This is an update of an extract of a report I wrote in 1998 – this extract covers the fundamentals of Vibrio parahaemolyticus isolation. Certainly, rapid and molecular methods have progressed in the past 22 years! …but the basic principles of isolation of this organism remain, such as saline conditions and TCBS Agar.

Vibrios are ecologically important marine organisms (Figure 1), and since the natural habitat of Vibrio species are marine environments, they do have a tolerance and sometimes preference for salty conditions, with regard to growth. As a rule, NaCl stimulates growth of vibrios and many cannot grow in the absence of it. For example, Baron et al. (1994) states that V. parahaemolyticus, V. vulnificus and V. hollisae are amongst those Vibrio species which will not grow when NaCl is absent from the culture medium. Although NaCl is required for growth of these species, there are limits, like all growth conditions. V. parahaemolyticus is less concerned with salt concentration, being able to grow from 0.5% – 10.0%, although the optimum is 3% (ICMSF, 1995). This tolerance to high salt concentrations is used in culture media for differentiation between Vibrio species, although most media used for routine isolation of V. parahaemolyticus (such as TCBS) is made up to 3% NaCl, as this is the amount of salt which allows for best growth of this species.

Figure 1: Importance of vibrios to the functioning of the marine ecosystem.
Figure 2: Prepared plates of TCBS agar

When vibrios are suspected in a sample, the best medium to isolate them on is Thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Figure 2). This medium has been used worldwide for over 40 years, and is the medium of choice when attempting to grow Vibrio species (AWWARF, 1997). A distinct advantage of TCBS is that it does not require sterilisation (autoclaving) prior to use. The medium is selective and differential for vibrios. Its selective property is based on the concentration of salt and high pH which allows Vibrio species to grow while being inhibitory to other genera likely to be present in foods or clinical samples where vibrios are present and clinically important. Differentiation between species of Vibrio is also simple when using TCBS. Sucrose is the largest single component of TCBS (Atlas, 1993), and this is fermentation of this sugar which is the basis for differentiating important species of Vibrio. Colonies of sucrose fermenters (V. cholerae) appear yellow while those of non-sucrose fermenters (V. parahaemolyticus and V. vulnificus) are green (Figure 3). The basis for this variance in colony colour is the acid alkaline conditions which result from the fermentation or non-fermentation of sucrose. The fermentation product must then be acidic, resulting in the yellow colour, while the colony colours are yellow and blue/green because the indicator incorporated into the medium is bromthymol blue. This indicator turns yellow at the slight acidic pH of 6.0 and blue at the slightly alkaline pH of 7.6 (Brady and Holum, 1993). Alternatives to TCBS include Bromthymol blue Teepol (BTBT) and Trypticase soy agar triphenyltetrazolium (TSAT) (Donovan & van Netten, 1995).

Figure 3: Colonies of Vibrio parahemolyticus on TCBS.

The TCBS plating method is the most traditional method for isolating vibrios, but like for all bacteria, a plethora of methods exist – cultural (such as chromogenic media), molecular, immunological and others. The reader is referred to a particularly comprehensive insight into testing methodologies by the FAO for further information (FAO/WHO, 2016). Despite their acceptance, stemming from reliability-based performance, traditional cultural methods (for any bacterium) have a major disadvantage in processing, especially if enrichment and confirmatory tests are required. In such cases, the entire process could take the best part of a week. Thus, a variety of rapid methods have been researched and developed. These include ELISA (Chen et al., 1992; Chen & Chang, 1995; Kumar et al., 2011), DNA hybridisation (Lee et al., 1992; McCarthy et al., 2000), PCR (Bej et al., 1999; Lee & Pan, 1993; Lee et al., 1995; Karunasagar et al., 1996; Wang et al., 1997; Ward & Bej, 2006) and fluorogenic assays (Miyamoto et al., 1990; Venkateswaran et al., 1996). Although all these techniques are rapid, they are of varying usefulness. PCR would probably be the best, with cost being the main drawback. It is an extremely sensitive method, being able to detect as few as four cells per millilitre (Wang et al., 1997) or ten cells in a fish sample of V. parahaemolyticus (Karunasagar et al., 1996) and offers tremendous time savings (Figure 4). Due to the very nature of PCR, it has an extremely high specificity, provided primers to species-specific proteins are used. Selection of such target proteins should not be a difficult task, with some options being the conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC), investigated by Wong and Lin (2001). Many other methods have low specificity, due to cross reaction with other closely related species (Chen et al., 1992; Chen & Chang, 1995; Venkateswaran et al., 1996) or are potentially unsafe due to radioactivity (Lee et al., 1992). These techniques are generally not as sensitive as PCR, although Miyamoto et al. (1990) found their fluorogenic assay to be very sensitive, as it was able to detect ten cells per gram. Although PCR is probably the best method for isolating V. parahaemolyticus, its universal and routine usage (and incorporation into The Australian Standards) is unlikely in the very near future due to the expense involved in purchasing a thermocycler.

Figure 4: Flowchart for culture-based confirmation of Vibrio parahaemolyticus compared with PCR.

