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JAN SCHMID,1* PAUL R. HUNTER,2 GRAHAM C. WHITE,3 ARITA K. NAND,4 .... against chemicals, the resistogram method of Hunter and Fraser (11) was ...
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1995, p. 2920–2926 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 11

Physiological Traits Associated with Success of Candida albicans Strains as Commensal Colonizers and Pathogens JAN SCHMID,1* PAUL R. HUNTER,2 GRAHAM C. WHITE,3 ARITA K. NAND,4 4 AND RICHARD D. CANNON Department of Microbiology and Genetics, School of Biological Sciences, Massey University, Palmerston North,1 and Department of Oral Biology and Oral Pathology, University of Otago, Dunedin,4 New Zealand, and Public Health Laboratory, Priority Care Unit, Countess of Chester Hospital, Chester,2 and Division of Hospital Infection, Central Public Health Laboratory, London,3 Great Britain Received 11 April 1995/Returned for modification 24 June 1995/Accepted 17 August 1995

DNA fingerprinting with the moderately repetitive sequence Ca3 has repeatedly identified groups of genetically similar strains of Candida albicans that are more frequently isolated than other groups of strains from human hosts in a geographical locale. Members of these groups are found in approximately 30% of healthy individuals and in up to 70% of patients suffering from candidiasis. The high prevalence of these strains implies that they are more successful in colonizing human hosts and in causing disease than other strains (J. Schmid, Clin. Adv. Treatment Fungal Infect. 4(6):12–16, 1993). In the present study, we have compared one such group of highly prevalent strains with other strains from the same locale to identify physiological traits associated with its success. We found that members of the group of highly prevalent strains were resistant to a larger number of chemicals than other strains in a resistogram assay. When resistance to individual chemicals used in the resistogram assay was analyzed, strains from the group of highly prevalent strains were significantly more often resistant to boric acid, cetrimide, chlorhexidine, 5-fluorocytosine, and high sodium chloride concentrations than other strains. Strains from the group of highly prevalent strains also adhered significantly (1.5 times) better to saliva-coated surfaces than did other strains. Because members of highly prevalent groups of strains are the most common infectious agents in candidiasis, these physiological traits may be involved in determining not only the success of C. albicans in colonizing human hosts in general but also its ability to cause disease. Sodium chloride resistance and increased adherence were also associated with infectious isolates outside the group of highly prevalent strains, indicating that they may be of particular importance in pathogenesis.

tify physiological traits which explain the success of these strains. Such traits should enhance the ability of C. albicans strains to colonize and to survive in human hosts and to cause disease. They should also enhance the chances of survival of strains during transmission between hosts. Since transmission may occur via inanimate objects (15, 16, 26, 31), such traits may include resistance to environmental stresses such as radiation, desiccation, or exposure to chemicals used as detergents, disinfectants, or antiseptics. These traits may be pathogenicity factors in the sense that their possession increases the probability of a strain’s causing disease. In an attempt to identify such physiological traits, we have compared the resistance to a variety of chemicals and the adhesion to saliva-coated surfaces of a group of genetically similar strains encountered with high frequency in both healthy individuals and candidiasis patients with the resistance and adhesion of other strains isolated from the same locale.

Candida albicans is an opportunistic pathogen existing in human hosts as a commensal organism but capable of causing serious and even life-threatening disease in compromised hosts (16). These infections may be caused by strains previously present on the hosts as commensals (16). However, a number of studies have indicated that C. albicans strains can be transmitted between individuals and that exogenous strains, replacing the previously present commensals, are often the etiological agents of candidiasis (8, 20, 21, 23, 31). Several studies using DNA fingerprinting with the moderately repetitive sequence Ca3 have demonstrated that a large proportion of the replacing, infection-causing strains in a given geographical area is often derived from a single group of genetically similar strains which are isolated from up to 70% of candidiasis patients (19, 20, 24). The same group of strains is also found in a large proportion of healthy individuals (approximately 30%) in the area, considerably more often than other groups of genetically similar strains (19, 20, 24). The high prevalence of such groups of C. albicans strains suggests that their members might be more successful in colonizing healthy human hosts than other strains and capable of replacing commensal strains on compromised hosts, subsequently causing candidiasis (19, 20, 23). If this hypothesis is correct, it should be possible to iden-

