and sometimes they are not able to distinguish between Aeromonas and Vibrio (Austin et al. 1997). As biomolecular methods are expensive and not yet suitable for routine analysis, it is necessary to set up alternative and more accurate biochemical methods. A possible solution to the problem exposed was suggested by Alsina and Blanch (1994a,b). Using statistical analysis of data obtained from other researchers (Baumann and Schubert 1984; Bryant et al. 1986), Alsina selected a set of biochemical keys, which provide accurate identification of Vibrio spp. However, to date, the real applicability of Alsina’s scheme has not yet been tested and working conditions such as temperature, time of incubation, composition of the biochemical test media have not yet been standardized. To have exact working conditions is very important in Alsina’s identification system because this, once standardized, can be applied by all microbiology laboratories without using expensive instruments and it can be easy to prepare culture media with a definite composition. The aim of this work was to set up a method based on biochemical tests to obtain an effective isolation, identification and discrimination of all clinically relevant species of Vibrio.
M A T E R I A LS A N D M E T H O D S Strain collection To standardize the isolation and identification protocol, the American Type Culture Collection (ATCC, Rockville, MD, USA) strains were used (Table 1). For the preparation of the culture media commercial reagents were used (SigmaAldrich, Milan, Italy). The stock cultures were prepared in slant test tubes of tryptone soy agar (TSA; Oxoid, Unipath, Basingstoke, Hampshire, UK) supplemented with 1% NaCl (w/v) incubated at room temperature and subcultivated every 3 months in fresh TSA. The work cultures were obtained by seeding the micro-organisms from TSA slant test tube to TSA plate and to nutrient broth (NB; Oxoid) with 1% NaCl (w/v) both stored at room temperature for not more than 1 week. Standardization of the isolation and identification protocols
Table 1 Reference and isolated strains used* Isolated strains (n)
Source
V. V. V. V. V. V. V. V. V. V. V. V. V. V.
alginolyticus (10) alginolyticus (10) alginolyticus (1) alginolyticus (10) alginolyticus (3) alginolyticus (1) alginolyticus (7) alginolyticus (10) alginolyticus (11) campbellii (1) cholerae (1) cholerae (1) diazotrophicus (1) diazotrophicus (1)
V. V. V. V. V. V. V. V. V. V. V. V. V. V.
fluvialis (1) fluvialis (1) furnissii (1) furnissii (1) harveyi (1) harveyi (3) harveyi (7) harveyi (2) harveyi (2) harveyi (1) mediterranei mediterranei mediterranei mediterranei
Water Vibrio spp. Anchovy V. vulnificus ATCC 27562 Crawfish V. diazotrophicus ATCC 33466 Mussel V. proteolyticus ATCC 15338 Cod V. alginolyticus ATCC 33787 Crawfish V. cincinnatiensis ATCC 35912 Cuttle-fish V. fluvialis ATCC 33809 Mackerel V. furnissii ATCC 33813 Clam V. harveyi ATCC 14126 Mussel V. hollisae ATCC 33564 Mussel V. mediterranei ATCC 43341 Crawfish V. metschnikovii ATCC 7708 Cod V. mimicus ATCC 33653 Clam V. parahaemolyticus ATCC 17802 Cod V. cholerae ATCC 9459 Clam V. natriegens ATCC 14048 Mussel V. logei ATCC 29985 Clam V. nereis ATCC 25917 Water V. carchariae ATCC 35084 Anchovy V. fischeri ATCC 25918 Mussel V. gazogenes ATCC 29988 Cod V. splendidus ATCC 33125 Mackerel V. ordalii ATCC 33509 Clam V. campbellii ATCC 33863 Cod V. orientalis ATCC 33934 Cuttle-fish V. aestuarianus ATCC 35058 Mackerel Clam Other seafood microbiological contaminants Crawfish Aeromonas hydrophila ATCC 7966 Sole A. veronii ATCC 9071 Mussel A. caviae ATCC 15468 Water Plesiomonas shigelloides ATCC 14029 Anchovy Staphylococcus aureus ATCC 25923 Mussel Pseudomonas aeruginosa ATCC 27853 Crawfish Enterococcus faecalis ATCC 29212 Cuttle-fish Proteus vulgaris ATCC 13315 Water Crawfish Mussel Cuttle-fish Mussel Mussel
(1) (1) (1) (1)
V. metschinikovii (1) V. metschinikovii (1) V. nereis (1) V. parahaemolyticus (3) V. parahaemolyticus (1) V. parahaemolyticus (11) V. parahaemolyticus (1) V. V. V. V. V. V. V.
parahaemolyticus (5) proteolyticus (1) proteolyticus (1) vulnificus B2 (2) vulnificus B2 (1) hollisae (2) mimicus (1)
Reference strains
*All Vibrio strains were identified with Willcox probability score >0Æ9.
