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Free-Living Microorganisms CHAPTER 5 Free-living microorganisms n addition to microorganisms introduced to recreational waters through human or Ianimal faecal contamination (see chapter 4), a number of pathogenic micro- organisms are indigenous to such areas or, (like the leptospires) once introduced are capable of colonizing the environment. This chapter describes the principal free- living microorganisms of concern; the diseases that they may cause associated with recreational water use; and potential control measures. 5.1 Human pathogenic Vibrio species Vibrio species are motile, non-spore-forming, slightly curved Gram-negative rods with a single polar flagellum. They are both aerobic and facultatively anaerobic. Vibrios require, or their growth is stimulated by, sodium chloride, and they are capable of respiratory and fermentative metabolism, with only a single species (V. furnissii) producing gas. All human pathogenic Vibrio species except V. metschnikovii reduce nitrate and are oxidase positive. Substantial evidence has been provided showing that Vibrio species are natural inhabitants of marine aquatic environments in both temperate and tropical regions, with most infections acquired by exposure to such environments or to foods derived from them (Kelly et al., 1991; Oliver & Kaper, 1997). Vibrio species have been iso- lated from a variety of environmental samples, including water, sediment, plankton, shellfish and finfish. Several studies suggest that the occurrence of vibrios does not correlate with the occurrence of the traditionally used faecal index organisms, although a positive correlation may be found in waters receiving human wastes from disease outbreaks (mainly cholera). There appears to be a positive correlation between water temperature and the numbers of human pathogenic vibrios isolated, as well as the number of reported infections. Seasonality is especially noted for V. vulnificus and V. parahaemolyticus (Oliver & Kaper, 1997). Vibrio species have been isolated in waters showing a broad range of salinities and varying pH values. V. cholerae and V. mimicus are the only species found in fresh water. Thus, due to the ubiquitous nature of Vibrio species in the aquatic environment, the presence of Vibrio species in bathing water cannot be controlled by water quality control measures such as wastewater treatment and disinfection. Human carriers and shedding appear to be of only limited importance in the epidemiology of Vibrio infections associated with recreational water use. While there exists considerable variation in the severity of the various Vibrio- associated diseases, the most severely ill patients generally suffer from pre-existing 102 illnesses, with chronic liver disease being one of the most common. An exception to this generalization is V. cholerae serogroups O1 and O139, the causes of cholera, which can readily cause disease in non-compromised individuals. The Vibrio species of medical importance grow well on common media, includ- ing blood, chocolate and Mueller-Hinton agars. A more thorough search for Vibrio species can be done by culture on a selective-differential plating medium, such as thiosulfate–citrate–bile salts–sucrose (TCBS) medium. However, not all Vibrio species of medical importance grow well on TCBS agar (Kelly et al., 1991). In fact, different brands of TCBS can select different species of vibrios and be less selective for the target microorganisms. Increased yields of vibrios from environmental samples may be obtained by enrichment in alkaline peptone water before subculture on plating media. Since Vibrio species vary considerably in pathogenicity and epidemi- ological significance, isolates should be identified to species level. Such identification should be performed by reference laboratories. Although commercial systems for the identification of Vibrio species have improved, misidentifications remain a problem. Currently, 12 Vibrio species are known to cause or to be associated with human infections: V. alginolyticus, V. carchariae, V. cholerae, V. cincinnatiensis, V. damsela, V. fluvialis, V. furnissii, V. hollisae, V. metschnikovii, V. mimicus, V. parahaemolyticus and V. vulnificus (Kelly et al., 1991). The infections can be classified as intestinal or extraintestinal, although this division is not absolute (Table 5.1). Studies of the infectious dose for vibrios able to cause gastrointestinal disease have been carried out mainly for V. cholerae, where the infectious dose appears high. For V. cholerae, 106 organisms or more are needed to cause cholera. In hypochlorhydric persons, the infectious dose is reduced to 104–105 organisms (Oliver & Kaper, 1997). Thus, it is unlikely that persons bathing or involved in other recreational water TABLE 5.1. VIBRIO SPECIES THAT MAY BE FOUND IN HUMAN CLINICAL