Letters in Applied Microbiology ISSN 0266-8254

Suspension of oysters reduces the populations of Vibrio parahaemolyticus and Vibrio vulnificus K.M. Cole1, J. Supan2, A. Ramirez1 and C.N. Johnson1 1 Department of Environmental Sciences, Louisiana State University, Baton Rouge, LA, USA 2 Louisiana Sea Grant College Program, Louisiana State University, Baton Rouge, LA, USA

Significance and Impact of the Study: This study found that oyster suspension significantly reduced some populations of potentially pathogenic vibrios. These results indicate that oyster suspension could be a viable approach for preharvest treatment to reduce illness in consumers of raw oysters.

Keywords benthic, Crassostrea virginica, oyster, suspension, Vibrio. Correspondence Crystal N. Johnson, Department of Environmental Sciences, Louisiana State University, Mailroom #1273, Energy, Coast and Environment Building, Baton Rouge, LA 70803, USA. E-mail: [email protected] 2015/0526: received 12 March 2015, revised 14 May 2015 and accepted 17 May 2015 doi:10.1111/lam.12449

Abstract Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) are associated with the consumption of raw oysters and cause illnesses ranging from simple gastroenteritis to life-threatening septicaemia. These halophilic bacteria are frequently found in marine and estuarine systems, accumulating within the tissues of a number of aquatic organisms and passing on to humans after consumption, through contaminated water, or via open wounds. As benthic organisms capable of filtering 40 gallons of water per hour, sediment is an important source of potentially pathogenic vibrios in oysters destined for raw consumption. This research used off-bottom oyster culture to reduce vibrio concentrations in oysters. Colony hybridization was used to enumerate Vp and Vv in bottom and suspended oysters. Vv and Vp concentrations were generally lower in oysters suspended off-bottom, and suspension decreased vibrio loads in oysters by an average of 13%. Suspension of oysters reduced vibrio concentrations.

Introduction Vibrio spp. are naturally occurring Gram-negative halophilic bacteria in aquatic environments (Okada et al. 2005; Su and Liu 2007; Teplitski et al. 2009; Lewis et al. 2010; Noriea et al. 2010; Johnson et al. 2012; Froelich and Oliver 2013; Johnson 2013). Most Vibrio spp. are nonpathogenic and can be beneficial to the environment by contributing to the carbon cycle and nutrient recycling (Pruzzo et al. 2008; Souza et al. 2011; Johnson 2013). However, pathogenic strains are of particular concern for the oyster industry, raw shellfish consumers, and immunocompromised individuals and can cause gastrointestinal symptoms and possible death (Mclaughlin et al. 2005; Bross et al. 2007; Jones et al. 2014; Young et al. 2015). Vibrio parahaemolyticus and Vibrio vulnificus are of concern for the majority of seafood-consuming countries worldwide (Morris 2003; Martinez-Urtaza et al. 2010). Vibrio parahaemolyticus is the leading cause of vibrio-

associated food-borne gastroenteritis in the United States, mainly due to raw shellfish consumption; wound infection and septicaemia have also been reported with less frequency (Yeung and Boor 2004; Givens et al. 2014). Vibrio vulnificus causes severe wound infections that are usually the result of exposing wounds to waters associated with vibrios (Jones and Oliver 2009). Oysters feed on phytoplankton and organic matter suspended in the water column by filter feeding. Particles and bacteria are taken in from the surrounding water column by cilia (Jones et al. 2001; Teplitski et al. 2009), accumulate internally, and may become highly concentrated in their digestive glands (Teplitski et al. 2009). The eastern oyster, Crassostrea virginica, can pump water through their gills at a rate of 40 gallons per hour, although this rate is decreased under turbid conditions (Jones et al. 2001; Froelich et al. 2013). As vibrio populations are very high in sediment (up to >83 000 CFU g 1 sediment for total V. parahaemolyticus and V. vulnificus,

Letters in Applied Microbiology 61, 209--213 © 2015 The Society for Applied Microbiology