MATERIALS AND METHODS Selection of strains and DNA fingerprinting with probe Ca3. Isolates for this study were randomly chosen from a larger sample of strains (collected in the Iowa City, Iowa, area between 1984 and 1990) which had been fingerprinted in earlier studies (10, 22, 24, 28) by probing EcoRI digests of total DNA with the moderately repetitive sequence Ca3 (24). Each strain was from a different individual. Twenty-nine strains were commensal isolates obtained from oral and anal sites. Eighteen isolates had been obtained from individuals suffering from candidiasis and had been isolated from oral lesions, blood cultures, or stool samples. Analysis of fingerprints for definition of groups of genetically similar strains. Fingerprints of strains, stored in a Dendron (24) database, were used to determine the genetic relationships between the strains as described by Schmid et al.

* Corresponding author. Mailing address: Department of Microbiology and Genetics, School of Biological Sciences, Massey University, Palmerston North, New Zealand. Phone: 64-6-350-4018. Fax: 64-6350-5637. Electronic mail address: [email protected]. 2920

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(24). The Dendron software on a Macintosh IIvi computer was used to quantitate the degree of similarity between patterns of isolates resulting in a similarity value (SAB) according to the following formula:

O k

SAB 5

i51

~ai 1 bi 2 ai 2 bi!

O k

~ai 1 bi!

i51

where ai and bi are the intensities of band i in patterns A and B, respectively, and k is the number of bands. The similarity value can range between 1.0 and 0.0. If the patterns of strains A and B are identical, SAB is 1; if they have no bands in common, the similarity value is 0. Relationships between groups of isolates were determined by using dendrograms constructed from a matrix of SABs with the unweighted pair group method (27). The branching point between strains in the dendrogram reflects their genetic similarity. Groups of genetically similar strains were defined as groups of isolates connected by branching points in the dendrogram at an SAB of $0.8. This value lies halfway between the average SAB found for multiple isolates from the same patients (0.96 [20, 23]) and the average SAB for isolates from unrelated healthy individuals in the community (0.66 [20, 23, 24, 28]). Determination of resistance to chemicals. To test the resistance of strains against chemicals, the resistogram method of Hunter and Fraser (11) was used. In brief, strains were serially subcultured on YM agar and finally suspended in distilled water to a density between McFarland standards 5 and 6. A 21-pin multipoint inoculator was used to inoculate with these suspensions YM agar plates containing one of the following (per liter): 0.6 g of sodium selenite, 1.2 g of boric acid, 0.14 g of cetrimide, 0.2 mg of malachite green, 1.2 g of cupric sulfate, 0.05 g of benzalkonium chloride, 0.12 g of chlorhexidine, 0.24 g of mercurochrome, 2.4 g of sodium arsenate, 35 mg of 5-fluorocytosine, or 94 g of sodium chloride. Strains were also plated on MacConkey agar no. 3. Strains were considered resistant to a chemical if they showed growth after 44 h of incubation at 378C (268C for cetrimide) on plates containing the substance. Determination of adhesion to saliva-coated surfaces. Adhesion was assessed by the method of Cannon et al. (3). In brief, C. albicans cells were first grown in liquid glucose-salts-biotin medium at 308C for 16 h. Portions of these cultures were then used to inoculate 2-ml cultures of the same medium, containing [35S]methionine, to give an optical density at 540 nm of 0.2. The cultures were grown at 308C until they reached an optical density at 540 nm of 1.0. Under these growth conditions, all strains grew exclusively in the yeast morphology. Cells thus radiolabelled were washed and incubated in KCl buffer with saliva-coated hydroxylapatite beads at room temperature for 90 min. The beads were then washed three times with KCl buffer, and the radioactivity associated with the beads was determined with a scintillation counter. The number of radioactive cells bound to the beads was expressed as a proportion of the adherence of strain ATCC 10261, included in each set of assays as a control for variation between experiments. In a standard assay, 36.5% of input (2.5 3 106) C. albicans ATCC 10261 cells bound to 12 mg of saliva-coated beads. Cannon et al. (3) have shown that the adherence in the assay is saliva dependent, since adherence to uncoated beads is negligible. The adherence value for each isolate was the mean of at least two experiments, each carried out in triplicate.

