Evaluation of the effect of pH on in vitro growth of Malassezia pachydermatis

Abstract

The purpose of this study was to evaluate the effects of pH on the growth of canine Malassezia pachydermatis isolates in vitro. Yeast growth was monitored by measuring the optical density with a spectrophotometer. The growth of American Type Culture Collection and field strains of M. pachydermatis was optimal between the pH values of 4.0 and 8.0, and inhibited at the ranges of 1.0 to 3.0 and 9.0 to 10.0. An analysis of covariance showed no significant differences among the growth curves at pH levels 5.0 to 8.0. Although specific contrast tests showed that the growth slope at pH 4.0 was significantly different from that at pH 5.0 to 8.0, only small, random differences were found when the growth slope at pH 4.0 was compared to the individual slopes at pH 5.0, 6.0, 7.0, and 8.0. The findings of this study suggest that topical acidifying products could be beneficial therapeutic options for cutaneous yeast infections in dogs.

Malassezia pachydermatis is a commensal yeast of mammals that causes a pruritic dermatitis in dogs and is a perpetuating cause of canine otitis externa (1). Topical antifungal therapies are routinely used to speed relief and resolution of Malassezia dermatitis and otitis. Frequent use of antifungal shampoos, conditioners, and ear medications may help to decrease the recurrence of yeast infections in some animals.

In addition to products with antifungal activity; such as, miconazole, topical products that lower cutaneous pH are also used as adjunctive therapies for canine Malassezia infections. The rationale for acidification of the skin is based on the acid mantle theory, which proposes that the relative acidity of human skin serves as a protective barrier against infection by cutaneous microorganisms (2). The pH of human skin typically ranges from 4.5 to 6.0, with higher pH values occurring in intertriginous areas (3,4). In accordance with the acid mantle theory, increased skin alkalinity in human subjects has been associated with a predisposition to cutaneous infection; such as, bacterial pyoderma, Candidiasis, and diaper rash (3).

Canine skin is more alkaline than human skin, with a pH range of 5.5 to 7.2 (5,6). We have observed even higher cutaneous pH levels on the dorsal thoracolumbar area of dogs, ranging from 6.4 to 9.1 (7). In a recent report, the mean pH of canine flank skin was 7.48 (8). Canine skin may be more alkaline than previously reported — a discrepancy which could be attributed in part to differences in the methods of pH measurement; sex, breed, coat color, anatomic site, general skin physiology, environmental climate, or geographical location (8). Additionally, an increase in cutaneous alkalinity was demonstrated in canine seborrheic dermatitis (5). The relative alkalinity of canine skin may be partly responsible for a higher predisposition to cutaneous infections in the dog compared to other species, such as cats or humans.

Based on the acid mantle theory and evidence of increased alkalinity in some cutaneous diseases, it is reasonable to speculate that topical acidifying products may be beneficial adjunctive therapies for cutaneous infections. However, it is interesting to note that slightly acidic conditions (pH 4.0 to 6.0) favor the growth of yeast (9). Therefore, in order to evaluate the potential use of products which decrease cutaneous pH for the treatment of fungal disorders, it is necessary to determine the pH levels that will inhibit the growth of these cutaneous fungal microorganisms optimally. The goal of this study was to test the effects of pH on the growth kinetics of M. pachydermatis in vitro.

An isolate of M. pachydermatis (#52682, canine ear) was obtained from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA), and rehydrated according to the manufacturer's instructions. In addition to the ATCC isolate, we isolated M. pachydermatis from the skin of a dog. Identification of the field strain was based on colony growth and microscopic characteristics of isolates on Sabouraud dextrose agar (10,11).

Using a glass electrode pH meter (Orion 520A, Boston, Massachusetts, USA), and drops of 2N NaOH and 6N HCl, the pH of ten 200 mL bottles of Sabouraud dextrose broth was changed to pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0. A bottle of Sabouraud dextrose broth with a fixed pH of 5.7 was used as the control. Quadruplicate 30.0 mL aliquots of each pH level were aseptically placed into separate test tubes and clearly marked with the respective pH value. Malassezia pachydermatis was cultured on Sabouraud dextrose agar with chloramphenicol (Remel, Lenexa, Kansas, USA) and incubated for 24 to 36 h in 6.7% CO₂ at 37°C. Individual colonies were then added to 5.0 mL of Sabouraud dextrose broth (pH 5.7, Remel) to make a turbid culture suspension equivalent, by visual comparison, to a 0.5 MacFarland standard. This standard is used for the estimation of bacterial numbers in a turbid solution, but is not optimal for estimating yeast numbers. It was, however, used as a guide to attain consistent turbidity in each sample. Next, 0.25 mL of the culture suspension was added to each of the prepared 30.0 mL tubes of Sabouraud dextrose broth (pH 1.0 to 10.0, and 5.7 control) and incubated in a shaker water bath at 35°C for 108 h. These procedures were performed in duplicate for each strain.