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Vibrio reduction in oysters

K.M. Cole et al.

respectively), with a higher detection frequency in sediment than in the overlying water column (89
7% of sediment samples contained tlh vs 69
5% of water samples) (Johnson et al. 2012), it is inferred that most vibrios in an ecosystem are located in the sediment. Thus, oysters residing on sediment surfaces would be exposed to vibrios at higher rates than would those suspended off-bottom closer to the surface of the water column. Although widely used globally, there has been little research carried out on the effectiveness of suspended aquaculture systems in reducing pathogenic vibrios in shellfish. Therefore, we hypothesized that the off-bottom suspension of oysters would decrease their vibrio concentrations because of the intensity of sediment as a vibrio source. Results and discussion Mean tlh concentrations in bottom water samples were significantly higher than mean tlh concentrations in surface water samples (P = 0
0041, Table 1). The tdh, trh and vvh concentrations in bottom water samples were consistently but not significantly higher than corresponding tdh, trh and vvh concentrations in surface water samples. There were surprisingly no statistically significant differences between the tlh, tdh, trh or vvh concentrations in suspended oysters and bottom oysters based on t-test results. Off-bottom suspended oysters carried vibrio concentrations that were lower than on-bottom oysters by an average of 13%. Specifically, suspended oysters carried vvh, tdh and trh concentrations that were lower by 3%, 41% and 57%, and there was a surprising increase in tlh concentrations in oysters of 48% associated with off-bottom suspension (Table 1). Overall, when surface-bottom pairs were compared, vibrio concentrations in bottom samples were consistently higher than those in surface samples (P = 0
0636), with the exception of tlh in oysters; tlh concentrations in suspended oysters were actually higher than those in bottom oysters in 67% of the samples in this study. Thus, most vibrio concentrations appeared to be lower closer to bottoms than at surfaces, and this appeared to be the case in both the water column and oysters. Vibrio concentrations in sediment samples for tlh, tdh, trh and vvh were 7790, 85, 241 and 114 CFU g 1 respectively. Water depth averaged 35
3″ (range of 23–49″) and was significantly correlated with concentrations of tdh in bottom oysters (mean of 87
9 CFU g 1) and vvh in suspended oysters (mean of 676 CFU g 1) (Table 1). Surface water samples did not appear to be a significant factor in predicting vibrio concentrations or the effectiveness of suspension in reducing vibrio concentrations (Table 1). Not surprisingly, both salinity and temperature were significantly correlated with multiple populations in multiple 210

sample types. In addition, vibrio concentrations in sediment samples were significantly correlated with those in overlying water and oyster samples. Increased vibrio concentrations in sediment were associated with increased vibrio concentrations in water and oysters. This study represents preliminary findings that support further studies examining the effectiveness of off-bottom culture as a means of decreasing vibrio concentrations in oysters. Specifically, although our study demonstrated increases in tlh concentrations as a result of off-bottom suspension, it also demonstrated decreases in potentially pathogenic populations of tdh and trh. It is not clear why this was the case. It is possible that although there was an increase in the total population of V. parahaemolyticus, off-bottom suspension may have selected against potentially pathogenic strains in the study site. It is well-documented that the percentage of total V. parahaemolyticus carrying the tdh and trh pathogenicity factors is not consistent (Zimmerman et al. 2007). It is also possible that fouling of the suspended cages over the length of the experiment and a well-mixed estuary, often visibly turbid with noticeable sediment suspension, may have led to such anomalies, although the tlh concentrations in water do not fully explain this. Future studies will address this phenomenon. While there was no surprise that salinity and temperature significantly correlated with certain vibrio subpopulations in certain sample types, it was very interesting that sample site depth was positively correlated with tdh in on-bottom oysters and with vvh in off-bottom water samples. The reasoning for the former is unclear, but it is possible that there is a sunlight role. It has been demonstrated that zooplankton carry vibrios and feed on phytoplankton (Kaneko and Colwell 1975; Tamplin et al. 1990; Rivera et al. 2013; Lewandowska et al. 2014). If phytoplankton concentrate in surface waters, at least during the day, then it is possible that vibrio-colonized zooplankton also concentrate in surface waters, leaving one less source of vibrios in the benthic areas, particularly given the high turbidity levels in coastal Louisiana, which have been previously demonstrated to be 37–295 mg l 1 suspended particulate matter (Johnson et al. 2012). It is also possible that the wind or tidal fluctuations that bring in the sea water also bring in tdh+ V. parahaemolyticus. The cause of the correlation between water depth and V. vulnificus in off-bottom water samples is even less clear. Our group has previously demonstrated that the correlations between turbidity and V. vulnificus in oysters are different from those between turbidity and V. parahaemolyticus in oysters (Johnson et al. 2012). Thus, the impacts of turbidity, sediment resuspension, water depth, wind, and tidal fluctuations on V. vulnificus concentrations are likely different from their impacts on V. parahaemolyticus

