Resident Vs Transient Microflora

It is generally accepted that resident microorganisms are those that multiply at a specific site, rather than simply survive. Transient organisms, on the other hand, arrive from an outside source, and are unable to compete successfully for a permanent home. While simple to state in principle, this difference is not easy to demonstrate in practice. There is an extensive body of literature concerning the microflora of the skin, but relatively little is known about the quantitative relationships among various microorganisms on various skin surfaces. Moreover, given the dichotomy between resident and transient microflora, quantitative data become difficult to interpret vis-a-vis the "normal" flora of a given site. Culturing a skin surface gives no indication whether the isolate represents resident or transient flora. It can be inferred from prevalence studies that an organism that is recovered repeatedly in large numbers is indeed a resident. However, minor residents are unlikely to be distinguishable from transients. Distinguishing residents from transients on the vulva is likely to be even more difficult because of the large number of transients contributed continuously by exogenous sources from the anus, urethra, and vagina. Thus, determining exactly what comprises the normal resident flora of the vulva will be difficult or impossible using traditional culture-based microbiological methods.

Culture-Based Studies

One of the first studies of vulvar microflora attempted to understand the relationship between urinary tract infections and the microflora of the vestibule (19). These researchers found that women with recurrent infections were more likely to be colonized with Gram-negative bacteria and speculated that the vestibule could serve as a reservoir for these potential pathogens. Moreover, the vestibules of normal healthy women were generally free from Gram-negative bacilli and were also found to have acidic pH more similar to the vagina than to that of other skin surfaces; the researchers suggested that this low pH might serve to inhibit the growth of Gram-negative enteric bacteria. Lactobacilli and coryne-bacteria were reported to constitute the predominant flora of the vestibule in this study. A more recent report (20) has shown a gradient in populations of enteric organisms from the perineum, through the vestibule, to the vagina. The pioneering study aimed at gaining an overall understanding of vulvar microflora was reported in 1979 (21), and remains the most comprehensive investigation to date. Eighteen normal healthy women with a mean age of 39 participated in this study, which compared vulvar skin with forearm skin, using the cup-scrub sampling method (22). Microbial counts were higher on the vulva (2.8 x 106/cm2) than on the forearm (6.4 x 102/cm2). Lipophilic diphtheroids, coagulase-nega-tive staphylococci, micrococci, nonlipophilic diphtheroids, and lactobacilli were the dominant microflora of the vulva, and streptococci, Gram-negative rods, and yeasts were also present. Most categories of bacteria found on the vulva were present at higher density and prevalence as compared with the forearm microflora. Exceptions were noted for micrococci and Bacillus, which tended to occur more frequently on forearm skin. This may reflect the better adaptation of these organisms to the drier environment found on the forearm. This study also reported a surprisingly higher incidence of S. aureus on the vulva (67%) than on the forearm (11%). Quantitative results from this study are shown in Table 2.

A more recent study (23) investigated the bacterial population of the epithelial surface of the labia majora during the menstrual cycle. Samples were obtained at days 2, 4, and 21 of the menstrual cycle, and the results essentially confirmed those of the earlier study with regard to incidence and densities of the microorganisms isolated and identified. While the authors expected vulvar counts of vaginally derived organisms (lactobacilli and Gardnerella vaginalis)

Table 2 Microbial Counts on Vulva and Forearm Skin (Mean of 18 Subjects)

Organisms

Vulva (cfu/cm2)

Forearm (cfu/cm2)

Staphylococcus aureus

4.1 x 104

1.4 x 10

Coagulase-negative staphylococci

5.7 x 105

1.8 x 102

Micrococci

5.1 x 105

2.9 x 102

Streptococci

3.7 x 102

0.48 x 10

Lipophilic diphtheroids

7.9 x 105

1.1 x 102

Nonlipophilic diphtheroids

4.6 x 105

1.1 x 10

Lactobacillus spp.

4.6 x 105

0.96 x 10

Bacillus spp.

Not detected

1.2 x 10

Gram-negative rods

1.8 x 103

0.12 x 10

Yeasts

8.2 x 10

0.8 x 10

Total count

2.8 x 106

6.4 x 102

Source: Adapted from Ref. 21.

Source: Adapted from Ref. 21.

