Chapter 17 Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples

Chapter 17 Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples

Handbook of Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 249 9 2003 Elsevier Science B.V. All rights reserved Chapter 17 Culture...

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Handbook of Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.)

249

9 2003 Elsevier Science B.V. All rights reserved

Chapter 17 Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples J a m e s D. O l i v e r

Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA

The genus Vibrio contains over 20 described species, of which 12 are known human pathogens, and of these, eight are food-associated. The vibrios are normal microflora in estuarine waters, and thus occur in high numbers in seafood. V. cholerae, V. parahaemolyticus, and V. vulnificus are the most important vibrios worldwide, causing diseases ranging from mild gastroenteritis to fatal infections. A large number of media have been developed over the last 40 years, both for the selective enrichment and for the isolation of these pathogens. Of these, alkaline peptone water (APW) and thiosulphate citrate bile salts sucrose agar are the most widely employed for the enrichment and isolation of V. cholerae and V.parahaemolyticus. For V. vulnificus, cellobiose polymyxin colistin agar is the most widely used selective medium. These media, numerous others which have been described, their modes of action, their use, and typical results, are the topic of this review.

Introduction At least twelve Vibrio spp. have been described which are human pathogens. Of these, eight are known to be directly food-associated. These include V. alginolyticus, V. fluvialis, V. furnissii, V. hollisae and V. mimicus. Of greatest significance, however, are V. cholerae, V. parahaemolyticus, and V. vulnificus, and culture media employed for these three pathogens are emphasized in this chapter.

The occurrence of pathogenic vibrios in foods Vibrios exist as a major component of the normal microflora of marine and estuafine waters, and thus occur in high numbers in seafood. V. cholerae is also found with some vegetables, including cooked rice, that become contaminated with sewagecontaminated water. As a result, not only foods but the potential sources (typically seawater and contaminated freshwater) of these pathogens need to be examined for their presence. The occurrence, identification, epidemiology, incidence of infections, susceptibility to physical and chemical treatments, and virulence mechanisms of the eight food-associated vibrios have recently been reviewed (Oliver and Kaper, 1998).

250 A few studies on the occurrence of pathogenic vibrios in foods are summarized here to illustrate the significance of this genus. Lowry et al. (1989) reported 100% of the raw oysters from Louisiana they tested contained V.parahaemolyticus and 67% harboured V. vulnificus. In a study of frozen raw shrimp from Mexico, China and Ecuador, 63% were reported to contain Vibrio spp., including V. vulnificus and V. parahaemolyticus (Berry et al., 1994). In a comprehensive study of pathogenic vibrios in tropical oysters, Matt6 et al. (1994) reported the presence of V. alginolyticus (81% of samples), V. parahaemolyticus (77%), V. cholerae non-01 (31%), V.fluvialis (27%), V.furnissii (19%), V. mimicus (12%), and V. vulnificus (12%). As vibrios typically prefer warm waters as their natural reservoir, their occurrence in waters in northern Europe is less common, and this probably accounts for the decreased incidence of the pathogenic vibrios in foods in these countries (see e.g. Dalsgaard et al., 1996 and references therein).

V. cholerae V. cholerae 01 is the causative agent of cholera, and one of the few food-borne pathogens capable of producing epidemic and pandemic outbreaks. While the majority of cases involve mild diarrhoea or are even asymptomatic, approximately 11% of patients develop the classic infection involving explosive diarrhoea. The resulting massive loss of fluid (500-1000 ml/h) leads to dehydration, tachycardia, hypotension, vascular collapse, and ultimately death. Essential to infection is the production of the cholera enterotoxin. While there are exceptions, production of the cholera toxin is limited to cells of the O1 and (more recently) the O139 serogroups; non-O1 serogroups generally do not produce the cholera toxin. The biology of this species has been recently reviewed by Kaper et al. (1995), and the role of food in cholera transmission by Kaysner and Hill (1994). Foods implicated in the spread of V. cholerae include seafood (crabs, shrimp, raw fish, mussels, cockles, squid, oysters, clams), rice, raw pork, street vendor food, frozen coconut milk, and raw fruits and vegetables (Oliver and Kaper, 1998), with greater survival occurring in cooked foods (Mintz et al., 1994). V. parahaemolyticus Numerous food-borne outbreaks of V. parahaemolyticus have been reported worldwide since this pathogen was first described in 1950. In Japan, up to 70% of all bacterial food-borne disease is caused by this species. Gastroenteritis is exclusively associated with seafood that is consumed raw or undercooked. Primarily implicated are raw fish (in Japan), crab, shrimp, lobster, and oysters. The largest outbreak in the United States occurred in 1978 and ultimately affected 1133 people. Boiled shrimp that had been returned into the original shipping boxes after cooking, and held for over 7 h in an unrefrigerated truck were implicated (Oliver and Kaper, 1998). Symptoms (typically beginning 16-24 h after ingestion and lasting 3-7 days) include diarrhoea, abdominal cramps, nausea and vomiting, and fever. As in V. cholerae, only certain strains of V. parahaemolyticus are involved in disease production, and these are cells