Letters in Applied Microbiology 61, 209--213 © 2015 The Society for Applied Microbiology

Letters in Applied Microbiology 61, 209--213 © 2015 The Society for Applied Microbiology

35
3″ 23
7°C 20
5 g l 1 9
6 CFU ml 1 16
8 CFU ml 1 970 CFU g 1 654 CFU g 1 7790 CFU g 1 1
0 CFU ml 1 1
2 CFU ml 1 52 CFU g 1 87
9 CFU g 1 85 CFU g 1 1
8 CFU ml 1 2
5 CFU ml 1 124 CFU g 1 290 CFU g 1 241 CFU g 1 2
1 CFU ml 1 2
9 CFU ml 1 676 CFU g 1 695 CFU g 1 114 CFU g 1

(23–49, 7
3) (16
3–31
5, 5
3) (8
4–31
3, 6
4) (0–26, 8
7) (1–46, 13
1) (68
2–5450, 1396) (63
6–4900, 1153) (2050–22 900, 6469) (0–5
5, 1
6) (0–5
5, 1
9) (0–346, 87
9) (0–727, 189) (0–380, 109) (0–11
5, 3
1) (0–9
0, 2
9) (0–659, 169) (0–1641, 475) (0–1380, 378) (0–6
5, 1
8) (0–9
0, 2
8) (114–2350, 632) (50–2159, 631) (0–550, 146) R = 0
28 R2 = 0
27

2

tlh wat bot

R = 0
34

2

tlh oys bot

R2 = 0
33

R2 = 0
44

tlh sed

R2 = 0
22

tdh oys sur R2 = 0
34

tdh oys bot

R2 = 0
3

R = 0
23

2

tdh sed

R2 = 0
33

R2 = 0
30

trh wat sur

R2 = 0
32

trh oys bot

R2 = 0
39

R2 = 0
70

R2 = 0
25

trh sed R2 = 0
29 R2 = 0
30

vvh wat sur

R2 = 0
60

R2 = 0
48

R2 = 0
28

vvh oys sur

R2 = 0
42

R2 = 0
38

R2 = 0
48

vvh oys bot

Depth, depth of water at sample collection; temp, water temperature; stdev, standard deviation; tlh, thermolabile hemolysin; tdh, thermostable direct hemolysin; trh, tdh-related hemolysin; vvh, Vibrio vulnificus hemolysin; wat, water; oys, oysters; sed, sediment; CFU, colony-forming units; sur, samples collected from sample site surface; bot, samples collected from sample site bottom. All parameters measured are represented in rows, but only those with statistically significant correlations are represented in columns. *Concentrations of tlh in bottom water samples were significantly higher than those in surface water samples (P = 0
0041). †Overall, vibrio concentrations in bottom samples were consistently higher than those in surface samples (P = 0
0636), with the exception of tlh in oysters.