Table 3 Bacterial Populations on Vulvar Skin (cfu/cm2) During the Menstrual Cycle (Mean of 20 Subjects)

Organisms Day 2 Day 4 Day 21

Table 3 Bacterial Populations on Vulvar Skin (cfu/cm2) During the Menstrual Cycle (Mean of 20 Subjects)

Organisms Day 2 Day 4 Day 21

Staphylococcus aureus

5.6 x

103

4.0 x 103

6.1

x

103

Coagulase-negative staphylococci

2.2 x

105

1.2 x 105

6.9

x

105

Micrococci

5.7 x

104

2.0 x 104

6.5

x

103

Lipophilic diphtheroids

3.1 x

105

3.3 x 105

4.5

x

105

Nonlipophilic diphtheroids

8.9 x

105

1.5 x 105

9.0

x

103

Beta hemolytic streptococci

1.0 x

102

Not detected

6.5

x

10

Alpha hemolytic streptococci

7.1 x

102

6.9 x 102

3.6

x

103

Nonhemolytic streptococci

3.1 x

105

1.6 x 102

1.2

x

102

Gram-negative rods

1.9 x

102

Not detected

3.5

x

102

Gram-positive rods

1.0 x

104

5.5 x 10

8.5

x

103

Nonpathogenic neisseria

Not detected

Not detected

1.9

x

103

Lactobacilli

1.8 x

105

2.9 x 103

3.4

x

105

Gardnerella vaginalis

5.7 x

102

2.2 x 105

8.0

x

104

Yeasts

Not detected

1.0 x 10

Not detected

Total count

2.0 x

106

8.9 x 105

1.6

x

106

Source: Adapted from Ref. 23.

Source: Adapted from Ref. 23.

to increase during menstruation, no significant changes in the microflora occurred at any of the three time points (Table 3).

A larger study involving 224 participants compared the frequencies and semiquantitative densities of selected microflora from the posterior vaginal fornix and the inner labial groove of the vulva (24). This study focused on aerobic and facultative species that are potentially pathogenic or otherwise have a known association with vaginal, vulvar, or urinary tract infections. Results (Table 4) revealed that the same organisms were generally found at both sites, but frequencies were significantly higher in the labial groove for a number of species, including S. aureus and other staphylococci, coliforms, and Gram-negative nonlactose fermenters, and Group D streptococci. G. vaginalis, in contrast, was more common in the vagina. The researchers also addressed the question whether daily wear of panty liners would increase the prevalence and/or density of clinically important species. No changes were detected that would suggest any adverse clinical outcomes. Similarly, more recent studies (25,26) have also concluded that tight-fitting underwear and panty liners are unlikely to increase microbial risk.

Nonculture-Based Studies

The microflora of a microbial community play many roles, such as resisting colonization by pathogens and nutritional interactions that shape and control the population (27). Adding to this complexity are the ecological pressures that the

Table 4 Comparison of the Frequencies and Densities of Selected Microorganisms Isolated from the Vagina and Vulva in 224 Women

Microorganisms

Vagina

Vulva

% Culture positive

Densitya

% Culture positive

Densitya

Candida albicans

12.1

1.3

8.5

1.3

Other yeasts

3.1

1.2

2.7

1.4

Gardnerella vaginalis

12.9

2.2

4.0b

1.5

Staphylococcus aureus

2.2

1.1

6.3b

1.8

Other Staphylococcus spp.

35.3

1.2

87.1b

1.9

Coliforms

17.0

1.7

37.9b

1.3

Gram-negative

2.7

1.2

7.1b

1.0

nonlactose fermenters

Proteus spp.

1.3

1.0

3.1

1.2

Pseudomonas spp.

Not detected

Not detected

Streptococcus Group A

0.9

1.0

1.3

1.5

Streptococcus Group B

8.9

1.8

10.3

1.7

Streptococcus Group D

19.6

1.5

30.8b

1.9

Streptococcus beta

Not detected

0.4

1.0

hemolytic, non-A,B,D

Viridans strep

15.2

1.8

19.6

1.7

Semiquantitative 0-4 scale.

bSignificantly different from vaginal site, p < 0.05.

Source: Adapted from Ref. 24.

Semiquantitative 0-4 scale.

bSignificantly different from vaginal site, p < 0.05.

Source: Adapted from Ref. 24.

host brings to bear on the community, which vary from one individual to another and over time. Understanding the diversity and role of individual microbes in the various human niches has, thus, been hampered severely by existing culture-based microbiological methodologies. The recent advent of molecular methodologies has been a boon to understanding the complex nature of the oro-gastrointestinal microflora (28,29). Future refinement and expansion of these molecular approaches potentially will unveil intricate details of the various ecological niches of the human.

Although tremendous strides have been made in community analyses of microbial populations, our knowledge of the ecology of the human microflora is largely still in its infancy. Many studies of environmental microbial communities have demonstrated clearly the limitations of culture-dependent techniques for analyses. Surprisingly, it has been estimated that more than 90% of the microbial communities are not amenable to culture-based analyses and thus, the composition (which species), species richness (number of species), and evenness (relative abundance of species) of microbial communities have been subjected to biased analyses using culture-dependent techniques for community analyses. Moreover, culture-based studies are limited fundamentally by their ability to grow and enumerate microorganisms on artificial culture media, where complex ecological and nutritional interactions found in natural habitats may be impossible to duplicate, even if such interactions were not so poorly understood.