251 that produce the so-called 'Kanagawa haemolysin'. Interestingly, whereas virtually all clinical isolates produce this toxin, only about 1% of environmental strains are Kanagawa positive (KP+). It is believed that selection of KP + cells occurs in the intestinal tract, and that this accounts for the predominance of this type in stool samples (Oliver and Kaper, 1998).

V. vulnificus While accounting for only about 1% of all food-borne infections in the United States, this species has the second highest rates of hospitalization and highest case fatality of all food-borne pathogens in that country (Mead et al., 1999). Indeed, this single species is responsible for 95% of all seafood-borne deaths in the United States, with a fatality rate exceeding 60% (Oliver and Kaper, 1998). V. vulnificus occurs as part of the normal microflora of warm estuarine waters worldwide, but occurs in especially high numbers in filter-feeding bivalve molluscs (oysters, clams, and mussels). When consumed raw or undercooked, these are the primary source of the infection. Unlike V. cholerae and V. parahaemolyticus, no outbreaks of V. vulnificus have been reported. This is probably because cases of this disease are largely restricted to persons who have an underlying chronic disease (Oliver, 1989), the most common being liver-related disorders such as alcohol-induced cirrhosis. An unusual aspect of the at-risk group is that over 80% of cases occur in males whose average age exceeds 50 years. Symptoms appear a median of 26 h after infection, with fever, chills, nausea, and hypotension being the most common (Oliver and Kaper, 1998). An unusual symptom occurring in most cases is the development of secondary lesions on the extremities. In fatal infections, death typically ensues within a few days. V. vulnificus is also able to produce potentially fatal infections when introduced into a wound (typically acquired when removing shrimp shells, stepping on a crab or oyster, or via a finfish puncture). Such infections generally occur in healthy persons, and carry a fatality rate of about 25% (Oliver, 1989).

Other vibrios Along with the species listed above, seafood-associated infections due to V. mimicus, V. fluvialis, V. furnissii, V. hollisae, and V. alginolyticus have also been reported. All of these vibrios cause disease whose primary symptoms are similar to those produced by V. cholerae, although of a milder nature. For a recent review, see Oliver and Kaper (1998).

The need for selective media for the enrichment and isolation of vibrios While vibrios typically comprise the major bacterial genus in estuarine waters (the source of many of the seafoods harvested worldwide), there are numerous competing bacteria which must be selected against in order to adequately characterize the vibrio species present in a sample. In addition, while vibrios such as V. parahaemolyticus, V. alginolyticus, and V. vulnificus may constitute a large population in seafoods, those

252 Table 1 Modes of action of selective agents in media for isolation of vibriosa. Agent

Mode of Action

Bile salts (including taurocholate) inhibit Gram-positive bacteria, some Gram-negative other than the enterics inhibits some Gram-positive bacteria Tellurite bactericidal towards many Gram-negative bacteria Polymyxins (including colistin) high pH values select for vibrios (optimum 8.4-8.6) pH NaC1 (0.5-1%) required by most vibrios; higher levels inhibit Salts many Gram-negative bacteria Sodium lauryl (dodecyl) sulphate membrane solubilizer; bactericidal for many bacteria aAdapted and extended from Donovan and van Netten (1995). such as V. cholerae generally occur at low numbers, and their presence is not easily determined unless the cells are allowed to grow preferentially over other naturally occurring bacterial groups. For this reason, enrichment and plating media have been described which take advantage of the resistance typically exhibited by vibrios to compounds such as bile salts, tellurite and certain antibiotics, as well as to elevated salt concentrations and pH values. The latter considerations are based on the fact that the pathogenic vibrios have a preference for alkaline conditions and, with the exception of V. cholerae, are all halophilic. These attributes are the basis for their frequent employment in media for the selection of vibrios, whether obtained from clinical, environmental, or food samples. Many media, both enrichment broths and plating agars, have been described over the last 40 years. For the most part, these have not been accepted by clinical or research investigators, the food industry, or government officials. This lack of acceptance is largely due to a lack of success of the various media for selectively enriching the various Vibrio spp. from the competing microflora, and the phenotypic similarity between many Vibrio spp. In the following sections, I have tried to mention most of these media, although this is primarily of historical interest only. Those few media that are widely accepted by the scientific community are emphasized. The reader is encouraged to review the previous edition of this chapter (Donovan and van Netten, 1995) for historical aspects and discussions of the principles on which the various media are based.

