Goals of the program are to:

• involve participants in the direct, hands-on collection of research data using current technologies,
• include them in the collation and analysis of data samples; and,
• allow each participant to prepare oral and written presentations of observations, interpretations and conclusions regarding the data.

The following document types are represented:

• Published Abstracts
• Student Seminars/Colloquia
• Research Project Reports

PUBLISHED ABSTRACTS:

BACON, J.J., D.A. IVERSON and G.M. WORTHAM . 1993. Antibiotic sensitivity testing at hyperbaric conditions (1.62 ATA). Proc. S.D. Acad. Sci. 72:286.

The increased and extended uses of undersea habitats as laboratories, observatories, and living quarters will subject diving researchers to prolonged hyperbaric and environmental conditions reported to have significant influence on the distribution of the normal bacerial flora of the body. Condsideration must be given to the epidemiology, pathology, and chemotherapy of bacterial diseases augmented by an redistribution of this flora.

This study examined the sensitivity of Escherichia coli, Pseudomona aeruginosa, and Staphylococcus epidermidis to anitbiotics administered in two hyperbaric microenvironments. The micorenvironments were maintained at 1.62 ATA and varied in ventilation rates (20 and 30 scfm). The Kirby-Bauer Method of antibiotic sensitivity testing was used to evaluate the bacterias' responses to ampicillin, erythromycin, gentamicin, polymyxin B, tetracycline, and vancomycin.

In both hyperbaric microenvironments, E. coli, P. aeruginosa, and S. epidermidis demonstrated antibiotic susceptibilities congruous (p < 0.01) with the normobaric Inhibition Zone Diameter Interpretive Standards (Difco Laboratories). Significant variations in individual bacteria's sensitivities within the susceptibility ranges were observed. E. coli 's sensitivities to erythromycin, gentamicin, and tetracycline increased in a 20 scfm microenvironment (p < 0.02). P. aeruginosa demonstrated no significant correlation between antibiotic sensitivities and hyperbaric conditions, exclusive of its increased sensitivities (intermediate) to tetracycline in both the 20 and 30 scfm microenvironments (p < 0.01).

Data analyses suggest the efficacies of protein sythesis inhibiting antibiotics (erythromycin, gentamicin, and tetracycline) may be modified by the increased and reapportioned gas partial pressures in hyperbaric environments.

DUBBELS, B.L., J.D. FRENCH, J.D. TRANAS, R.T. DICKEY, D.J. DINGSOR AND W.J. SOEFFING . 1996. The effects of hyperbaric air on the population growth rates of the common etiological agents of otitis and the development of a buffered general growth meidum for use in compressed air environments. Proc. S.D. Acad. Sci. submitted.

The redistribution of normal bacterial flora of the human body has been implicated with the prevalence of auditory canal infections in saturation divers living in and working from undersea habitats. This study examined the influence that hyperbaric air environments have on the population growth parameters of three bacterial species (Escherichia coli, Pseudomonas aeruginosa and Staphylococcus epidermidis) associated with epidemiology and pathology of otitis externa and media. As a result of preliminary data analyses, the need for the development of a general growth medium capable of minimizing the influence of carbon dioxide gas absorption on the pH of experimental conditions was identified. Subsequent experimentation into the characterization of that medium was pursued.

Standardized bacterial subcultures were incubated at 37 degrees Celsius in a humidified compressed air microenvironment (hyperbaric chamber). Standard plate count methods were used to evaluate population growth as influenced by normobaric, 151, 162, 182 and 202 kPa pressures. Ancillary experimentation was conducted in a manned undersea habitat (MarineLab, Key Largo, FL) pressurized at 162 kPa and population growth was evaluated as aforementioned.

The carbon dioxide absorption profiles of phosphate and bicarbonate buffered and non-buffered media were indirectly evaluated by measuring the change in pH of 50 ml samples during four hour exposures to the manned undersea habitat environment. The buffering capacities of suitable buffered and non-buffered general growth media were evaluated by standardized acid-base titrations at normobaric conditions.

Results obtained form experimentation conducted in the hyperbaric chamber were reproducible and statistically significant. E. coli and S. epidermidis demonstrated predictable declining population growth rates along increasing pressure gradients. Conversely, P. aeruginosa initially demonstrated an increasing population growth rate along the pressure gradient to 162 kPa and subsequently declined. Results obtained from experimentation conducted in the manned undersea habitat were inconsistent but appeared to be associated (r<-0.90) with abnormally excessive (486 +/- 7 Pa: mean +/- sem) and fluctuating partial pressures of atmospheric carbon dioxide (385 - 571 Pa range).

Preliminary screening of buffered and non-buffered general growth media revealed that only phosphate buffered nutrient broth possessed the initial pH (pH 7.09) and buffering capacity (0.007 M/pH unit) appropriate for ensuing studies. Furthermore, the buffered media did not demonstrate the transitory pH changes observed in the non-buffered media.

The implications of varying population growth rates along a pressure gradient, contributing to a redistribution and reapportionment of bacterial flora, might alter symbioses that are proportion-dependent and promote opportunistic pathogenesis. To advance the comparison of microenvironmental in vitro experimentation, a phosphate-buffered general growth meidum has been developed and is being characterized.

