Differential Response of Vibrio vulnificus Isolates from South Texas Waters to Oxidative Stress

* Individuals contributed equally and should be considered as joint first authors.

‡ Corresponding author and primary advisor

ABSTRACT

Vibrio vulnificus, a common human pathogen, is autochthonous to warm estuarine and coastal waters where it can undergo different kinds of environmental stresses. Very little is known about how environmental isolates from Texas waters cope with extreme conditions; our objective was to study the response of these isolates under oxidative stress. Four environmental isolates were individually exposed to 880 μM and 2000 μM of H2O2, and their growth measured by spectrophotometry at an absorbance of 600 nm, and compared to untreated cells. Two of the isolates, PR1 and RP4, had a significant decrease in growth of one order of magnitude at the stationary phase at both concentrations of H2O2. Two other isolates, CP1 and MI4, revealed a decrease in growth during log phase, but ultimately recovered to show normal growth in stationary phase at 880 μM of H2O2. Both isolates demonstrated a prolonged log phase at 2000 μM of H2O2, and an overall decrease of ~20% at the start of stationary phase. These results suggest that resistance to H2O2 may be determined by factors other than the environment, for which future studies would involve a gene expression focus.

Introduction

The capacity of an organism to respond to its environment is perhaps the most important factor contributing to its potential viability. Coastal south Texas waters are perennially warm and with slightly saline conditions favorable for growth of Vibrio vulnificus. This organism, a gram-negative, asporogenous curved bacillus, is autochthonous to these surroundings (Holt et al. 1994). This species is grouped into three biotypes based on pathogenesis. Biotype 1 is principally responsible for being harmful to humans, while biotype 2 causes disease in both humans and eels. A third biotype may cause bacteremia and wound infections (Maugeri et al. 2006). Exposure to Vibrio vulnificus in seawater may cause severe infections of open wounds, while the consumption of uncooked shellfish has been shown to cause primary septicemia (Lin and Schwarz 2003; Maugeri et al. 2006).

V. vulnificus is most persistent in water temperatures exceeding 20°C and salinities ranging from 5 to 25 ppt (Lin and Schwarz 2003). However, upon entering a more variable or demanding environment, Vibrio vulnificus may enter what is known as a viable but nonculturable (VBNC) state, in which the bacteria no longer exhibit typical growth in the laboratory but may still maintain characteristics, such as virulence, of functioning bacteria (Desnues et al. 2003). By doing so, Vibrio vulnificus ensures its potential for survival in hostile conditions. However, not all environmental stresses induce entry into the VBNC state.

Bacteria may undergo oxidative stress when the action of UV radiation on water produces reactive oxygen species (ROS), including the superoxide radical (•O21-), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH) (Yildiz and Schoolnik 1998; Friedberg et al. 2006). Since the bays and estuaries in south Texas are continuously exposed to sunlight, it is expected that bacteria in these environments will be under continuous stress from ROS. Hydrogen peroxide alone is relatively stable, but, in the presence of iron (Fenton reaction), H2O2 readily converts into the hydroxyl radical (•OH), which in turn is the chief instigator of DNA damage by producing an electrophilic attack on DNA bases, either by abstracting hydrogen atoms from bases, or by cleaving double bonds within nucleobases (Freidberg et al. 2006).

Bacterial responses to multiple stress events are controlled globally by one specific gene, which in turn mediates actions of disparate, unrelated groups of genes (Savageau 1983). These groups are termed "regulons." One such regulon involves the alternative sigma factor (σS ), which has been shown to induce or repress various genes or operons during periods of stress, such as starvation, osmotic stress, extremes in either temperature or pH, and oxidative stress in many bacteria, including Pseudomonas aeruginosa, Escherichia coli, Vibrio cholerae, and V. vulnificus (Yildiz and Schoolnik 1998; Hulsmann et al. 2003). A study done by Park et al. (2004) revealed that Vibrio vulnificus employs the RpoS regulon to persist in growth conditions, which contain a small concentration of H2O2. Consequently, one may surmise that Vibrio vulnificus maintains survivability under stress through a saltatory response that enables the bacteria to be resistant to a number of environmental conditions.