Mode of action of agents employed in vibrio-selective media The reagents employed in many of the media designed to be selective for Vibrio spp., along with their modes of action, are shown in Table 1.

253 Enrichment broths for vibrios

Whereas quantitative methods (MPN and direct plating) for the isolation of vibrios are often used, many investigators prefer a qualitative enrichment step prior to plating to various selective media. Of these, only alkaline peptone water (APW) has been widely accepted. This broth, which has been employed since 1887 (Donovan and van Netten, 1995) takes advantage of the fact that vibrios thrive in moderately alkaline environments. While many variations have been suggested during that time (Donovan and van Netten, 1995, and see below), APW (1% tryptone peptone and 1% NaC1 at a final pH of 8.6) has been, and continues to be, most commonly employed for the isolation of vibrios from foods and other natural sources. In one study, Sloan et al. (1992) compared the effectiveness of APW with four other enrichment broths, and found APW to provide the greatest detection of V. vulnificus (see below). Some investigators have employed additions to this basic broth in attempts to enrich various Vibrio spp. (Table 2). Among these, NaC1 additions have been the most common. For example, Arias et al. (1998) recommended the use of APW with 3% NaC1 (APWS) for the isolation of V. vulnificus. Recently, Hr et al. (1998a) proposed the addition of 2x 104 U/liter polymyxin B (APWP) for the enrichment of V. vulnificus. While such additions may be of value in the isolation of certain species, other laboratories have yet to confirm these modifications. None of the suggested additions, however, have proven to be of value (or to be adopted) in the case of V. cholerae or V. pa rahaemo lytic us. V. cholerae The American Public Health Association (APHA) analytical method (Clesceri et al., 1998) for V. cholerae recommends a 6-8 h enrichment in APW at 35~ followed by isolation on TCBS agar (see also Madden et al., 1989 for a detailed description). This simple enrichment is used worldwide, and while several modifications or alternate enrichments have been proposed, no other enrichment medium has found acceptance among investigators or government authorities. Some proposed enrichments, such as gelatin-phosphate-saline (GPS; Madden et al., 1989) have been shown to be of no value for the selective enrichment of V. cholerae (Spira, 1984). Others, such as Monsur's taurocholate tellurite peptone (TTP), have been found by some investigators to be as good or better than APW in supporting growth of V. cholerae (Furniss et al., 1978), but have not been adopted by investigators. Similarly, starch gelatin polymyxin broth (SGP), developed by Kitaura et al. (1983) for the isolation of V. cholerae, has not been accepted by the research community. However, one simple modification of the APW enrichment appears to warrant consideration. An elevated temperature (42~ enrichment method for recovery of V. cholerae from oysters was found by DePaola et al. (1988) to give a significantly (p<0.05) higher recovery and a greater specificity (P<0.01) than the standard enrichment protocol at 35~

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V. parahaemolyticus As with V. cholerae, a number of enrichment broths, including glucose salt teepol broth (GSTB), salt polymyxin broth (SPB), and salt colistin broth (SCB), have been proposed for the enrichment of this pathogen (see Joseph et al., 1982, Karunasagar et al., 1986, and Twedt, 1989 for reviews). To date, however, none has been found to be preferable over APW. While SPB and SCB were reported by Nakanishi and Murase (1974) to be of value in the enrichment of V. parahaemolyticus from raw fish, Karunasagar et al. (1986) found direct plating to TCBS to yield better recovery than the MPN technique using GSTB, SPB, or two other marine broths. Further, Hagen et al. (1994) compared SPB and APW for the isolation of V. parahaemolyticus from crab, oysters, shrimp, lobster, and shark, and found APW to be significantly more efficient than SPB. Similarly, APW was found by Eyles et al. (1985) to be the most effective for isolating this species from oysters and prawns. Hagen et al. (1994) also found APW to be more effective for the isolation of V. parahaemolyticus from seafood samples which had been refrigerated at 2-4~ for up to 7 days, or frozen at-15~ for up to 28 days. An interesting method for enriching and quantifying V. parahaemolyticus in foods has been described by Miyamoto et al. (1990). They employed an arabinose glucuronate enrichment medium in which they incubated samples at 37~ overnight. They then examined the trypsin-like activity of the bacteria, measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethyl-coumarin. They reported that, even in the presence of > 105 cells of V. alginolyticus, 20 cells of V.parahaemolyticus could be detected after 6 h. In a study of 14 seafoods (50 different samples), they found a 0.95 correlation after a 6 h incubation when comparing this method with a conventional assay (bromothymol blue teepol agar and the MPN method). The presence of 10 cells of V. parahaemolyticus could be detected in the seafoods after a 10 h detection period. No fluorogenic activity was detected with eight other Vibrio spp., or members of Pseudomonas, Bacillus, Staphylococcus, Aeromonas, or a variety of Enterobacteriaceae.