Depth Temp Salinity tlh wat sur*,† tlh wat bot*,† tlh oys sur tlh oys bot tlh sed tdh wat sur† tdh wat bot† tdh oys sur† tdh oys bot† tdh sed trh wat sur† trh wat bot† trh oys sur† trh oys bot† trh sed vvh wat sur† vvh wat bot† vvh oys sur† vvh oys bot† vvh sed

Mean (range, SD)

Table 1 Mean, range and standard deviation of measured parameters, and coefficients of determination (R2) among measured parameters

K.M. Cole et al. Vibrio reduction in oysters

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Vibrio reduction in oysters

K.M. Cole et al.

concentrations. Unfortunately, water depth at the time of sample collection is merely a snapshot of a very complex ecosystem, and more detailed studies would be required to fully understand the interactions. Overall, the ubiquitous and persistent nature of potentially pathogenic Vibrio spp. has proven difficult when attempting to eliminate them from oysters intended for raw consumption. Although many techniques have been employed, vibrio infections remain an issue for the oyster industry and a concern for consumers. Suspension culture of oysters offers another approach for bacterial reduction by growing oysters off-bottom to avoid contamination via sediment; however, while intuitive, more research is needed to determine the long-term effectiveness and sustainability of these systems. Further studies are needed, specifically related to pathogenic vibrio populations in C. virginica and to allow for a better understanding of the dynamics of their relationships. Materials and methods In January 2012, 300 eastern oysters (C. virginica from Collins Oyster Company, Golden Meadow, LA) were distributed in floating cages (OysterGro, Bouctouche, New Brunswick) that floated on the surface of the water while 300 were placed in identical on-bottom cages following a 1-month acclimation period. The Louisiana Sea Grant Oyster Research and Demonstration Farm in Grand Isle, LA (N29°14’358″W90°00’208″) served as the sampling site for this study. The depth in this location varies seasonally, averaging 5 m with lower depths occurring in the winter due to dominant northerly winds following frontal passages. Samples of 12–25 individual oysters were collected once monthly for a total of 18 months from February 2012 to August 2013. During each sampling, environmental parameters were measured, and water, oyster, and sediment samples were transported on ice from Grand Isle, LA to Louisiana State University in Baton Rouge, LA for processing within 3 h. Temperature and salinity were measured using a YSI probe (Yellow Springs Instruments, Yellow Springs, OH). Water was shaken vigorously as previously described (Andrews and Hammack 2003); oysters were scrubbed, shucked and homogenized; and pore water was decanted from sediment, which was then diluted 1 : 1 and shaken (Johnson et al. 2010). Concentrations of thermolabile hemolysin indicating total V. parahaemolyticus (tlh), thermostable direct hemolysin indicating pathogenic V. parahaemolyticus (tdh), tdhrelated hemolysin indicating pathogenic V. parahaemolyticus (trh), and V. vulnificus hemolysin indicating total V. vulnificus (vvh) in sediment, surface water, bottom water, surface oysters, and bottom oysters were determined by direct plating/colony hybridization as described 212

previously (Johnson et al. 2012). Subsamples including 1 ml water, 0
01 and 0
1 g oyster homogenate, and 0
01 and 0
1 g sediment were spread onto T1N3 agar (1% tryptone, 3% NaCl, 2% agar, pH 7
2) and incubated at 33°C for 16–18 h. All colonies on all plates were lifted, treated with proteinase K and other buffers, and probed with alkaline phosphatase-conjugated oligonucleotide probes specific for tlh, tdh, trh, and vvh as described previously (Nordstrom et al. 2006). Positive purple markers were counted up to an upper limit of 250 colony forming units per 85-mm filter. Statistical analysis methods including t-tests and analysis of variance were carried out on log10-transformed data using MATLAB (Matlab and Statistics Toolbox Release 2012a; The MathWorks, Inc., Natick, MA). Acknowledgements This manuscript was written in partial fulfilment of the requirements of a master’s degree for Krystal Cole from the Louisiana State University Department of Environmental Sciences in Baton Rouge, LA. This review and portions of research presented were made possible by funding from the Louisiana Sea Grant program, award number R/OVV-02. The authors thank the reviewers for their thorough and detailed review of the research described in this manuscript. Conflict of Interest The authors declare no conflict of interest associated with this research.

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