In this study, the behavior of Vibrio vulnificus environmental isolates from south Texas to environmental perturbations was examined by measuring bacterial growth in the presence of two different concentrations of H2O2. It is clear that several other studies have monitored the growth of Vibrio vulnificus under oxidative stress; however, those studies concentrated on the bacteria's gene expression under oxidative stress (Hulsmann et al. 2003; Park et al. 2004). Moreover, previous inquiries used environmental isolates from the Chesapeake Bay and North Korea (Hulsmann et al. 2003; Park et al. 2004). The south Texas environment is categorically different from these sites because bacteria are exposed to perennially higher temperatures and higher levels of UV radiation. The hypothesis was that Vibrio vulnificus environmental isolates from south Texas would show higher rates of survival when subjected to oxidative stress, as compared to V. vulnificus isolates originating from temperate climates. Furthermore, since it is not clear if, under similar conditions, separate isolates of Vibrio vulnificus respond differently to oxidative stress, investigating this could indicate a genetic role for resistance to oxidative stress, possibly due to differential RpoS expression. Additionally, analyzing the growth of Vibrio vulnificus under oxidative stress may help explain the patterns of growth under stress of the bacteria in their native environment.

Materials and Methods

Bacterial isolates and sites. Samples were collected from Corpus Christi Bay at Cole Park (CP1) and Ropes Park (RP4), the Gulf of Mexico at Mustang Island (MI4), and the Laguna Madre off Park Road 22 (PR1) between the months of September and October 2005, between 8-11 a.m. (Table 1). Fig. 1 and Fig. 2 represent the study sites.

Collection and preparation of environmental isolates. Sampling of isolates was conducted using sterile 1L polypropylene bottles at approximately 0.6 m deep and were maintained at a temperature of 1-4 °C. For each sample, bottles were capped and uncapped under water to ensure sterility; water temperature was measured using a thermometer (VWR) and salinity was measured using a Vista AE66ATC refractometer. Instrument calibration was performed according to National Institute of Standards and Technology criteria. Each sample was stored in an ice chest at 1-4 °C for no more than six hours before analysis in the lab.

Samples were withdrawn in graded volumes of 0.3 mL, 1.0 mL, 3.0 mL, and 10 mL, diluted in 10 mL of phosphate buffered saline (PBS) and passed through 0.45 µm membrane filters (VWR International). In order to detect Vibrio vulnificus, filtered samples were incubated on Vibrio vulnificus agar (VVA) plates. Vibrio vulnificus agar (VVA) is a selective complex medium that enables detection of that organism, as colonies of Vibrio vulnificus appear yellow (BAM, US FDA, 2004). Per liter, the medium contains 20 g peptone, 30 g NaCl, 0.06 g bromthymol blue and 10 g of cellobiose in 2.5% agar at pH 8.2 (BAM, US FDA, 2004). Yellow isolates (presumptive V. vulnificus) were then confirmed with a probe specific for the V. vulnificus gene, vvhA, by colony blot hybridization (BAM, 2004). Colonies that appeared purple on membranes were confirmed to be V. vulnificus. These confirmed colonies were subcultured onto Marine agar slants (final pH 7.6 ± 0.2). Marine Broth 2216 (Becton-Dickinson/Difco) is a complex broth base used for growth of marine bacteria. Per liter, the broth contains 5 g peptone, 1 g yeast extract, 0.1 g FeCl3, 19.45 g NaCl, 5.9 g MgCl2, 3.24 g MgSO4, 1.8 g CaCl2, 0.55 g KCl, 0.16 g NaHCO3, 80 mg KBr, 34 mg SrCl2, 22 mg H3BO3, 4 mg Na2SiO3, 2.4 g NaF, 1.6 mg (NH4)NO3 and 8 mg HNa2O4P, with 1.5 % agar added to make Marine agar. Seven mililiters of Marine agar solution was dispensed into each test tube (Difco Manual, 1984).