V. vulnificus Until recently, the United States FDA recommended the use of glucose salt teepol broth (GST) as an enrichment for V. vulnificus in an MPN procedure, with positive tubes being streaked onto TCBS agar. However, teepol is no longer commercially available, and other enrichment broths, primarily APW, have been proposed. Along with their study on V.parahaemolyticus, Hagen et al. (1994) compared SPB and APW for the isolation of V. vulnificus from a variety of fish and shellfish, and found APW to be significantly more efficient than SPB. As they also observed with V. parahaemolyticus, APW was also found to be superior when the seafood samples had been cold stressed. In a more comprehensive study, Sloan et al. (1992) compared five selective enrichment broths for the isolation of V. vulnificus from seeded oysters. APW, Marine broth (Difco), Horie's arabinose-ethyl violet broth (HAE; Horie et al., 1964), Monsur's TTP broth (Monsur, 1963; Kaysner et al., 1987), and glucose salt

256 teepol broth (GSTB; U.S. Food and Drug Administration, 1984) were examined. They found that APW and marine broths yielded significantly higher recovery than the others, with APW being the most successful. Similarly, Kaysner et al. (1989) found that an 18 h MPN enrichment step in APW gave higher recovery levels of V. vulnificus from two species of oysters than did direct plating to TCBS or CPC (see below for a discussion of this medium). Hsu et al. (1998) recently described PNC enrichment broth for V. vulnificus, containing 5% peptone, 1% NaC1, and 0.08% cellobiose, the concentrations of which were said to be optimized for this species. A further modification of this broth (PNCC) included 1.0-4.1 U of colistin methanesulfonate per ml, which the authors found increased the growth of low levels of V. vulnificus while suppressing non-target bacteria. To date, this medium has not been tested outside the laboratory. In a study examining vibrios in shellfish from coastal waters of Spain, Arias et al. (1998) concluded that the best combination of methods was enrichment in APWS (APW with 3% NaC1) for three hours at 40~ followed by plating onto CPC agar. Dalsgaard and Hr (1997) and Hr et al. (1998 a,b) described the addition of polymyxin B (2.0xl 04 U) to APW to give APWE Following incubation at 37~ for 18-24 h, a loopful of the surface pellicle was streaked onto CPC agar (or one of its modified versions; see below) which was then incubated for an additional 18-24 h at 40~ These authors have employed this modified enrichment for the isolation of V. vulnificus from Danish mussels and from shrimp products imported into Denmark, noting that this amendment appears to be of value in the pre-enrichment of samples that contain large numbers of bacteria capable of growing under alkaline conditions.

Other vibrios Nishibuchi et al. (1983) described FEM (Vibriofluvialis enrichment medium), containing novobiocin as an inhibitor, for the enrichment and enumeration of this species from environmental sources. In a test of 177 samples (including crabs), they reported that FEM was more effective than APW in enriching this species, particularly from samples taken from waters of low (<6%) salinity.

Plating media for vibrios A variety of plating media has been proposed for the isolation of vibrios from clinical and environmental sources (Table 3), and these are reviewed in Donovan and van Netten (1995). Of these, however, only thiosulphate citrate bile salts sucrose (TCBS; Kobayashi et al., 1963) is routinely employed. All of the pathogenic vibrios, with the exception of V. hollisae, grow on this medium, which depends on ox bile and an alkaline pH to suppress growth of other bacteria. V. damsela does not grow on TCBS when incubated at 37~ but will grow at lower temperatures (e.g. 28~ see e.g. Song et al., 1993). Differentiation between Vibrio spp. depends on their ability to ferment sucrose and turn the bromothymol blue indicator from green to yellow (Table 4). The medium should not be autoclaved, requiting only boiling for its preparation, and is available

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258 Table 4 Sucrose reactions of the pathogenic vibrios. Sucrose +