Optical density (OD) was measured every 12 h with a spectrophotometer (Genesys 5; Spectronic Instruments, Rochester, New York, USA). One mL of the culture solution was placed into a semimicro methacrylate cuvette (Fisher Scientific, Pittsburgh, Pennsylvania, USA) and percent transmittance of light was measured at a wavelength of 600 nm and a pathlength of 1 cm. The percent transmittance was determined by comparing the absorbance of the sample (yeast suspension) to a control (Sabouraud dextrose broth) at the same pH level.

Intermittent evaluations of the culture suspensions were performed, both to monitor the viability of the yeast in suspension and to screen for contaminants. First, 10 mL of the culture suspensions was cultured on Sabouraud dextrose agar with chloramphenicol (Remel) and incubated for 24 to 36 h in 6.7 % CO₂ at 37°C. Second, 10 mL of the culture suspensions were stained by the gram method, and evaluated cytologically for evidence of yeast or bacteria.

The null hypothesis was that pH would not alter the yeast growth curves. An analysis of covariance was used to determine if the slopes of the growth curves at different pH levels were parallel (SYSTAT, version 9; SPSS Inc., Chicago, Illinois, USA). The level of significance was chosen as P < 0.05. Duplicate measurements from the yeast were averaged, and the ATCC strains were tested separately from the field strains.

No growth occurred on the intermittent cultures of the culture suspensions, and no yeast or bacteria were identified cytologically at any time during the study in tubes at pH 1.0 to 3.0 and 9.0 to 10.0. Correspondingly, the spectrophotometer did not detect a change in turbidity at pH 1.0 to 3.0 and 9.0 to 10.0. For both strains, the slopes at pH 1.0 to 3.0 and 9.0 to 10.0 were different from slopes at pH 4.0 to 8.0 (Figure 1). Only the parallelism of these latter slopes (those showing a logarithmic growth phase) was compared statistically, using an analysis of covariance. Within the pH values of 4.0 to 8.0, at least 1 growth slope was significantly different from the other slopes (ATCC strain: F = 6.2, P ≤ 0.001; field strain: F = 6.5, P ≤ 0.001). When the pH of 4.0 was not included in the analysis, no significant differences were found between the growth curves at pH levels 5.0 to 8.0 (ATCC strain: F = 0.8, P > 0.5; field strain: F = 2.2, P > 0.1). Specific contrast tests showed that the growth slope of pH 4.0 was significantly different from the slopes at pH 5.0 to 8.0 (ATCC strain: F = 25.0, P ≤ 0.001; field strain: F = 11.5, P ≤ 0.002). Although the overall difference was significant, only small, random differences were found when the pH 4.0 slope was compared to the individual slopes of pH 5.0, 6.0, 7.0, and 8.0.

Figure 1. Graph demonstrating the growth curve of Malassezia pachydermatis (ATCC strain) with altered pH in hours versus percent transmittance.

The growth curves of M. pachydermatis in broth were consistent with the expected growth curve for a microorganism in liquid media. A normal growth curve includes a lag phase during which the organisms are not actively multiplying as they acclimatize to a new environment. This phase seemed prolonged for M. pachydermatis at pH 4.0 compared to pH 5.0 to 8.0 (Figure 1), which may account for some of the statistical differences found. The lag phase is followed by an exponential (logarithmic) growth phase, occurring until the nutrients are exhausted or toxic metabolites accumulate, at which time growth is slowed. During the next (stationary) phase, there is a balance between cell multiplication and death. The decline phase occurs when the magnitude of cell death exceeds cell growth, and may persist for a long time. In this study, the effect of the pH changes of M. pachydermatis in vitro was evaluated during the exponential growth phase, thus allowing the determination of the inhibitory effects of different pH levels.

Measuring the optical density of a yeast suspension (spectrophotometry) is a widely accepted method for evaluating yeast growth. Transmittance measured with a spectrophotometer is fast and easy, but it does not evaluate the viability of the organisms in the suspension. Instead, it is a measure of the accumulation of live and dead cells over time. Techniques to assess viable cell counts of M. pachydermatis were considered, but deemed less accurate due to a large degree of cell clumping, which was visible microscopically.