Overnight cultures were prepared in triplicate by inoculating 2.5 mL of Marine Broth in a 10 mL test tube with a well-isolated colony of bacteria grown on a Marine agar slant; the overnight cultures were incubated for 15-20 hours at 35-37 °C and agitated in a rotary shaker at 175 rpm. The Marine Broth was prepared by dissolving 37.4 g of Difco Marine Broth in 1L deionized water; the expected pH was 7.6 ± 0.2.

Growth of isolates under oxidative stress. 500 mL flasks containing 25 mL sterile marine broth were placed in a pre-warmed shaker for 20-30 minutes at 35-37 °C. Once the 500 mL flasks reached an equilibrium temperature, 250 µL of overnight culture were added. Immediately, 1 mL samples were taken from the 500 mL flasks and placed in 1 mL quartz micro cuvettes, with a 10 mm light path, for measurement of absorbance at 600 nm. Additionally, 250 mL flasks containing 25 mL sterile Marine broth were placed in the shaker concurrently.

The absorbance was recorded every thirty minutes according to this procedure until the O.D.600 nm was between 0.15 and 0.3. This O.D. range marks the transition between the lag and the exponential phases of growth, as described by Park et al (2004) and corroborated empirically using ATCC strains of V. vulnificus (27562, 33817) and V. parahaemolyticus (17802). Consequently, oxidative stress was not introduced to the cells until the log phase began. At this time, the warmed 250 mL flasks were removed from the shaker, and 3% H2O2 was added to the prewarmed media to obtain a concentration of either 1760 µM or 4000 µM. An equal volume of marine broth containing H2O2 was added to the cells in the 500 mL flask to achieve a final concentration of either 880 µM or 2000 μM of H2O2. Correspondingly, an equal amount of sterile marine broth was added to the control flasks. Aliquots of 1 mL were immediately taken from all flasks, and the absorbance was summarily recorded. Aliquots were taken at thirty minute intervals for five hours. If the O.D.600 nm was greater than 0.7, the samples were diluted 1:5 or 1:10 with sterile marine broth. The experiments were conducted in triplicate with independent samples. The results were graphed on Microsoft Excel using a semi-log scale, and the mean and standard error were calculated.

Results

For all environmental isolates grown in the absence of H2O2, growth curves showed characteristic lag, log, and stationary phases typical of Vibrio species. These phases were also seen with ATCC strains of V. vulnificus (27562, 33817) and V. parahaemolyticus (17802) grown in marine broth (data not shown). The mean temperature of the water of the sampling locations was 28.6 °C, with a standard deviation of 3.04 °C, while the mean salinity was 42.75 ppt with a standard deviation of 2.63 ppt. Of the environmental isolates tested in this experiment, PR1 and RP4 were shown to be significantly impaired by oxidative stress, as evidenced by nominal growth of these two samples in the presence of both concentrations of H2O2. For PR1, the experiments under stress exhibited a final 92% decrease in growth at 880 μM of H2O2 (see Figure 3) and a 91.7% decrease at 2000 μM of H2O2 (see Figure 4). Similarly, RP4 under these conditions had a final 94.3% decrease in growth at 880 μM of H2O2 (see Figure 5) and a 93.8% decrease at 2000 μM of H2O2 (see Figure 6), as compared to controls. Untreated controls showed growth curves similar to those seen for the V. vulnificus and V. parahaemolyticus when grown in marine broth (data not shown), and CP1 and MI4 showed stronger resistance to growth conditions at 880 μM H2O2 and a more noticeable decrease in growth at 2000 μM of H2O2. CP1 at 880 μM of H2O2 had a less than 5% decrease in growth between treated and untreated cultures (see Figure 7). Furthermore, at 2000 μM of H2O2, CP1 had a final 25.4% decrease (see Figure 8). MI4 behaved in a comparable manner, with a 4.13% decrease in growth at 880 μM of H2O2 (see Figure 9) and a 20.2% decrease at 2000 μM of H2O2 (see Figure 10).