V. cholerae SucroseV. metschnikovii ~ V. cincinnatiensis a V. fluvialis V. furnissii V. alginolyticus V. carchariae b

V. mimicus V. hollisae V. damsela b V. parahaemolyticus V. vulnificus

a The route of infection of these pathogens has not been established. b Infections appear to result solely from wound infections. from commercial suppliers. As with all fermentable carbohydrate-containing media, one drawback of TCBS is the inability to perform the oxidase test (crucial for differentiating vibrios from the Enterobacteriaceae) directly on colonies. Variations in effectiveness of TCBS obtained from different suppliers has also been documented by several investigators (McCormack et al., 1974; Morris, 1982). The major disadvantage of this medium, however, is its lack of selectivity and differentiation among vibrios. A number of non-vibrios are known to grow on TCBS (McCormack et al., 1974; Morris et al., 1976; West et al., 1982; Lotz et al., 1983; Spira, 1984). Indeed, Lotz et al. (1983) found that 177 of 188 strains from 15 genera, including three Gram-positive genera, were able to grow on TCBS. These included Acinetobacter, A e r o m o n a s (47 of 47 strains tested), Alcaligenes, Enterobacter, Escherichia coli (5 of 7 strains), Pasteurella, Pseudomonas spp. (12 of 12 strains), Salmonella, and Proteus. Some non-vibrios produce black colonies (due to FeS precipitation as a result of H2S production from thiols). On the other hand, TCBS appears to be the best medium currently available for the isolation of vibrios (Morris et al., 1979; Rennels et al., 1980; Karunasager et al., 1986). In a study of several media designed for the isolation of vibrios, Bolinches et al. (1988) found TSAT (trypticase soy agar sucrose bile triphenyltetrazolium; Kourany, 1983), TTGA (modified gelatin taurocholate tellurite agar; O'Brien and Colwell, 1985), GS (glucose-salts; Simidu and Tsukamoto, 1980), and GSTC (glucose salt tellurite crystal violet; Bolinches et al., 1988) agars recovered only 55, 13, 12, and 0%, respectively, of the vibrios present in three estuarine water samples. In contrast, their study reported TCBS provided an 83 % recovery of vibrios from these same natural samples. To cite just two examples of its usage, Barbieri et al. (1999) employed TCBS (following enrichment in APW) to examine estuarine waters along the Italian Adriatic coast for the presence of pathogenic vibrios. They readily isolated V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. Similarly, Hanharan et al. (1995) employed TCBS to characterize the microbial flora of mussels and oysters from Canada. They isolated V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. damsela, V. cholerae (non-01), and V. metschnikovii, along with eight additional species of non-pathogenic vibrios. Such studies clearly indicate that this medium is capable of cultivating a variety of Vibrio spp. from natural waters and shellfish. Indeed, the medium has been employed extensively in characterizing the vibrio flora of seafood

259 (e.g. see Kelly, 1982; Oliver et al., 1982; Oliver et al., 1983; West et al., 1982; Karunasagar et al., 1986). Thiosulphate chloride iodide (TCI) agar, which employs potassium iodide in lieu of bile as an inhibitory agent, was described for the isolation of pathogenic Vibrio spp. by Beazley and Palmer (1992). These authors reported that the plating efficiency of five Vibrio spp. was superior to that of TCBS, and because it contains no fermentable carbohydrate, colonies can be directly tested for oxidase activity. In a more extensive examination of TCI, Abbott et al. (1993) reported that 102 strains of vibrios grew well on this medium. Of interest is that none of the Aeromonas spp. (14 strains), Plesiomonas shigelloides (10 strains), or three species of non-pathogenic vibrios grew on TCI agar. On the negative side, extremely poor (<1%) plating efficiencies were reported for V. vulnificus and V. damsela, and most of the pathogenic vibrios developed into relatively small (typically <3 mm) colonies. We (Pfeffer and Oliver, unpublished) recently compared this medium to TCBS in isolating vibrios from estuafine water samples. We found that 61% of the colonies developing on TCBS could be presumptively identified as being Vibrio spp., whereas only 46% were so identified on TCI agar. Further, over twice as many presumptive vibrio colonies developed on TCBS compared to TCI. Thus, this medium does not appear to have great potential in isolating vibrios from environmental samples.

Plating media for V. cholerae The primary plating medium of choice for V. cholerae continues to be TCBS, and this medium has been recommended by the World Health Organization for the identification of this species (Morris, 1982) from stool samples. While few organisms in clinical samples, other than vibrios, grow on this medium, numerous genera found in foodstuffs are capable of growth, and this has been discussed above. Being sucrosepositive, V. cholerae produces large yellow colonies on TCBS (Table 5). A variety of solid media has been proposed for the cultivation of V. cholerae. Monsur (1961) developed gelatin taurocholate tellurite (GTT) agar, taking advantage of the resistance to tellurite, taurocholate, and high pH of this species. V. cholerae produces small, translucent colonies surrounded by a cloudy zone of gelatinase activity on this light brown medium (Morris, 1982; Donovan and van Netten, 1995). Morris et al. (1979) found this medium to be comparable to TCBS for the isolation and identification of this species. Sucrose tellurite teepol (STT) agar (Chatterjee et al., 1977) also employs tellurite as a selective agent, but substitutes teepol for the bile salts found in TCBS. The latter are said to be the cause of the variation that is often reported in TCBS from different vendors (Donovan and van Netten, 1995). STT is also salt-free, which allows the preferential growth of V. cholerae over other vibrios which are more halophilic. Morris et al. (1979), however, found this medium to be the poorest in performance among the four plating media they tested. Polymyxin mannose tellurite (PMT) agar was developed specifically to differentiate V. cholerae O1 from non-O1 strains (Shimada et al., 1990). The medium owes its selectivity to tellurite and polymyxin B. Its differentiating abilities depend on O 1