RESULTS Fingerprinting with probe Ca3 defines a group of genetically similar isolates frequently isolated from both healthy individuals and candidiasis patients. Figure 1 shows the genetic relationships among 47 C. albicans strains (each from a different individual) used in this study (examples of fingerprints are shown in Fig. 2). The isolates fell into 22 groups of genetic similarity, defined as containing isolates with SABs of $0.8 between them (numbered bars in Fig. 1). The groups differed in size; most of them contained only one or two isolates. Members of one group (group 13) were isolated more frequently than members of any other group. They were present in 40% of all individuals tested and accounted for 31% of all commensal isolates and 56% of all candidiasis isolates. Group 13 therefore represents a cluster of genetically similar strains more frequently found in humans and thus presumably more successful in colonizing human hosts and at the same time more likely to cause candidiasis than other groups of genetically similar strains. Members of group 13 are more frequently resistant to chemicals. To determine if increased resistance to chemical stress

FIG. 1. Dendrogram showing the genetic relationships between C. albicans strains based on Ca3 fingerprints. Numbered bars represent groups of genetically similar strains, i.e., groups of strains whose genetic similarity to each other exceeds 80% (SAB $ 0.8). Strains matched with no other similar isolate represent rare groups, isolated only once in the sample. Commensal isolates (●) hp11an, hp12an, hp31an, hp38an, hp50an, and hp56an were cultured from anal specimens; all other commensal isolates were from oral sites. Candidiasis isolates (h) gaymc-c, hol-c, livo-c, sim-c, and svobo-c were cultured from oral lesions; isolates crr-1c, gee-2c, jam-2c, ko-2c, and pra-1c were from stool samples; and all other candidiasis isolates were from blood cultures.

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FIG. 2. Examples of Ca3 fingerprints of C. albicans strains used in this study and of the laboratory strain 3153A, included in each Southern hybridization as a standard. Molecular sizes shown (in kilobases) are those of the bands of strain 3153A.

factors was associated with the success of the group 13 strains, we compared their resistance to 12 chemicals with that of all other strains, using the resistogram method (11). Eleven of the chemicals were fungicides, some of which are used in human health care as disinfectants or antiseptics and in one case (5-fluorocytosine) as an antifungal agent. One additional test exposed strains to 9.4% NaCl, an environment of low water activity. Figure 3a shows a comparison of the number of chemicals to which group 13 strains were resistant and the number of chemicals to which other strains were resistant. All strains belonging to group 13 were resistant to at least six of the chemicals tested. In contrast, half of the remaining strains were resistant to fewer than six chemicals. The average group 13 strain was resistant to 6.79 6 0.98 (mean 6 standard deviation) chemicals, whereas the average strain not belonging to group 13 was resistant to only 5.07 6 2.19 chemicals. In a t test, this difference was significant, with a P of ,0.003, verifying that group 13 strains were on average resistant to more chemicals than other strains were. Because candidiasis isolates were overrepresented in group 13, we tested whether the increased frequency of resistance was associated with candidiasis isolates rather than with group 13 as a whole. We therefore compared the number of resistances per strain between candidiasis isolates and commensal isolates in the sample as a whole (Fig. 3b) and within group 13 (Fig. 3c). No statistically significant differences were observed. On average, commensal isolates were resistant to 5.86 6 1.96 chemicals and candidiasis isolates were resistant to 5.61 6 2.06 chemicals. Commensal strains belonging to group 13 were on average resistant to 6.77 6 0.97 chemicals, compared with 6.80 6 1.03 for candidiasis isolates in group 13. Chemical resistance profile of group 13 strains. Figure 4a shows the frequency of resistance to individual chemicals of group 13 strains compared with the resistance of other strains. Members of group 13 were resistant significantly more often to four of the fungicides tested, namely, boric acid, cetrimide, chlorhexidine, and 5-fluorocytosine, as well as to high sodium

FIG. 3. Comparison of the number of chemical resistances per strain between group 13 strains (black bars) and all other strains (unfilled bars) (a), between all candidiasis isolates (black bars) and all commensal isolates (unfilled bars) (b), and between candidiasis isolates in group 13 (black bars) and commensal isolates in group 13 (unfilled bars) (c). Resistances were determined as described in Materials and Methods.