STUDENT SEMINARS/COLLOQUIA:

McINTOSH,K . 1994. The effect of carbon dioxide on the effacacies of protein sythesis inhibiting antibiotics on bacteria cultured in hyperbaric conditions. USF Colloquium Abstract

The increased utilization and extended use of underwater habitats as research facilities, observatories, and living quarters, will subject divers to hyperbaric and extreme environmental conditions identified as significantly influencing the distribution of the normal bacterial flora of the body. This redistribution has led to the frequent occurrence of otitis media and otitis externa in underwater inhabitants. Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis are consistently found to be causal agents of external and middle ear infections. The specific purpose of this study was to test the effacacies of protein synthesis inhibiting antibiotics on these species of bacteria in hyperbaric conditions and to determine the effect of carbon dioxide gas absorption by microbiological media on these results. A modified Kirby-Bauer method was used to predict the effectiveness of the antibiotics on a specific bacterial sample. Carbon dioxide absorption was evaluated indirectly through pH testing and directly by carbon dioxide concentration testing. It is believed that the absorption of carbon dioxide causes a decrease in pH of the microbiological growth media. This decrease in the pH of the culture medium, due to elevated atmospheric carbon dioxide concentrations, influences the activities of protein sythesis inhibiting antibiotics. In the clinical application of this studies' results, it will be important for researchers to consider the effects that hyperbaric conditions have on in vitro experiments.

RESEARCH PROJECT REPORTS:

IVERSON, D.A. . 1990. The distribution and preponderance of coliform, pseudomonal, and staphylococcal species in the vicinity of a shallow water habitat. HE2RP Project Report

The qualitative knowledge of microbial biocoenoses of oceans is insufficient (ZoBell, 1963; Odum, 1971; and , Barnes and Hughes, 1982). Microbiological studies in the oceans are sporadic, being conducted by a small number of scientists and lagging behind other oceanogarphical disciplines (ZoBell, 1963). The results of these studies typically represent bacterial distributions at isolated stations. No extensive microbiological studies have been conducted that establish quantitative or qualitative baseline bacterial disbributions for all marine communities.

However, these isolated studies have indicated large bacterial populations in the vicinity of watersheds, coral reefs, plankton blooms, convergences, or areas of high organic matter concentrations. In the littoral zone, where most observations have been made, biological activity is the greatest. ZoBell (1963) reports the presence of 100 to 1000 bacteria per millilitre of water; with extremes ranging from <1 to 100,000,000 bacteria per millilitre. Bacterial populations ranging from <10 to 100,000,000 per gram of wet sediment have been detected in benthic samples (mean values ranging from 100 to 100,000 bacteria per gram of sediment).

Generally, bacteria are most abundant in the topmost layers of bottom sediments under shallow organic-rich waters (ZoBell, 1963; Odum, 1971, and APHA, 1985). The abundance of bacteria decreases with sediment depth (ZoBell, 1963; and , APHA, 1985). Bacteria are scarcest in pelagic areas particularly at depths exceeding 1000 meters (ZoBell, 1963). Wood (1967) attributed the distribution of baceria to the selective pressures of the marine environment. Jones (1963) reported the sea water varies in its ability to support the growth of bacteria, depending upon where and when it is collected, and the species concerned. Liston and Colwell (1963) identified specific bacterial types (based on metabolic, thermal, and biochemical requirements) associated with particular geographical locations. Obviously, envirionmental variability necessitated an investigation into the distribution of bacteria in the research locale.

This study set out to examine the distribution and preponderance of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis in the vicinity of a shallow water habitat. Since these three species have been associated with the etiology of external ear infections, it was necessary to examine the potential for environmental contributions to the occurrence of these species in the divers' external auditory canals.

Methods and Procedures: Two water samples were collected at each of two locations within the immediate vicinity of the undersea habitat. Collection Station #1 included that area circumscribed by the habitat's hatch at a depth of 10-20 cm below the air-water interface (approximately 21 ft. of water depth). Station #2 was immediately beneath a suspended oyster culture bed and 20-30 cm above the bottom sediments (approximately 17 ft. of water depth). Water samples were collected aseptically with 60 ml presterilized syringes. Precautions were taken against bacterial contamination or cross-contamination of the syringes to insure the integrity of the collected sample. Water sampling coincided with each diving exposure.

Water samples were processed using positive-pressure, presterilized, 0.22 micron ultrafiltration sytems. The ultrafiltration system was assembled aseptically and affixed via a Lur-Lock (TM) attachment to the sample syringe. Three 25 ml samples were filtered from each collection station, one sample (filter disc) for each media type.

The filter discs were aseptically transferred, organism side up, to a prepared medium which would select and/or differentiate the sample's bacteria (mannitol salt agar--Staphylococcus spp., M-Pseudomonas Agar--Pseudomonas spp., and Eosin Methylene Blue Agar--coliforms). The media plates containing the filter discs were incubated for 16 to 20 hours in a 35 degree celcius incubator at 1.62 ATA.

Results and Discussion: Enumeration and identification of colonies were intially unsuccessful. The ambient humidity of the habitat and the media moisture content impeded the production of discrete colonies. This situation dictated a modification in the filtration method. A brief exposure of the filter disc to a forced filtered-air drying produced superior results to that previously performed.

Upon examining the first set of incubated samples, it was found that the colorimetric indicators of the Eosin Methylene Blue Agar had not responded under hyperbaric conditions according to the normobaric standards. Further investigation revealed the methylene blue indicator had been reduced by the increased partial pressure of the oxygen associated with the hyperbaric environment of the habitat. The selective characteristics of the media appreared to be unaffected by the hyperbaric environment. Subsequent collaborative studies, conducted under normobaric conditions, confirmed that the selection, but not differentiation, had occurred. For that reason, the identification of specific coliform species could not be made.

Data, collected after the modification of methods, indicated an increased preponderance of Staphylococcus spp. in the vicinity of the undersea habitat and an association of coliforms with the oyster bed. These findings are based on an extremely small sample size and suggest statistical unreliability.