260 Table 5 Colony size and appearance of the major pathogenic vibrior Medium Vibrio spp.

Size (mm) Appearance

TCBS b

V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus

>5 >5 3-5 >5

TCI

V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus

<1-1.5 2.5-3.5

2 3

GTT

V. cholerae

2-3 2-5 ND 2-5

PMT

V. parahaemolyticus V. vulnificus V. alginolyticus V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus

1-2 ND

SPS

V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus

1-3 2-3 1-3 1-2

yellow with halo purple-green purple green with halo yellow

CPC

V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus other Vibrio spp. V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus

2-3 NG e 2-3 NG NG

purple surrounded by blue zone

3-5 3-5 ND 3-5

yellow blue

STT

TSAT

VV

VVE

2-3

3-4

transparent with surrounding halo and faint black centre opaque grey with surrounding halo and black centre opaque grey with surrounding halo and black centre yellow or dark violetd yellow yellow

flat, yellow with darker centre

yellow

ND

2-4

red

ND 1-3

white (possible pink centre)

V. cholerae V. parahaemolyticus V. vulnificus V. alginolyticus

NG

V. vulnificus

ND NG

other Vibrio spp.

yellow green green yellow ND c ND ND ND

Vf 2-4

light grey, translucent with black centre

ND blue to greenish-blue

Adapted and expanded from Donovan and van Netten (1995). b For a more complete list of the various pathogenic vibrios on TCBS agar, see Table 4. c No description. d Mannose-fermenting strains are yellow; mannose non-fermenting strains are violet. e NO growth. f Variations in size and appearance. a

261 strains fermenting the mannose in the medium, resulting in yellow colonies, whereas non-O 1 strains yield dark violet colonies. However, mannose fermentation has proven to be a poor indicator of serogroups, and this medium has not found acceptance for the isolation of V. cholerae. VP agar, originally developed by De et al. (1977) for the isolation of V.parahaemolyticus, contains the selective agents sodium taurocholate and sodium lauryl sulphate. The medium was reported to have value in isolating V. cholerae, but Morris et al. (1979), comparing this medium to several others for its ability to isolate V. cholerae from patients with cholera symptoms, reported that TCBS and GTT were superior to both VP agar and STT agar, based on the total number of positive specimens obtained. Sodium dodecylsulphate polymyxin sucrose (SPS) agar, developed by Kitaura et al. (1983) and cellobiose polymyxin colistin (CPC) agar, developed by Massad and Oliver (1987), were both said to be of value in the isolation of V. cholerae as well as V. vulnificus. To date, however, these media have not been examined outside the laboratory for this purpose.

Plating media for V. parahaemolyticus Many solid media have been proposed for the isolation of V.parahaemolyticus, but none has proven to be effective. Furniss et al. (1978) reported that Monsur's GTT, originally developed for the isolation of V. cholerae, was of value in isolating V.parahaemolyticus, but this has not been corroborated. Trypticase soy agar triphenyltetrazolium (TSAT), developed by Kourany (1983), employs sucrose and triphenyltetrazolium to differentiate V. parahaemolyticus from V. alginolyticus. V. parahaemolyticus colonies are red on this medium, as compared to white colonies for the latter. Unfortunately, the medium does not appear to be very selective, and Proteus spp. grow with colonies similar to V. parahaemolyticus. Escherichia spp. and other Enterobacteriaceae also grow on the medium. VP agar was developed by De et al. (1977) for the isolation of V.parahaemolyticus. However, when this medium was tested with samples of seawater, oysters and clams by Cleland et al. (1985), only 22% of the colonies developing on this medium could be identified as vibrios. Further, whereas 19.2% of the colonies developing on TCBS from these samples were subsequently identified as V. parahaemolyticus, only 4% of those on VP medium were identified as this species.