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chloride concentrations (z test, P # 0.05). Group 13 strains were not significantly less frequently resistant to any of the remaining chemicals than were the other strains. Because candidiasis isolates were overrepresented in group 13, we wanted to test whether any of the increased frequencies of resistance described above were associated with candidiasis isolates rather than with group 13 as a whole. For this purpose, we searched for statistically significant differences (P , 0.05 in a z test) between all candidiasis and all commensal isolates (Fig. 4b) and between commensal and candidiasis isolates within group 13 (Fig. 4c). In the sample as a whole, sodium chloride resistance was significantly more frequent in candidiasis isolates than in commensal isolates (94 versus 62%). Within group 13, the frequency of sodium chloride resistance was 100% for both commensal and candidiasis isolates (Fig. 4c). For candidiasis isolates not belonging to group 13, we calculated a frequency of resistance of 75%, compared with 45% for commensal isolates outside group 13 (not shown in Fig. 4). Sodium chloride resistance was thus more frequent among group 13 commensals than among other commensals and more frequent among group 13 pathogens than among other pathogens and therefore an attribute associated with group 13. Nevertheless, it is a feature which is also more pronounced in candidiasis isolates than in commensal isolates not belonging to group 13. We also found that in the sample as a whole, candidiasis isolates were significantly less often resistant to cetrimide than were commensal isolates (61 versus 86%; Fig. 4b). Since candidiasis isolates are overrepresented in group 13, this result does not call into question that increased cetrimide resistance is a feature associated with group 13. Members of group 13 adhere more strongly to saliva-coated surfaces. Because adherence to saliva-coated surfaces is considered to be important for the colonization of the oral cavity and thus of the human host in general (3, 16), we tested whether the strains belonging to group 13 were superior to other strains in this respect. Adherence of group 13 strains to saliva-coated surfaces was on average 1.5 times greater than that of other strains (Table 1), and this difference was statistically significant (t test, P , 0.005). No significant differences in adherence existed between candidiasis isolates and commensal isolates either in the sample as a whole or in the comparison of only pathogenic isolates and commensal isolates belonging to group 13 (Table 1). Characteristics of isolates from the bloodstream. It has been reported that C. albicans isolates from invasive infections differ from superficial isolates in morphotype and have an increased frequency of phenotypic switching (12, 13). We therefore determined whether the eight bloodstream isolates included in our study differed from other isolates in terms of resistance or adhesion. No statistically significant differences in terms of the number of resistances or in the frequencies of resistance to most of the individual chemicals were found (data not shown). The only exception was chlorhexidine resistance, which was significantly less frequent in bloodstream isolates than in other candidiasis isolates (50 and 100%, respectively; t test, P , 0.05). Isolates from the bloodstream adhered to saliva-coated

FIG. 4. Comparison of the frequency of resistance to individual chemicals between group 13 strains (black bars) and other strains (shaded bars) (a), between all candidiasis isolates (black bars) and all commensal isolates (shaded bars) (b), and between candidiasis isolates (black bars) and commensal isolates within group 13 (shaded bars) (c). Statistically significant differences (P , 0.05 in a double-sided z test) are marked with an asterisk. Resistances were determined as described in Materials and Methods.

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TABLE 1. Adherence of groups of C. albicans isolates to saliva-coated surfaces Mean adherence 6 SDSa

Isolate group

Total isolates Group 13 .......................................................................... Non-group 13 ................................................................... Candidiasis ....................................................................... Commensal.......................................................................

1.09 0.72 0.94 0.83

6 6 6 6

0.33 0.46 0.45 0.43

Group 13 isolates Candidiasis ....................................................................... 1.16 6 0.29 Commensal....................................................................... 1.02 6 0.37 Bloodstream candidiasis isolates Group 13 .......................................................................... 1.34 6 0.13 Non-group 13 ................................................................... 1.06 6 0.54 Total .................................................................................. 1.16 6 0.44 Non bloodstream isolates Total .................................................................................. 0.81 6 0.43 Candidiasis ....................................................................... 0.75 6 0.38 a Adherence was determined for individual isolates as described in Materials and Methods and expressed as multiples of the adherence of strain ATCC 10261. Adherence values for groups were obtained by averaging the adherences of isolates in the groups specified.