Optimization of the conditions for the Vibrio strain growth. The parameters, which have been investigated, ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1277–1284, doi:10.1046/j.1365-2672.2003.02105.x
IDENTIFICATION OF VIBRIO IN SEAFOOD
were the concentration of NaCl, temperature and time of incubation. The NB overnight cultures of single ATCC Vibrio strains were inoculated in both fresh NB and in alkaline peptone water (APW; Oxoid) changing the NaCl concentration (1–6%; w/v). The same experiments were performed on the overnight cultures after 2 weeks of refrigerated storage (4C). After incubation at 20, 26, 30 and 37C, the bacterial counts were assayed every 8, 24, 48 and 72 h. In the conventional plate count method 0Æ1 ml of decimal dilutions of each broth culture was plated in duplicate onto the surface of TSA plates and the cultures incubated at the same temperatures of the enrichment phase for 24 h. After 24 h incubation, the optimal growth of both uninjured and injured microorganisms was, in NB and in APW, obtained with salt concentrations ranging from 1 to 4% (with the exception of V. cholerae for which the best conditions were at 1% NaCl after 8–24 h of incubation). The optimal range of temperature was 26–30C for some species and 30–37C for the others. In all the cultures, taking into account the behaviour of V. cholerae, we conventionally used a standard concentration of 1% NaCl and an incubation of 30C for all tests. Isolation phase. To standardize the protocol of isolation, 25 g of sterile seafood (Ray et al. 1978) were artificially contaminated using, for each ATCC Vibrio strain, concentrations of 10, 100 and 1000 CFU ml)1, obtained by dilutions from a suspension with 0Æ5 McFarland opacity standard (100 000 000 CFU ml)1) (Biomerieux, Marcy l’Etoile, France). To investigate the interference power of other seafood autochthonal bacterial strains, to each sample contaminated with the target micro-organism, one of the microbiological contaminants (Table 1) was added at a fixed concentration (1000 CFU ml)1). To evaluate selectivity and recovery, the method was also applied to samples contaminated with the target micro-organism in the presence of one of the other Vibrio strains at the same concentrations (10–1000 CFU ml)1). The isolation phase included the following steps. The contaminated substratum was diluted in a 1 : 10 ratio with the enrichment broths tested: APW (Elliot et al. 2001) and salt polymyxin broth (SPB) (ISO 8914 1990) and incubated at 30C for 24 h. The cultures obtained in each enrichment medium, after 8 and 24 h of incubation, were plated on the surface of the following selective media: thiosulphate citrate bile salt sucrose agar (TCBS; Oxoid) (Elliot et al. 2001) and triphenyltetrazolium chloride soy tryptone agar (TSAT) (ISO 8914 1990). To evaluate the growth in the enrichment media of each reference strain, the APW and SPB cultures were also plated on the TSA. From the TCBS, TSAT and TSA plates, the colonies were selected and subcultivated in TSA incubating at 30C for 24 h. The above-mentioned
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subcultures were submitted for the identification of genus and species. Identification phase. All tests of identification of genus and species were incubated for 24–72 h. To standardize the identification of genus Vibrio, the following biochemical tests were selected (Elliot et al. 2001). The vibrios were discriminated from i micro-organisms, which were not tolerant to strong salt concentration (like Proteus); the capability of growth at 3% NaCl was tested; ii alophilic micro-organisms, which were cytocrome oxidase negatives (like Enterococcus and Staphylococcus); the production of cytochrome oxidase was tested using commercially available kit (Oxoid); iii alophilic micro-organisms, which were cytochrome oxidase positives, but with oxidative metabolism (like Pseudomonas); the production of acid from glucose fermentation in Kligler iron agar was tested; iv micro-organisms with fermentative metabolism, but insensible to vibriostatic (like Aeromonas and Plesiomonas); the sensitivity to 150 lg vibriostatic discs (Oxoid) was tested. The identification of species was assayed with a scheme (Alsina and Blanch 1994a,b) that, using six blocks of biochemical reactions, enables the exact classification of all known genospecies of Vibrio. Among the six blocks of reactions, only one was selected for species identification by using the following preliminary tests: arginine dihydrolase (ADH), lysine decarboxylase (LDC), ornithine decarboxylase (ODC) (Table 2). All the reactions (with the exception of carbohydrate utilization, nitrate reduction and luminescence tests) were performed according to standardized techniques (Andrews and Hammack 2001; Elliot et al. 2001); conventional formulations for all biochemical media included 1% NaCl. Carbohydrate utilization capability was investigated as follows: from TSA plates a loopful of bacterial culture was inoculated in broth containing carbohydrate as the sole source of carbon (bromocresol purple 0Æ02 g, carbohydrate 5 g, NaCl 10 g; distilled water 1 l; pH 6Æ8 ± 0Æ2; sterilization by autoclaving at 115C for 10 min) and, after incubation, the test was positive at the yellow colour change of culture broth. The micro-organisms unable to use the carbohydrate failed in growth. In the nitrate reduction test from TSA plate a loopful of bacterial culture was drawn and seeded in a tube of nitrate agar (beef extract 3 g, peptone 5 g, KNO3 1 g, agar 15 g, NaCl 10 g; distilled water 1 l; pH 7Æ00 ± 0Æ2; sterilization by autoclaving at 121C for 15 min) and, after incubation, 0Æ1 ml of Griess reagent (Oxoid) was added. The test was positive at the red colour change of the colourless medium. The luminescence was assayed irradiating the TSA cultures with a UV lamp.