Plating media for V. vulnificus V. vulnificus is the only Vibrio species for which there exists a widely adopted, selective medium. While many media have been proposed for the isolation of V. vulnificus (Table 3, and see Donovan and van Netten, 1995, for a historic treatment), the cellobiose polymyxin B colistin (CPC) agar described by Massad and Oliver (1987) is the only medium generally used for this purpose. The medium has also been adopted by the United States FDA for isolation of this bacterium. This medium takes advantage of the colistin and polymyxin B resistance of V. vulnificus to eliminate most other

262 pathogenic vibrios, high temperature incubation (40~ to eliminate many marine bacteria, and the fermentation of cellobiose as a differential agent. In the initial study (Massad and Oliver, 1987), 136 strains representing 19 species of Vibrio, as well as marine isolates of three other genera, were tested for growth on this medium. CPC agar was found to be highly selective for V. vulnificus (which ferments cellobiose and produces yellow colonies) and for V. cholerae (which does not ferment the sugar and produces purple colonies). Of the 79 strains of the remaining 17 species of Vibrio, only one out of nine V. parahaemolyticus strains tested grew on CPC agar. Similarly, no growth was observed for 17 strains of Photobacterium, Pseudomonas, or Flavobacterium. Subsequent field testing of this medium, involving direct plating of oyster and clam homogenates (followed by gene probe and monoclonal antibody technologies), confirmed the value of CPC agar, finding it superior to SPS agar and TCBS (Oliver et al., 1992). A later study (Sun and Oliver, 1995) compared CPC agar and VVE agar for isolation of V. vulnificus from 224 oysters, with gene probe hybridization used to confirm identification. Of over 3,500 cellobiose-positive colonies tested, 28.7% of those on CPC were identified as V. vulnificus on the basis of the probe whereas only 2.8% of 19,000 colonies developing on VVE agar could be identified as this species. When colony morphology (flat, with a darker central area) as well as colony colour was considered, 81.6% of over 1000 colonies developing on CPC agar proved to be V. vulnificus. Using these same criteria, Sloan et al. (1992) found 81% of the 'typical' V. vulnificus colonies on CPC to be identified as this species. The observation that only yellow colonies with a flat, darker yellow central area represent V. vulnificus is clearly very important in the use of this medium. Kaysner et al. (1989) examined V. vulnificus in shellstock and shucked oysters, and concluded that CPC gave the best recovery (following APW enrichment), and that the background microflora level on CPC agar was much lower than other selective media, and that in most instances V. vulnificus was in pure culture on CPC agar. Several modifications of CPC agar have been proposed, primarily involving reductions or deletions of the antibiotics. Tamplin et al. (1991) described mCPC, with the colistin level lowered to 400,000U/1. This medium has been employed by DePaola et al. (1994) to isolate V. vulnificus from the intestines of fish from the U.S. Gulf Coast, by Parker et al. (1994) for examining the levels of V. vulnificus in frozen and vacuumpackaged oysters, and by Hr et al. (1998a) in a study on the occurrence of V. vulnificus in Danish mussels and fish. CC agar, recently described by Hr et al. (1998b), was reported to give a significantly higher isolation rate of V. vulnificus from water and sediment samples than did mCPC, and to have a statistically higher plating efficiency than TCBS agar. CC agar has the same composition as CPC agar, but with no polymyxin B and a slight reduction in colistin concentration. Cerd~-Cu611ar et al. (2000) recently described another medium, termed VVM agar, for the isolation of V. vulnificus. This medium, which contains a number of minor modifications from the original CPC, was combined with a 16S rDNA probe to isolate and identify this species. Like CPC, the medium appears to be selective for V. vulnificus, but several other Vibrio spp. (V. campbellii, V. carchariae, and V. navarrensis) also produced yellow colonies. Several other vibrios produce yellow colonies, as does P. aeruginosa. The medium has not been tested in the field or by other laboratories. Indeed, with all of these media, more