surfaces on average 1.4 times better than all other isolates and 1.6 times better than other candidiasis isolates (Table 1). Both differences were statistically significant (t test, P , 0.05). The three bloodstream isolates from group 13 apparently adhered on average 1.3 times better to saliva-coated surfaces than the five remaining bloodstream isolates (Table 1), but this difference was not significant as determined by a t test. DISCUSSION Traits associated with the general success of C. albicans strains. Previous studies have shown that in a given geographical area, a large percentage (up to 70%) of candidiasis isolates frequently belongs to a single group of genetically similar strains and that the same strains are also more frequent as commensals than other groups of strains, being isolated from approximately 30% of healthy hosts (19, 20, 24). This has led us to propose that these groups are more successful in colonizing human hosts than other groups of strains and can replace other commensal strains in compromised hosts and can subsequently cause candidiasis. The reason these successful strains are more frequent in compromised hosts than in healthy hosts would, according to our model, be mainly the increased vulnerability of compromised hosts to colonization attempts by exogenous strains. In other words, compromised hosts may provide successful strains with an opportunity to compete with other strains more frequently than healthy hosts (19, 20). The evidence for this model before this study rested entirely on our DNA fingerprinting data. We therefore undertook the current study to provide additional evidence by testing one of its predictions, namely, that these highly prevalent groups of strains should possess attributes which give them a selective advantage over other strains. Using a collection of strains from a single geographical locale, we found that a group of genetically similar strains, highly prevalent in both healthy and compromised hosts, indeed possesses traits that would conceivably give these strains selective advantages over other strains. Survival of C. albicans strains in populations of human hosts

probably depends on their resistance to a broad variety of stress factors which are not limited to chemicals alone but also include factors such as heat, starvation, radiation, and desiccation. Our goal in testing strains for chemical resistance was therefore not so much the identification of resistance to specific chemicals but the use of chemical resistance as an indicator of the general ‘‘toughness’’ of a strain under environmental stress. To assess this toughness, we chose the resistotyping method, because it was likely to differentiate between strains. We found that members of the highly prevalent group 13 were indeed tougher, i.e., resistant to a larger number of chemicals than other strains. When resistances to individual chemicals were compared, members of this group were significantly more often resistant to 5 of the 12 chemicals tested. For none of the remaining seven chemicals was their resistance significantly inferior to that of other strains. The observed increased frequency of resistance among group 13 isolates may be attributable to merely an enhanced ability of successful C. albicans strains to cope with environmental stress in general. We note, however, that group 13 strains were more often resistant to four of the six fungicides used in human health care which are part of the resistogram (boric acid, cetrimide, benzalkonium chloride, chlorhexidine, mercurochrome, and 5-fluorocytosine). In contrast, no increased frequency of resistance of group 13 strains against the remaining fungicides (sodium selenite, malachite green, cupric sulfate, sodium arsenate, and bile salts plus crystal violet) was found. The rapid development of chemical resistance in C. albicans is well documented for antifungal agents including 5-fluorocytosine (2, 16, 18). It is therefore possible that exposure to antibiotics, antiseptics, or disinfectants in the evolutionary history of the strains surveyed may have played a role in the initial development of increased resistance against these agents specifically and in the continued selection for these resistances. Group 13 strains were also more frequently resistant to high concentrations of sodium chloride. This resistance is most likely an adaptation to survival in environments of low water activity. The ability to survive in environments of low water activity could be of considerable importance in determining the success of transmission of C. albicans strains to new hosts. Food has been identified as a possible vehicle of transmission of C. albicans (16, 31), and the ability to survive under conditions of low water activity would greatly enhance the ability of strains to use this vehicle. The NaCl concentration used in the assay creates a water activity of 0.95, comparable to that in many major food items, such as bread, cheese, canned fruits, lightly salted fish, pork, and beef products (5). Only strains able to tolerate water activities in this range can use these food items as vehicles. Other strains would be restricted to items with higher water activity, such as fresh fruits, vegetables, and milk (5). The observed resistance of group 13 strains to environments of low water activity may in addition indicate increased resistance to desiccation. Since transmission may occur via inanimate surfaces (16, 31), resistance to desiccation would certainly enhance the chances of survival of a strain until contact with another host is made. The importance of adherence to host tissues is frequently discussed as an important factor in both commensal colonization of human hosts by C. albicans and pathogenesis (6, 14, 17, 30). The oral cavity is one of the main reservoirs of C. albicans in the human host (16), and the ability to adhere to salivacoated surfaces, which is of paramount significance in colonization of the mouth (3), can therefore be expected to play a decisive role in the ability of C. albicans to establish itself in human hosts. The increased adherence of group 13 strains to