The addition of 0.1% Triton X-100, a nonionic surfactant, can be used to decrease yeast cell aggregation (12). Faergemann (12) found that this detergent was superior to other detergents, with only 10% growth inhibition of a Malassezia yeast species after 30 min of incubation. The use of stains that differentiate between live and dead cells were considered, but the most viable stains are pH sensitive, and the design of this study did not permit the pH of the media to be controlled within acceptable pH ranges.

The results of this study support the hypothesis that an acidic environment can be inhibitory to the growth of yeast. It is interesting to note that the amount of acidification required to achieve a significant decrease in yeast growth is greater than that required for bacteria. In our study, a pH less than 4.0 was required to inhibit the growth of M. pachydermatis in vitro.

These results differ slightly from those obtained by Sharma (11), who subjectively evaluated M. pachydermatis growth at pH 2.0 to 11.0 by visually assessing changes in turbidity. He noted that there was variable growth at pH level 3.0 by day 7. In that study, no growth was identified at pH 2.0 after 15 d. There are some differences between the studies that may account for the growth differences of M. pachydermatis at pH 3.0. First, our study evaluated growth for 108 h, and it is possible that we would have identified growth at pH 3.0 if the measurements had been taken for longer. However, in our study, there was no evidence of viable yeast cells in the culture suspension at pH 3.0 for the entire duration of the study.

Secondly, it is likely that different strains of M. pachydermatis were tested. Sharma (11) reported good M. pachydermatis growth at pH 4.0 to 6.0 on day 3, whereas turbidity at the same pH levels was visually evident earlier (by 24 to 36 h) in our study. It is possible for strains of the same organism to have different characteristics. For example, 2 phenotypically different colonies of M. pachydermatis have been identified; the smaller colony type is often more difficult to subculture on Sabouraud dextrose medium (13). A difference in M. pachydermatis strains could account for the discrepancy in growth rates, and even the difference in growth at pH 3.0. This is one of the reasons why we tested strains of M. pachydermatis obtained from 2 different sources. Lastly, different culture conditions, such as temperature, aeration, and exposure to CO₂, may have had different effects on the growth of yeast.

From the longer lag phase observed on the growth curve at pH 4.0, it appears that M. pachydermatis adapted to lower pH levels. Some species of bacteria have demonstrated increased tolerance for acidic environments when they were acclimated to a mild acid prior to exposure to a stronger acid (14). It would be interesting to determine if M. pachydermatis would have a greater ability to survive at low pH levels (for example, ≤ 3) if the changes occurred gradually, as opposed to the sudden alterations done in this study. Although the ability to adapt to lower pH levels should not affect the use of topical acidifiers for the adjunctive treatment of an acute infection, it may limit their usefulness for long term prevention of cutaneous infections.

The degree of cutaneous acidification required for potential therapeutic effects is dependent upon the target organism. It seems that the inhibition of yeast organisms will require a larger cutaneous pH change than bacterial organisms. Malassezia yeast and Staphylococcus intermedius are able to utilize each other's metabolic products, thus they are capable of enhancing each other's growth. For example, Kiss et al (15) found that the growth of some species of Malassezia yeast is dependent on the presence of nicotinic acid produced by S. intermedius. It is possible that lowering cutaneous pH to a level sufficient to decrease S. intermedius populations could lead to a concurrent decrease in cutaneous Malassezia yeast.

Since cutaneous pH plays an integral role in the barrier function of the skin, one concern about such acidification of the skin is that it will lead to cutaneous irritation. Recent studies demonstrate that irritation to human skin is usually due to the product ingredients, not the product pH (16,17). However, dogs have more alkaline skin than people, and the potential for irritation with cutaneous acidifiers in dogs warrants further investigation.

The alkaline pH levels of 9.0 and 10.0 also inhibited yeast growth, but alkalinization of the skin may be associated with unacceptable side effects. Increasing the alkalinity of human skin has been associated with irritation, increased transepidermal water loss, and increased bacterial counts (18). Studies in hairless mice demonstrated impairment of barrier recovery when the skin was exposed to a neutral or alkaline pH (19). This may be partially due to the presence of pH-dependent enzymes in the stratum corneum, which may not function normally in more alkaline pH levels (19,20). It appears that therapeutic cutaneous alkalinization is not a likely option at this time, but these pH-dependent processes have primarily been evaluated in human skin, where the surface is acidic. Since differences in cutaneous pH exist between species, the results found in humans may not directly pertain to other animals.

In conclusion, from this in vitro study, it appears that a pH of less than 4.0 is required to inhibit M. pachydermatis growth. Further research is required to determine the effects of cutaneous acidification on canine M. pachydermatis flora in vivo. It would be important to determine whether decreasing the cutaneous pH of dogs to 4.0 would lead to a decrease in the cutaneous yeast flora.