The enrichment step is an important part of the isolation process, and aims to ensure all viable cells present, do in fact grow on the isolation medium. Some may be injured or otherwise non-culturable but still potentially viable under ideal conditions. It is these cells which the enrichment process attempts to recover, or revive. The traditional and standard method of enrichment of V. parahaemolyticus is with alkaline peptone water (APW). This medium is listed in Donovan et al. (1995) alongside others considered suitable for use as enrichment, such as salt polymyxin broth (SPB) or glucose salt teepol broth (GSTB). Researchers who have looked at the various enrichment broths have found APW to give the best results. Eyles et al. (1985) found that APW was 111% more effective than GSTB while Hagen et al. (1994) investigated APW and SPB, and found APW to be the superior enrichment medium. The benefits afforded by APW are reflected by its inclusion in The Australian Standard method.

References

Atlas, R. (1993). Handbook of Microbiological Media. Boca Raton, FL, United States: CRC.

AWWARF/AWWA Research Foundation. (1997). Drinking Water Inspectorate Fact Sheet–Vibrio. [Online]. URL: http://www.awwarf.com/newprojects/pathogens/vibrio.html [Accessed 16 October 1998].

Baron, E., Peterson, L. & Finegold, S. (1994). Bailey & Scott’s Diagnostic Microbiology. (9th ed.), Sydney: Mosby.

Bej, A. K., Patterson, D. P., Brasher, C. W., Vickery, M. C., Jones, D. D., & Kaysner, C. A. (1999). Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. Journal of microbiological methods, 36(3), 215-225.

Brady, J. & Holum, J. (1993). Chemistry: The Study of Matter and its Changes. Brisbane, QLD, Australia: Wiley.

Chen, C. H., & Chang, T. C. (1995). An enzyme-linked immunosorbent assay for the rapid detection of Vibrio parahaemolyticus. Journal of food protection, 58(8), 873-878.

Chen, D., Hanna, P. J., Altmann, K., Smith, A., Moon, P., & Hammond, L. S. (1992). Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish. Appl. Environ. Microbiol., 58(11), 3694-3700.

Donovan, T. J., & Van Netten, P. (1995). Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples. International journal of food microbiology, 26(1), 77-91.

Eyles, M. J., Davey, G. R., Arnold, G., & Wane, H. M. (1985). Evaluation of methods for enumeration and identification of Vibrio parahaemolyticus in oysters. Food Technology in Australia 37 : 302-304.

FAO/WHO [Food and Agriculture Organization of the United Nations/World Health Organization]. (2016). Selection and application of methods for the detection and enumeration of human-pathogenic halophilic Vibrio spp. in seafood: Guidance. Microbiological Risk Assessment Series No. 22. 74p

HAGEN’, C. J., Sloan, E. M., Lancette, G. A., Peeler, J. T., & Sofos, J. N. (1994). Enumeration of Vibrio parahaemolyticus and Vibrio vulnificus in various seafoods with two enrichment broths. Journal of food protection, 57(5), 403-409.

ICMSF/International Commission on Microbiological Specifications for Foods. (1996). Microorganisms in foods 5: Characteristics of microbial pathogens (Vol. 5). Springer Science & Business Media.

Karunasagar, I., Sugumar, G., Karunasagar, I., & Reilly, P. J. A. (1996). Rapid polymerase chain reaction method for detection of Kanagawa positive Vibrio parahaemolyticus in seafoods. International journal of food microbiology, 31(1-3), 317-323.

Kumar, B. K., Raghunath, P., Devegowda, D., Deekshit, V. K., Venugopal, M. N., Karunasagar, I., & Karunasagar, I. (2011). Development of monoclonal antibody based sandwich ELISA for the rapid detection of pathogenic Vibrio parahaemolyticus in seafood. International journal of food microbiology, 145(1), 244-249.

Lee, C., Chen, L. H., Liu, M. L., & Su, Y. C. (1992). Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters. Appl. Environ. Microbiol., 58(10), 3419-3422.

Lee, C., & Pan, S. F. (1993). Rapid and specific detection of the thermostable direct haemolysin gene in Vibrio parahaemolyticus by the polymerase chain reaction. Microbiology, 139(12), 3225-3231.

Lee, C. Y., Pan, S. F., & Chen, C. H. (1995). Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR. Appl. Environ. Microbiol., 61(4), 1311-1317.

McCARTHY, S. A., DePaola, A., Kaysner, C. A., Hill, W. E., & Cook, D. W. (2000). Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of Vibrio parahaemolyticus. Journal of food protection, 63(12), 1660-1664.

Miyamoto, T., Miwa, H., & Hatano, S. (1990). Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods. Appl. Environ. Microbiol., 56(5), 1480-1484.

Venkateswaran, K., Kurusu, T., Satake, M., & Shinoda, S. (1996). Comparison of a fluorogenic assay with a conventional method for rapid detection of Vibrio parahaemolyticus in seafoods. Appl. Environ. Microbiol., 62(9), 3516-3520.

Ward, L. N., & Bej, A. K. (2006). Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes. Appl. Environ. Microbiol., 72(3), 2031-2042.

Wang, R. F., Cao, W. W., & Cerniglia, C. E. (1997). A universal protocol for PCR detection of 13 species of foodborne pathogens in foods. Journal of applied microbiology, 83(6), 727-736.

Wong, H. C., & Lin, C. H. (2001). Evaluation of typing of Vibrio parahaemolyticus by three PCR methods using specific primers. Journal of clinical microbiology, 39(12), 4233-4240.