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saliva-coated surfaces is thus a trait expected to occur in such a group of successful colonizers and pathogens. Traits of particular importance in pathogenesis. Our study has identified physiological traits which are associated with groups of strains responsible for a large proportion of Candida infections. All of these traits may be pathogenicity factors in the sense that their possession enhances the probability of a strain’s causing disease. It is unlikely, though, that any of them are of advantage to a strain only when it is causing disease, since they are also associated with the success of commensal colonization. Indeed, it is unlikely that we would find any traits which are of benefit only in pathogenesis among group 13 strains or any other groups of strains of C. albicans which are highly prevalent in candidiasis patients and at the same time successful colonizers of healthy individuals. Any adaptation of an organism to a specific environment must be seen as costly, and the resources used up in this adaptation are not available for use in another environment (7). Evolution of specific traits which benefit a C. albicans strain only when it causes disease in the compromised host would thus diminish the strain’s fitness in healthy hosts. Its frequency in healthy hosts would decrease as a result, and its ecological niche would tend to be restricted to hosts in which it can cause disease. This phenomenon has been observed for Escherichia coli, a species whose interactions with human hosts, ranging from commensal colonization to invasive disease in uncompromised hosts, are the subject of intense study. Specific virulence traits in this species are restricted to a limited number of clones which are highly prevalent in invasive diseases but which are absent or rare in the commensal intestinal floras of healthy hosts (1, 9, 25, 29). To our knowledge, for C. albicans, groups of disease-specific clones have never been observed. We note that this does not preclude that certain C. albicans strains may have traits which are of particular advantage in pathogenesis as long as they are also, albeit to a lesser degree, beneficial in commensal colonization. Two possible examples of such traits from this study are resistance to low water activity, as assessed by resistance to sodium chloride, and adherence. Both traits were associated with the group of generally successful strains. At the same time, sodium chloride resistance was also more frequent in candidiasis isolates in general and increased adherence was more frequent in isolates causing infections in the bloodstream. Two other examples of traits that could be of greater benefit to a strain in pathogenesis than in commensal colonization are an increased frequency of phenotypic switching and increased levels of proteinase secretion. Both the switching frequency and the level of proteinase secretion have been reported to be higher in candidiasis isolates than in commensal isolates (4, 13). However, without knowing the genetic relationships between the isolates used in these studies, it cannot be resolved whether these traits are of particular importance in pathogenesis: the reported apparent differences between commensal and candidiasis isolates may have been only a secondary result of the fact that a higher proportion of the infectious isolates than of the commensal isolates belonged to groups of generally successful genetically similar strains, all of whose members display increased proteinase secretion and phenotypic switching. Comparative studies aimed at the identification of traits of specific relevance to pathogenesis should therefore incorporate determinations of the genetic relationships between strains or use sets of strains for which these relationships have already been determined. We invite researchers to make use of the set of strains used in this study or of other strains which are