The introduction of culture-independent technologies—in particular, those based on ribosomal RNA (rRNA) and their genes (rDNA)—are rapidly replacing conventional detection and enumeration methods and can provide insights into the phylogenetic diversity of communities. At present, the 16S rRNA molecule is the measure of diversity used most commonly because it is most amenable to DNA sequence analyses. By simply retrieving rDNA sequences from microbial samples, for example, using 16S rRNA-specific oligonucleotide primers and the polymerase chain reaction, the biodiversity and population dynamics of the ecosystem can be investigated rapidly. Large-scale cloning and sequencing of 16S rRNA from feces has revealed that microbial diversity has been grossly underestimated (29). Designing of specific probes to the 16S rRNA sequences allows estimation of the microbiota diversity by dot-blot hybridization techniques (30). More accurate enumeration of the microbiota can be achieved by fluorescent in situ hybridization (31).

Fingerprinting techniques for complex communities including denaturing/ temperature gradient gel electrophoresis have been applied to human intestinal samples. A recent study that analyzed 13,355 prokaryotic rRNA gene sequences from multiple intestinal sites revealed that each individual's microbiota is remarkably stable and unique (29). Further improvements in molecular methods will involve the analysis of larger numbers of samples with greater speed and ease using high-throughput techniques such as DNA arrays. Table 5 provides a brief comparison of some culture-independent techniques used for analysis of microbial communities.

Table 5 Diversity Indices for a Hypothetical Community

Method

Richness

Evenness

LH-PCR

26

0.814

ARISA

68

0.951

DGGE

32

0.900

T-RFLP (RsaI)

42

0.904

T-RFLP (MspI)

40

0.885

T-RFLP (HhaI)

38

0.881

Ideal species level

41

1.00

Note: Calculations for the ideal values were based on a model community with all populations at equal abundances.

Abbreviations: LH-PCR, length heterogeneity polymerase chain reaction; ARISA, automated ribosomal intergenic spacer analysis; DGGE, denaturing gradient gel electrophoresis; T-RFLP, terminal restriction fragment length polymorphism with restriction enzymes RsaI, MspI, and HhaI. Source: Adapted from Ref. 32.

Note: Calculations for the ideal values were based on a model community with all populations at equal abundances.

Abbreviations: LH-PCR, length heterogeneity polymerase chain reaction; ARISA, automated ribosomal intergenic spacer analysis; DGGE, denaturing gradient gel electrophoresis; T-RFLP, terminal restriction fragment length polymorphism with restriction enzymes RsaI, MspI, and HhaI. Source: Adapted from Ref. 32.

Culture-Independent Analyses of Vaginal-Vulvar Communities

Some of these molecular techniques for community analyses have been applied to analyze samples obtained from the urogenital tracts of healthy female participants. Results indicated that the diversity and kinds of organisms that comprise the vaginal microbial community varied among women studied (33,34). Species of Lactobacillus dominated the communities in most of the vaginal samples analyzed. However, as an unexpected and surprising result, Atopobium sp. was identified as a dominant member in one woman and appreciable numbers of Megasphaera spp. and Leptotrichia spp. were identified in two women; none of these species has been shown previously to be common members of this ecosystem (34). Some progress has been made regarding the analysis of human vulvar microbial communities by molecular techniques (35). The results indicate that the microbial communities are more complex than previously thought and the complexity of the microbial communities of the labia majora and minora varies among women. In some cases, the communities are comparatively simple and contain few numerically dominant populations, whereas others are more complex. A further analysis of vulvar samples via nonculture-based techniques will undoubtedly provide more insight into these complex bacterial communities. Moreover, molecular techniques may open the door to the discovery of entirely new groups of microorganisms, independently of whether they can be cultured in the laboratory.

An unusual group of organisms was described in the late 1970s and was noted for its ability to grow at extreme temperatures (36). DNA sequence analyses showed that these organisms, which as a group exist typically at high temperatures and/or produce methane, clustered together well away from known bacteria (eubacteria) and eukaryotes. This observation led to the proposal that life be divided into three domains: eukaryotes, eubacteria, and archaea. Not only have these organisms been isolated from extreme environments (such as icebergs or hot sulfur springs), they have also been identified in human clinical samples. The methanogenic Archaea subsequently have been isolated from the human oral cavity (37) as well as from the human gut (38) and the vagina (39). Their potential presence on the vulva and their overall role in human microbial ecology are yet to be determined.

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