Discussion and Conclusions

This study was focused on the phenotypic manifestations of stress in V. vulnificus environmental isolates. This is the first report done on isolates from south Texas exposed to oxidative stress throughout the entire log phase and focused on bacteria's phenotypic behavior. While other studies focused on gene expression in stationary phase, these studies differed from prior investigations in that isolates of V. vulnificus were exposed to media containing H2O2 at the beginning of log phase, which marks the period of the most rapid reproduction in bacteria, and, as a result, this period provided the best measurable contrast for growth under stress. In Vibrio vulnificus, the transition from lag to log phase has been established to occur at O.D.600 nm of 0.15-0.3 (Park et. al. 2004)

An analysis of the growth behavior of PR1 and RP4 versus CP1 and MI4 shows that with both PR1 and RP4, oxidative stress seemed to mark a decrease in overall growth at stationary phase. On the contrary, CP1 and MI4 under stress were characterized by a decreased log phase but a predominantly concurrent stationary phase at 880 μM of H2O2, and a prolonged log phase at 2000 μM of H2O2. These results suggest that the isolates were not dying due to lack of nutrients or co-factors normally seen by bacteria in the environment.

South Texas presents different environmental disturbances and tends to be comparatively warmer than other regions. Therefore, it may be postulated that the isolates used in this study would have similar stress responses. However, our results suggest differently since PR1 and RP4 behaved differently from the CP1 and MI4 isolates. A comparison of the ambient temperature and salinity values for these samples (Table 1) did not vary significantly enough to mark such a drastic change in behavior, as reflected by mean and standard deviation values. These experiments showed that bacteria collected from different areas but with no remarkable differences in temperature or salinity may give dramatic differences in growth under oxidative stress. Consequently, the resistance to oxidative stress seen in CP1 and MI4 may be attributed to more than surroundings. One possibility is that several biotypes of V. vulnificus exist in south Texas waters. An inquiry into the different biotypes represented by the isolates in this study could provide an explanation for the observed behavior. A second possibility is that alternate controls of RpoS, the stress response regulon of Vibrio vulnificus (Hülsmann et al., 2003) enable these bacteria to survive increased levels of exposure to UV radiation or to ROS. Park et al. (2004) found that V. vulnificus isolates were generally more sensitive to H2O2 than other enteric bacteria, but they did find that one isolate showed a RpoS-independent resistance to peroxide. While this work does not confirm that phenomenon, other factors such as environment may play a role. Because the temperature and salinity differences were slight among the environmental isolates, one may hypothesize instead that differential expression of the RpoS regulon is a contributor to the overall persistence under oxidative stress. A study of RpoS expression and other gene determinants may elucidate the behavioral differences among isolates. Moreover, as environmental conditions for each of the samples were comparable, a study of other intracellular factors such as enzyme production and co-factor interaction, and additional regulatory proteins acting over RpoS may provide insight into its variable expression.

In addition, the association between RpoS and virulence determinants has been noted for many vibrio species, including V. vulnificus (Hülsmann et al., 2003) and V. cholerae (Yildiz and Schoolnik, 1998). Such a suggestion lends itself to the possibility that differential gene expression of RpoS affects the virulence of the bacteria. Further virulence inquiries, in conjunction with RpoS expression studies, are important considerations for the understanding of the south Texas environmental isolates. Also, a phenotypic analysis of V. vulnificus in analogous conditions, such as the waters off the coast of Puerto Rico, may allow for a more comprehensive insight into genotypic studies.

Acknowledgements

This work was funded by a Summer Undergraduate Research Fellowship (National Science Foundation/Dept. of Defense #DBI0453329) to C.G.R., a Texas Excellent Funds grant (#34014) to Drs. Buck and Mott, and a Coastal Bend Bays and Estuaries Foundation grant (#0624) to Drs. Mott and Buck. We thank all of the members of the Environmental Microbiology Laboratory team led by Gabriel Ramirez for providing isolates and demonstrating proper collection techniques, as well as Ms. Amanda Smith and Mrs. LaDonna Henson for their assistance. We also thank Dr. José M. Planas from the University of Puerto Rico at Aguadilla, Dr. Jun Kelly Liu of Cornell University, and Amit Chowdhry for their editorial suggestions.

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