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Table 2 Tests to be performed for each of LDC/ODC/ADH combinations* LDC+/ODC+/ ADH+
Tests Growth at 0% NaCl Growth at 6% NaCl Growth at 8% NaCl Growth at 10% NaCl Growth at 4C Growth at 35C Growth at 40C Resistance to vibriostatic 10 lg Sucrose fermentation Amigdalyn fermentation Arabinose fermentation Arbutin fermentation Inositol fermentation Mannitol fermentation Salicin fermentation Sorbitol fermentation D-glucosamine utilization a-ketoglutarate utilization D-glucose utilization L-arabinose utilization Lactose utilization Mellobiose utilization Voges–Proskauer Indole Citrate utilization Nitrate reduction Gelatinase Luminescence Beta-galactosidase reaction Urease Oxidase Resistance to amplicillin 10 lg
LDC+/ODC)/ ADH+
LDC+/ODC+/ ADH)
• •
•
LDC+/ODC)/ ADH)
LDC)/ODC)/ ADH)
LDC)/ODC)/ ADH+ • •
• •
• • • •
• •
•
•
•
•
•
•
• •
•
•
• •
• • •
•
•
• •
• •
•
•
• •
• •
• • • • • •
• •
• • • • •
•
•
• •
LDC, lysine decarboxylase; ODC, ornithine decarboxylase; ADH, arginine dihydrolase. *The tests are to be interpreted in accordance with Alsina’s keys (Alsina and Blanch 1994a,b).
Evaluation of the standardized protocol
Probabilistic identification
In the second part of the study, after the standardization of each set of tests using the reference strains (Table 3), the complete protocol for genus and species identification was also tested in 134 strains from refrigerated and frozen seafood (Table 1) isolated following the standardized isolation method. The ratio between the frozen and refrigerated samples was 1 : 1.
Identification was made with the Bayes Theorem by Willcox (Lapage et al. 1973; West et al. 1986) using the free software Probabilistic Identification of Bacteria (Bryant 1995). An identification score at the Willcox probability (P) was calculated for identification thresholds of P > 0Æ9 (positive) and P < 0Æ1 (negative) for all the isolates and ATCC Vibrio strains. In few cases, the percentages were reduced to P ‡ 0Æ8 and P £ 0Æ2 (Alsina and Blanch 1994a,b).
Reproducibility test Each biochemical scheme was performed twice independently for all the strains, using different batches of standardized media.
RESULTS The preliminary evaluation of the enrichment and isolation phases for Vibrio spp. gave the following results. In the
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 1277–1284, doi:10.1046/j.1365-2672.2003.02105.x
Evaluation of the bacterial growth of APW and NB ATCC Vibrio cultures at different temperatures of incubation
Standardization procedures used
30C
30C
Optimum conditions identified
Temperature of incubation
Evaluation of a clear reading of results in the selective media seeded with the ATCC Vibrio strains at different time of incubation
Evaluation of the bacterial growth of APW and NB ATCC Vibrio cultures at different time of incubation
Standardization procedures used
Time of incubation
24 h with the exception of LDC ODC ADH 48 h
24 h
8–24 h APW 24 h TCBS TSAT
Optimum conditions identified Evaluation of the ATCC strains recovery in the selective media from experimentally contaminated seafood* Evaluation of the discrimination between Vibrio and the interfering genera in the selective media seeded with the ATCC strains Evaluation of a clear reading of results in the selective media seeded with the ATCC Vibrio strains
Standardization procedures used
Formulation of media
Standard with the exception of sugar utilization, nitrate and luminescence tests§
Standard
Standard
Optimum conditions identified
KIA, Kligler iron agar; TSA, tryptone soy agar; NB, nutrient broth; APW, alkaline peptone water; TCBS, thiosulphate citrate bile salt sucrose agar; TSAT, triphenyltetrazolium chloride soy tryptone agar; ADH, arginine dihydrolase; LDC, lysine decarboxylase; ODC, ornithine decarboxylase; ATCC, American Type Culture Collection. *Vibrio target (10, 100, 1000 CFU ml)1) + interfering genus (1000 CFU ml)1), or Vibrio target (10, 100, 1000 CFU ml)1) + interfering Vibrio (10, 100, 1000 CFU ml)1). Elliot et al. (2001). ISO 8914 (1990). §In the Simmon’s citrate medium (Andrews and Hammack 2001) to be addeed 0Æ1% of yeast extract.