SPECIMENSa Occurrence in human clinical specimensb Species Intestinal Extraintestinal V. alginolyticus -++ V. carchariae -+ V. cholerae O1 ++++ + Non-O1 ++ + V. cincinnatiensis -++ V. damsela -+ V. fluvialis ++ - V. furnissii ++ - V. hollisae ++ - V. metschnikovii ++ V. mimicus ++ + V. parahaemolyticus ++++ + V. vulnificus + +++ a Modified from Kelly et al. (1991) b The symbols +, ++, +++ and ++++ give the relative frequency of each organism in specimens from implicated infections; -=not found. CHAPTER 5. FREE-LIVING MICROORGANISMS 103 activities would ingest vibrios in numbers high enough to cause gastrointestinal disease in the absence of extreme contamination. However, the risk of extraintesti- nal infections associated with human pathogenic Vibrio species, especially wound and ear infections, during recreational activities in water is of health importance, although the infectious doses for such infections are unknown. 5.1.1 V. alginolyticus V. alginolyticus is very common in the marine environment. The organism does not cause diarrhoea but may cause soft tissue infections following exposure to seawater (Kelly et al., 1991). Ear, wound and eye infections have been reported most often. It appears that a majority of patients with otitis associated with V. alginolyticus have predisposing conditions, including chronic otitis media and rupture or tubulation of the tympanic membrane. Infections are usually self-limiting and of moderate sever- ity and short duration, and antibiotic treatment is only occasionally necessary. 5.1.2 V. cholerae Among the vibrios, special attention has focused on the identity of those causing cholera. V. cholerae has been divided into more than 150 serological types on the basis of the O or somatic antigens. Numerous outbreaks of cholera involving drinking-water and foods have been documented. Although V. cholerae O1 and O139 are occasionally isolated from the aquatic environment, especially in areas with cholera outbreaks, no confirmed cholera cases caused by activities in recreational waters, including bathing, seem to have been reported. Thus, probably because of the high infectious dose required to cause cholera, it appears that the isolation of V. cholerae O1 and O139 from marine waters represents a very low health risk to persons bathing or participating in other recre- ational activities in such waters. Other non-O1 serotypes cause gastroenteritis, with the range of symptoms found to vary, but common features include diarrhoea and occasional vomiting with abdominal cramps. Although isolates have been recovered from wounds, ears and a variety of other sites, the clinical significance of V. cholerae as a cause of extraintestinal infections is uncertain, as other potential pathogens are often also isolated. 5.1.3 V. parahaemolyticus V. parahaemolyticus is an agent of food poisoning, associated with the consumption of raw or insufficiently cooked seafood. It has also been associated with pneumonia (Yu & Uy-Yu, 1984), resulting from inhalation of contaminated aerosol, and wound infection. V. parahaemolyticus has been associated with severe life-threatening infec- tions these, however, were subsequently found to be due to V. vulnificus (Kelly et al., 1991). 5.1.4 V. vulnificus V. vulnificus is a foodborne pathogen, causing a rapidly fatal infection in persons with underlying liver diseases. Generally implicated is the consumption of raw or under- 104 GUIDELINES FOR SAFE RECREATIONAL WATER ENVIRONMENTS cooked oysters, although raw clams, octopus and other marine fish and shellfish have also been associated with the disease. In addition to being foodborne, V. vulnificus causes wound infections following entry into a skin lesion. Such infections are almost always associated with seawater and/or shellfish. Puncture wounds generally result from utensils used to clean shellfish or from the hard shell or fins of shellfish and fish. Symptoms, which develop after about 16h, include intense pain, redness, swelling and rapidly developing tissue destruction. Although the pathogenesis of these infections has yet to be elucidated, it is likely that one or more of the several exoenzymes (e.g., collagenase, protease, elastase, phospholipase, cytotoxic haemolysin) produced by this species are essential for its ability to invade and cause tissue destruction. Surgical removal of tissue, skin grafting and even amputation are generally required. Mortality rates average 20–25%. Wound infections with V. vulnificus typically occur in healthy persons and remain localized, although the bacterium may become systemic in people with chronic liver disease, and this carries a high fatality rate. In the past, several reports of severe, life-threatening infections associated with V. parahaemolyticus were described; however, subsequent examination
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