Course:EOSC 475/ResearchProject/TheIdealIndicator
IN SEARCH OF THE IDEAL VIBRIO VULNIFICUS INDICATOR
By María Alejandra Molina Letelier
Figure 1 and 2. Images retrieved from http://article.wn.com/view/2013/10/01/Vibrio_vulnificus_precautions_for_Flagler_and_Volusia_Counti/ and http://acceleratingscience.com/food/fact-sheet-on-vibrio/
Background and Introduction
Vibrio vulnificus is a bacterium that occurs naturally in warm coastal areas, such as the Gulf of Mexico (FDA, 2015). It comprises two biotypes, biotypes 1 and 2, with biotype 1 strains being pathogenic to humans (Cerda-Cuellar et al., 2000). It is found at higher concentrations in summer as the water becomes warmer. Since the 1980s, Vibrio infections have resulted in severe morbidity and high mortality being the area of focus for many public health investigators. A study performed in the State of Florida alone reported a total of 690 Vibrio infections in 675 persons during 1981-1993 (4.3 patients/1,000,000 population/year) (Hlady and Klontz, 1996). The same study concluded that most infections resulted in gastroenteritis (51%), wound infections (24%) or primary septicemia (17%) with case fatality rates of 1% for gastroenteritis, 5% for wound infections and 44% for primary septicemia. In terms of risk factors, Vibrio strains are remarkably virulent in people with pre-existing conditions such as liver disease, alcoholism, diabetes mellitus, gastrointestinal surgery and immune disorders (Hlady and Klontz, 1996). Another risk factor is seafood consumption, with oysters being the most common seafood associated with infections. It is important to mention that Vibrio vulnificus bacteria is not the result of pollution (FDA, 2015). Therefore, eating raw or undercooked shellfish like oysters from or swimming in “cleaner” waters does not reduce the risk of infection. Hlady and Klontz (1996) suggest the urgent need for occupational, educational and marketing interventions aimed at preventing Vibrio infections.
It is clear that people using the marine environment are in high risk of Vibrio infections, therefore it is important to develop a detection method that is both sensitive and specific. In their paper, Griffin et al. (2001) state that over 50 years, public health agencies have been relying on microbiological analysis of water samples and have defined a water quality indicator based on the presence or absence of bacteria in a given body of water. The microbes are counted through a media based assay, in which certain nutrients and chemicals are used to identify colony forming units (CFU) of bacteria in the water sample (Griffin et al., 2001). Griffin et al. (2001) describe the use of Fecal Coliform (FC) bacteria as indicator for pathogenic Vibrio species, given FC ability to grow at elevated temperatures. Other methods described include the use of molecular techniques such as antibiotic resistance and also the use of bacteriophages, viruses that infect specific bacteria strains, as an alternative to FC. Some problems, however, regarding the described methodology include the lack of correlation between pathogenic Vibrio presence and density with that of fecal indicators, the lack of nationwide consensus among public health officials and researchers regarding the indicator to be used and the presence of false positive results in media based assays (Griffin et al., 2001). Given the limitations of current microbiological indicators, Griffin et al. (2001), propose a re-evaluation of the system and emphasis for baseline monitoring and assessment for several types of indicators and pathogens in conjunction with abiotic variables. The long term goal is integration of the data to develop statistical and dynamic models, that along with real-time measurements, will predict when high levels of a particular organism may be present; thus, preventing human exposure to a variety of harmful agents.
Figure 3. Image retrieved from http://noticiasrepublicadominicana.blogspot.ca/2014_08_07_archive.html
In this document, I will describe the development of Vibrio vulnificus molecular techniques as indicators since 1980 to actuality. The paper of focus and analysis is "A Selective Medium and a Specific Probe for Detection of Vibrio vulnificus " by Cerda-Cuellar et al., 2000. The paper has been chosen given that it marks a middle point in the development of molecular techniques as Vibrio indicators to what is currently used today.
Cerda-Cuellar et al., 2000 developed a selective medium (VVM) and a specific 16S rRNA gene (rDNA) probe (V3VV) for detection of Vibrio vulnificus. The medium contains D-(+)-cellobiose as the main carbon source and electrolytes (MgCl2-6H2O and KCl) that stimulate bacterial growth. VVM is also composed of Polymyxin B, colistin, which is an antibiotic, and moderate alkalinity and salinity which provides selectivity properties. VVM is specific to Vibrio vulnificus given that other Vibrio species do not grow in the medium or show unwanted coloration. V. vulnificus grows on VVM as flat bright yellow colonies, with a yellow diffusion halo, as a result of fermentation of the D-(+)-cellobiose. Higher V. vulnificus colony count result on VVM agar than on cellobiose-colistin agar or on modified cellobiose-polymyxin B-colistin agar with 89% mean platting efficiency in VVM; 37% and 67% in mCPC and CC, respectively (Cerda-Cuellar et al., 2000). Cerda-Cuellar et al. (2000) evaluated the specificity of the probe by colony hybridization and dot blot hybridization with PCR amplified 16S rDNA from a collection of strain and environmental isolates. V. vulnificus was the only strain that showed positive hybridization with the oligonucleotide.
Relationship to Literature
Since some pathogenic Vibrio strains are present in the environment with bacteria, isolation of V. vulnificus is usually performed by enrichment in alkaline peptone water (APW) or in APW supplemented with polymyxin B. However, the enrichment also causes bacteria to grow; thus, further inoculation of the sample in selective media like V. vulnificus agar, sodium dodecyl sulfate (SDS)-polymyxin B-sucrose (SPS) agar, cellobiose-polymyxin B-colistin (CPC) agar, V. vulnificus enumeration agar, modified CPC (mCPC) agar, cellobiose colistin (CC) agar or thiosulfate-citrate-bile salts-sucrose (TCBS) is required. A probe is then used to identify the presumptive V. vulnificus recovered (Cerda-Cuellar et al., 2000).