1%
Tests of species identification
1%
1% KIAs TSAs
Evaluation of bacterial growth of APW and NB ATCC Vibrio cultures changing NaCl concentration of media
Optimum conditions identified
Tests of genus identification
Tests of isolation phase
Set of tests
Standardization procedures used
Concentration of NaCl
Table 3 Standardization procedures used and optimum conditions identified for each set of tests
IDENTIFICATION OF VIBRIO IN SEAFOOD
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range of microbial concentrations assayed, all the Vibrio spp. grew in APW when incubated at 30C for 24 h with the only exception of V. cholerae that grew faster and was detectable even after 8 h. However, in SPB, growth has inhibited in many micro-organisms. All micro-organisms showed a good growth in TCBS and in TSAT after 24 h when incubated at 30C, with the exception of V. hollisae, which was unable to grow in TCBS. All the microorganisms tested as interferences developed in the APW. In the isolation phase, Aeromonas and Plesiomonas were not able to grow in TCBS and Plesiomonas failed in growth also in TSAT where Aeromonas developed colonies indistinguishable from vibrios. Whereas Staphylococcus, Proteus, Enterococcus and Pseudomonas were able to grow in TCBS and in TSAT but the colonies were smaller (0Æ9. DISCUSSION The availability of standard operative protocols for Vibrio spp. is an important determinant in food and environmental microbiology taking into account that these micro-organisms, for a long time considered nonpathogens, have remarkable clinical importance. The few official protocols for the isolation and identification of vibrios are specific for V. cholerae or V. parahaemolyticus, and they cannot be used for other vibrios. In fact the strain-specific antimicrobial susceptibility of Vibrio spp. makes it difficult to isolate these micro-organisms. Moreover, the temperatures commonly used (35–37C) do not enable the optimal growth of some species of Vibrio that grow faster and more effectively at lower temperatures. APW and TCBS are appropriate media for the enrichment and isolation of Vibrio spp., with a good recovery of uninjured and injured vibrios, when incubated at 30C. To obtain an efficient recovery of V. cholerae in the mixed cultures, it is important to plate the enrichment culture APW on TCBS after 8 and 24 h. The TSAT, even if unable to discriminate among vibrios, could be used together with TCBS to recover the micro-organisms unable to grow in TCBS as V. hollisae. As it is not always possible to distinguish between the different vibrios on the basis of colony morphology, to obtain an efficient recovery it is necessary to isolate, at 8 and/or 24 h, at least five colonies from TCBS or TSAT. The identification of Vibrio spp. is problematic because of phenotypic similarity of some species. However, the development of a practical system for the identification has involved the use of commercial kits, which work properly, only in the identification of vibrios of medical importance, but their utility in detecting new species of veterinary importance is limited. Moreover, there are no commercial
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APW 225 ml 30°C
Food 25 g 8h
TCBS
8h
24 h
TSAT
TSAT
24 h
Enrichment
TCBS
Isolation 24 h 30°C
TSAs
Five colonies from TCBS
TSAs
Purification TSAT to be used only in absence of growth in TCBS
Identification of genus (24 h)
Typical response of Vibrio
Medium Growth at 3% NaCl Oxidase
+ +a
Kligler iron agar
Slant K or A; Butt A b
Sensitivity to vibriostatic (150 µg) + a If the oxidase test results are negative while all the other genus tests give typical response for vibrios the presence of Vibrio metschnikovii should be confirmed. b K, alkaline; A, acid.
Identification of species (72 h) LDC/ODC/ADH
Tests of Alsina’s Keys
Fig. 1 Schematic diagram of the vibrios isolation and identification method
kits that include the whole set of tests selected in Alsina’s scheme. This protocol would provide biochemical identification for environmental vibrios and should be applicable in different fields of microbiology (clinical, ecological, taxonomical, animal health and environmental monitoring). It is always necessary to standardize methods to have comparable results and more accurate informations about identification and characterization of Vibrio spp. Up to now, the results reported in the literature are rarely comparable, because the tests were not performed by standard methods.
The results show that the standardized identification scheme is perfectly suitable as regards culture media composition, temperature and time of incubation. We obtained a clear identification of species with both reference and isolated strains. Working with field isolates, we succeeded in discriminating new recent vibrios impossible to distinguish using classical miniaturized identification systems. To sum up, the complete protocol we proposed allows an efficient isolation and identification of Vibrio spp. and could be applicable even in basic microbiology
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