Survival of Pathogenic Bacteria Under Nutrient Starvation Conditions 901381

The purpose of this research is to determine the survival of human pathogens within a water distribution system proposed for the orbiting space station. Initially we investigated the survival of opportunistic pathogenic microorganisms in water under nutrient limiting conditions. A strain of Pseudomonas aeruginosa and two strains of Staphylococcus aureus were grown to mid-log phase then transferred to a starvation regime of sterile deionized water. Cultures were incubated at 10°, 25° or 37° C and were sampled at 24 hr, 1 week, 4 weeks and 6 weeks. The viable cell density was determined by enumerating colony forming units and by directly counting cells stained with acridine orange. Neither of the Staphylococcus strains tested were detected after 1 week of starvation. Our data indicate that Pseudomonas aeruginosa can survive in deionized water at all three temperatures tested at levels exceeding 10⁴ cells per ml. The bacterial surface characteristics associated with attachment under nutrient limiting conditions are currently being examined.

The formation of microbial biofilms in the pipes and filters of the water reclamation system (WRS) aboard the orbiting space station (OSS) poses a potential health risk. Reclamation of shower, laundering, and dish washing water will be necessary for long duration space flight. This waste water will be heavily contaminated with bacteria including opportunistic pathogens. Such a closed loop reclamation system will provide an ideal environment for the development of a biofilm, which is composed of bacterial cells in close association with extracellular polymers. Once a biofilm has formed within the piping and filtering systems of the WRS, an infectious reservoir of opportunistic pathogens can develop.

Our experience working with surface films of microorganisms has shown that colonization is extremely rapid, even when the inoculum is small. After initial colonization, build-up of an extensive biofilm occurs. Many opportunistic pathogens readily attach to surfaces and will survive and proliferate within films. The nature of biofilms is such that they resist physical cleaning techniques and penetration of biocides. They are therefore extremely difficult to eliminate and will continually be a source of potentially pathogenic microorganisms.

Biofilms have the potential not only to decrease the diffusion rate of biocides ILLEGIBLEbut ILLEGIBLEalso to react with chemical oxidants (e.g. iodine) intended to destroy living cells. The effective concentration of a biocide needed to remove possible pathogens will be dependent to some degree on the thickness of the biofilm. The removal of this organic layer on the surface would also eliminate the source of free-living (planktonic) cells that can slough off to reinoculate the water distribution system downstream (1).*

The unique nature of the WRS, based within the OSS where the human immune system will be compromised, necessitates extremely high sanitary standards. These standards are, however, currently unobtainable without the addition of chemicals at high concentration which themselves pose a serious health risk. In order to develop an effective method of water purification within the WRS that does not involve high levels of toxic chemicals, it is necessary to improve our understanding of basic mechanisms of initial attachment and subsequent film formation. This information will permit the development of non-toxic “biofilm inhibitors” working perhaps at the level of attachment prevention or “biofilm matrix” degradation.

The sequence of events that leads to the irreversible adhesion of bacteria to a surface is summarized in Table 1 (2). Important factors that contribute to the adhesion process include a) the characteristics of the substratum, b) the presence and composition of a molecular film on the substratum, and c) the physiological state of the bacterium. These different surface characteristics and the methods used to measure them have been discussed recently in detail (3,4,5).

Pseudomonas aeruginosa is a common bacterium capable of causing severe infections in compromised hosts. The irreversible attachment of P. aeruginosa to stainless steel (304) can occur within the first minute of exposure. Furthermore, pH, cation concentration and cell motility are important factors involved in the adhesion of this bacterium (6). P. aeruginosa can produce large amounts of exopolysaccharides as well as form biofilms on various surfaces (e.g. PVC, stainless steel, copper) (7,8,9). The exopolysaccharide material may help to irreversibly bind the bacteria to certain surfaces while also protecting this bacterium from biocides. P. aeruginosa has recently been reported to survive (i.e. 10⁶ cell ml^-1) for up to 15 months in an iodine “germicide” solution, a proposed disinfectant for the WRS (10).

Despite our knowledge of the the mechanisms of attachment of bacteria to surfaces, we know little about the role that nutrient limition has on initial adhesion and biofilm development. This paper describes an investigation on the survival of a specific opportunistic pathogen under starvation conditions at three different temperatures. The purpose of this study was to determine the survival of human pathogens and subsequent adsorbtion to the inner surfaces of the water distribution system on the orbiting space station.