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part of our fingerprint database for their research on pathogenicity factors. ACKNOWLEDGMENTS This work was supported by a grant from the Massey University Research Fund to J.S. A.K.N. gratefully acknowledges the receipt of an Elisabeth Kerr Crow studentship. We thank Richard Hollis for providing some of the isolates used and Paul O’Toole for reading and evaluating the manuscript. Photographic work was carried out by Tony Watts. REFERENCES 1. Achtman, M., and G. Pluschke. 1986. Clonal analysis of descent and virulence among selected Escherichia coli. Annu. Rev. Microbiol. 40:185–210. 2. Bart-Delabesse, E., P. Boiron, A. Carlotti, and B. Dupont. 1993. Candida albicans genotyping in studies with patients with AIDS developing resistance to fluconazole. J. Clin. Microbiol. 31:2933–2937. 3. Cannon, R. D., A. K. Nand, and H. F. Jenkinson. 1995. Adherence of Candida albicans to human salivary components adsorbed to hydroxylapatite. Microbiology 141:213–219. 4. Cassone, A., F. De Bernardis, F. Mondello, T. Ceddia, and L. Agatensi. 1987. Evidence for a correlation between proteinase secretion and vulvovaginal candidosis. J. Infect. Dis. 156:777–783. 5. Christian, J. H. B. 1980. Reduced water activity, p. 70–91. In J. H. Silliker, R. P. Elliott, A. C. Baird-Parker, F. L. Bryan, J. H. B. Christian, D. S. Clark, J. C. Olson, Jr., and T. A. Roberts (ed.), Factors affecting life and death of microorganisms. Academic Press, Inc., New York. 6. Cutler, J. E. 1991. Putative virulence factors of Candida albicans. Annu. Rev. Microbiol. 45:187–218. 7. Dawkins, R., and J. R. Krebs. 1979. Arms races between and within species, p. 55–77. In J. M. Smith and R. Holliday (ed.), The evolution of adaptation by natural selection. University Press, Cambridge. 8. Doi, M., M. Homma, S.-I. Iwaguchi, K. Horibe, and K. Tanaka. 1994. Strain relatedness of Candida albicans strains isolated from children with leukemia and their bedside parents. J. Clin. Microbiol. 32:2253–2259. 9. Hartl, D. L., and D. E. Dykhuizen. 1984. The population genetics of Escherichia coli. Annu. Rev. Genet. 18:31–68. 10. Hellstein, J., H. Vawter-Hugart, P. Fotos, J. Schmid, and D. R. Soll. 1993. Genetic similarity and phenotypic diversity of commensal and pathogenic strains of Candida albicans isolated from the oral cavity. J. Clin. Microbiol. 31:3190–3199. 11. Hunter, P. R., and C. A. M. Fraser. 1989. Application of a numerical index of discriminatory power to a comparison of four physiochemical typing methods for Candida albicans. J. Clin. Microbiol. 27:2156–2160. 12. Hunter, P. R., C. A. M. Fraser, and D. W. R. Mackenzie. 1989. Morphotype markers of virulence in human candidal infections. J. Med. Microbiol. 28: 85–91. 13. Jones, S., G. White, and P. R. Hunter. 1994. Increased phenotypic switching in strains of Candida albicans associated with invasive infection. J. Clin. Microbiol. 32:2869–2870. 14. Matthews, R. C. 1994. Pathogenicity determinants of Candida albicans: potential targets for immunotherapy? Microbiology 140:1505–1511. 15. Moro, M. L., C. Maffei, E. Manso, G. Morace, L. Polonelli, and F. Biavasco. 1990. Nosocomial outbreak of systemic candidosis associated with parenteral nutrition. Infect. Control Hosp. Epidemiol. 11:27–35. 16. Odds, F. C. 1988. Candida and candidosis, 2nd ed. Bailliere Tindall, London. 17. Odds, F. C. 1994. Candida species and virulence. ASM News 60:313–318. 18. Sandven, P., A. Bjørneklett, A. Maeland, and the Norwegian Yeast Study Group. 1993. Susceptibilities of Norwegian Candida albicans strains to fluconazole: emergence of resistance. Antimicrob. Agents Chemother. 37: 2443–2448. 19. Schmid, J. 1993. Candidiasis: conclusions from DNA fingerprinting. Clin. Adv. Treatment Fungal Infect. 4(6):12–16. 20. Schmid, J., F. C. Odds, M. J. Wiselka, K. G. Nicholson, and D. R. Soll. 1992. Genetic similarity and maintenance of Candida albicans strains from a group of AIDS patients, demonstrated by DNA fingerprinting. J. Clin. Microbiol. 30:935–941. 21. Schmid, J., M. Rotman, B. Reed, C. L. Pierson, and D. R. Soll. 1993. Genetic similarity of Candida albicans strains from vaginitis patients and their partners. J. Clin. Microbiol. 31:39–46. 22. Schmid, J., and D. R. Soll. Unpublished data. 23. Schmid, J., Y. P. Tay, L. Wan, M. Carr, D. Parr, and W. McKinney. 1995. Evidence for nosocomial transmission of Candida albicans obtained by Ca3 fingerprinting. J. Clin. Microbiol. 33:1223–1230. 24. Schmid, J., E. Voss, and D. R. Soll. 1990. Computer-assisted methods for assessing strain relatedness in Candida albicans by fingerprinting with the moderately repetitive sequence Ca3. J. Clin. Microbiol. 28:1236–1243. 25. Selander, R. K., and J. M. Musser. 1990. Population genetics of bacterial pathogenesis, p. 11–36. In B. H. Iglewski and V. L. Clark (ed.), Molecular

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