In the 1980, the common method for isolation of Vibrio vulnificus used thiosulfate-citrate-bile salts-sucrose (TCBS) agar media. However, this technique was criticized for not not resulting in adequate recovery and growth of the species compared to a reference medium (Brayton et al., 1983). Brayton et al. (1983) developed a new plating medium (VV agar) as an alternative to thiosulfate-citrate-bile salts-sucrose (TCBS) agar for the isolation of Vibrio vulnificus in response to the critics. V. vulnificus appears in VV agar as large grey colonies with black centers; while other non-vibrio strains, produce smaller colonies. The recovery and growth of V. vulnificus proved superior in VV agar compared to TCBS agar (Brayton et al., 1983). In 1987, another method was developed, which seemed promising for direct enumeration of Vibrio vulnificus from shellfish and other environmental sources (Bryant et al., 1987). The technique was based on the alkylsulfatase activity of the species using sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS). The mean platting efficiency for V. vulnificus strains resulted 39% on SPS (Bryant et al., 1987).
Figure 4. Vibrio cholerae and Vibrio parahaemolyticus in TCBS Agar media
A procedure that facilitated the enumeration and identification of V. vulnificus in molluscan oyters was developed in 1993. The then new method consisted on direct platting medium (VVM medium) for isolating the organisms followed by biochemical test for identifying the isolates (Miceli et al., 1993). In 2004, an new protocol aimed at more specific serovar E isolation of Vibrio vulnificus was developed. The key element of this new protocol was the new broth containing eel serum as the nutritive and selective component (Sanjuán and Amaro, 2004).
Problems and gaps
In the 1980s, the techniques for isolation and identification of Vibrio vlunificus were not that specific, only capable of isolating Vibrio genus organisms from non-vibrio species. A lot of research has been devoted since then, to develop Vibrio vulnificus specific isolation and identification techniques. It was in the year 2000 that Cerda-Cuellar et al. 2000 developed the method which showed that VVM agar in conjunction with the V3VV probe is a useful way to identify Vibrio vulnificus among a mixed bacterial suspension and spiked mussel samples. The new method was a revolution at that time yet in 2007, Warner and Oliver published a paper were they criticized the selectivity of the Cerda-Cuellar proposed methodology. V. vulnificus can be divided into allelic variants, E-type and C-type, based in virulence correlated gene (vcg) (Warner and Oliver, 2007). They stated that Cerda-Cuellar methodology is C-genotype selective and proposed a modified formulation. The new formulation (CPC +) consisted of modified total salt concentration of CPC agar, added Mg+ and K+ and adjusted antibiotic concentrations (Warner and Oliver, 2007). CPC + showed no selectivity to either C/E genotype. The C-Genotype selective advantage when cells are taken from nutrient-rich environment (like that found in oysters) to nutrient poor environemnt (seawater), and then enriched in APW and plated to selective media was removed in CPC+ plates (Warner and Oliver, 2007).
Importance and Impact of Paper in the Field
Sanjuán and Amaro (2004) used the method developed by Cerda-Cuellar et al., (2000) to develop a method of their own with improved selectivity. Research following the Cerda-Cuellar et al. (2000) paper, used the positive results of previous papers and identified what ultimately lead to the improvement of Vibrio vulnificus selectivity methods. Given the threat that Vibrio vulnificus poses to public health, this area is currently seeking improvement and increase efficiency of results. The combined use of a selective medium and a specific probe provided a feasible method for the detection and identification of V. vulnificus for epidemiological and ecological studies in 2001 (Cerda-Cuellar et al. 2001). More contemporary research is focused on integrating abiotic parameters for better understanding of the Vibrio community. Froelich et al. (2014) created the best fitting linear regression models that uses a combination of salinity and water temperature in water and oysters. These models are used to generate matrix tools that provide easy to interpret visual references about the potential concentrations of V. vulnificus in North Carolina Oysters.
The paper, "A Selective Medium and a Specific Probe for Detection of Vibrio vulnificus " by Cerda-Cuellar et al. ( 2000), constitutes a piece in the great puzzle of identifying the best technique as indicator of V. vulnificus. It has opened the doors to totally new areas of research. As was mentioned in EOSC 475 course, molecular techniques such as Denaturing Gradient Gel Electrophoresis (DGGE) and Terminal Restriction Fragment Length Polymorphism (TRFLP) have shown more accurate in the identification of microbial organisms. DGGE, which differentiates DNA fragments based on different melting temperatures, followed by sequence, has proven to be an effective way to identify microbiological species. TRFLP, which uses restriction enzymes to produce fluorescently labeled DNA fragments and analysis of product based on frequency of fragment size, provides accurate quantification of microorganisms abundance. Additionally, the next generation sequencing of metagenomics, currently under development, provides new insights on the total genetic diversity of the microbiological world.
Figure 5. Introducing metagenomics
Retrieved from http://www.scigenom.com/metagenomics
Bibliography
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Bryant et al. 1987. Use of Sodium Dodecyl Sulfate-Polymyxin B-Sucrose Medium for Isolation of Vibrio vulnificus from Shellfish. American Society of Microbiology. 52: 1556-1559.
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U.S. Food and Drug Administration. 2015. Vibrio vulnificus Health Education Kit Fact Sheet. Retrieved from http://www.fda.gov/Food/ResourcesForYou/HealthEducators/ucm085365.htm
Warner, E, Oliver, J. 2007. Refined Medium for Direct Isolation of Vibrio vulnificus from Oster Tissue and Seawater. Appl. Environ. Microbiol. 73: 3098-3100.