263 studies are required to determine if any show sufficient improvement on the original to justify replacing CPC as the recommended plating medium for V. vulnificus. Other media have been developed for the isolation of V. vulnificus. VV agar (Brayton et al., 1983) was tested by Dinnuzzo et al. (1984) and Cleland et al. (1985) who reported that only 9-10% of the colonies could be identified as being V. vulnificus. In another field study, Tilton and Ryan (1987) also found VV agar to be inadequate for the isolation of V. vulnificus. SPS (sodium dodecylsulphate polymyxin B sucrose), developed by Kitaura et al. (1983) is selective for V. vulnificus through its polymyxin B resistance, and is differential through the sulphatase activity of V. vulnificus. The latter, in the presence of sodium dodecylsulphate, results in haloes around colonies of this species. However, growth and halo production also occurs with V. cholerae and V. anguillarum. The medium was found by Kitaura et al. (1983) to isolate V. vulnificus from a variety of shellfish and Bryant et al. (1987) found it to be of value for the direct isolation of V. vulnificus from shellfish. Oliver et al. (1992), however, in comparing CPC to SDS and TCBS for their ability to select and differentiate V. vulnificus from background vibrios in shellfish, reported CPC to be superior to both these media. Using monoclonal antibody and gene probe technology, as well as classic taxonomic methods, to verify the identity of presumptive V. vulnificus colonies, these authors reported that approximately twice as many colonies on CPC agar could be identified as this species when compared to TCBS, with none of the sulphatase-positive colonies taken from SDS agar being so identified. VVA agar, described by Wright et al. (1993), was designed to be non-selective, but to be used in combination with a V. vulnificus-specific gene probe ('VVAP'). However, in a study examining seawater, sediment, plankton and oysters for the presence of V. vulnificus, they reported that LB agar yielded higher counts than did TCBS agar, which yielded higher levels than did VVA. VVE medium, described by Micelli et al. (1993) for the direct isolation of V. vulnificus, contains lactose as well as cellobiose, and several inhibitors (oxgall, sodium cholate, sodium taurocholate, and tellurite). A field test using VVE to determine the levels of V. vulnificus in oysters found 104 to > 10 6 cells/100g oyster. A study by Sun and Oliver (1995) on 224 oysters, however, reported only 2.8% of 19,000 colonies developing on VVE agar could be identified as this species. The use of this medium has not been reported by other laboratories.

Plating media for other vibrios TSAT was designed by Kourany (1983) to differentiate V. parahaemolyticus from V. alginolyticus, employing sucrose, bile salts, and triphenyltetrazolium chloride as selective and differential agents. The medium has not been used routinely for the isolation of V. alginolyticus.

264 Recommended culture media

The only medium that has proven of value for the culture of Vibrio spp. from clinical, environmental and food sources, continues to be TCBS. Despite its limitations, this medium remains the medium of choice by investigators worldwide.

V. cholerae A combination of alkaline peptone water (APW) enrichment (with incubation at 35-37~ for 18 h) and subsequent plating to TCBS agar as the selective/differential medium remains the most commonly employed (and recommended) method for isolation of V. cholerae. When large numbers of background bacterial populations exist, it is advisable to subculture APW at 2 h as well as 18 h. SPS and CPC agars may have value in the isolation of V. cholerae, especially following APW enrichment, but further testing is required to establish these media for this purpose.

V. parahaemolyticus Despite advocacy for numerous alternate enrichment and plating media for this species, APW coupled with plating onto TCBS agar is the only generally accepted method for the isolation of this pathogen.

V. vulnificus Unlike the case with V. cholerae and V. parahaemolyticus, a medium exists which is highly selective for this pathogenic species, and which has found wide acceptance for use. The general recommendation is a 16 h enrichment at 35-37~ in APW followed by plating a loopful from the top cm of the broth to CPC agar. For quantitative determinations, direct plating of samples onto CPC agar or one of its derivatives (e.g. mCPC) is recommended. Enrichment in APW with colistin (Hr et al., 1998b) may have merit, and should be examined further.

Identification of vibrios

Because of the phenotypic variation exhibited by the vibrios, and the need to determine which isolates are toxin producers, it has become commonplace for investigators to supplement isolation media with a variety of molecular techniques for identification of presumptive vibrio isolates. The reader is referred to studies by Koch et al. (1993) and Popovic et al. (1994) for identification of cholera toxin (ctx)-producing strains of V. cholerae in foods. Similarly, since only Kanagawa haemolysin-positive (tdh +) strains of V. parahaemolyticus are pathogenic, some investigators now use gene probes or PCR primers against these genes to differentiate K § and K- strains (DePaola et al., 1990; Beasley et al., 1994; McCarthy et al., 1999). Identification of V. vulnificus

265 presents special problems, as significant phenotypic variation exists among isolates. Further, and as is the case with V. cholerae and V. parahaemolyticus, not all strains of V. vulnificus may be of concern in food safety (Warner and Oliver, 1999). For these reasons, a probe (VVAP) developed by Wright et al. (1993) against the haemolysin/ cytotoxin of V. vulnificus has proved to be of great value in identifying this species. The probe was shown in laboratory studies to have 100% specificity against this species (Morris et al., 1987), and has subsequently been used in numerous studies (e.g. Oliver et al., 1992; Kaspar and Tamplin, 1993; Dalsgaard and Hr 1997; DePaola et al., 1997).

Acknowledgement I am deeply indebted to Drs. Donovan and van Netten for their contributions to the previous edition of this chapter.

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