MAJOR ARTICLE

Human α-amylase Present in Lower-Genital-

Tract Mucosal Fluid Processes Glycogen to
Support Vaginal Colonization by Lactobacillus
Gregory T. Spear,1 Audrey L. French,2 Douglas Gilbert,1 M. Reza Zariffard,1 Paria Mirmonsef,1 Thomas H. Sullivan,1
William W. Spear,1 Alan Landay,1 Sandra Micci,2 Byung-Hoo Lee,3 and Bruce R. Hamaker3
1
Department of Immunology/Microbiology, and 2CORE Center of Cook County Health and Hospitals System, Rush University Medical Center, Chicago,
Illinois; and 3Whistler Center for Carbohydrate Research, Department of Food Science, Purdue University, West Lafayette, Indiana

Lactobacillus colonization of the lower female genital tract provides protection from the acquisition of sexually
transmitted diseases, including human immunodeficiency virus, and from adverse pregnancy outcomes. While
glycogen in vaginal epithelium is thought to support Lactobacillus colonization in vivo, many Lactobacillus iso-
lates cannot utilize glycogen in vitro. This study investigated how glycogen could be utilized by vaginal lacto-
bacilli in the genital tract. Several Lactobacillus isolates were confirmed to not grow in glycogen, but did grow in
glycogen-breakdown products, including maltose, maltotriose, maltopentaose, maltodextrins, and glycogen
treated with salivary α-amylase. A temperature-dependent glycogen-degrading activity was detected in genital
fluids that correlated with levels of α-amylase. Treatment of glycogen with genital fluids resulted in production
of maltose, maltotriose, and maltotetraose, the major products of α-amylase digestion. These studies show that
human α-amylase is present in the female lower genital tract and elucidates how epithelial glycogen can support
Lactobacillus colonization in the genital tract.
Keywords. α-amylase; female genital tract; glycogen; Lactobacillus; maltose.

Colonization of the lower genital tract with Lactobacil-
lus is an important component of the reproductive
health of women. Lactobacillus inhibits other microbes
in the genital tract, primarily by creating a low genital
pH through the production of lactic acid [1–4]. This in-
hibition results in a genital microbiota that is predom-
inantly composed of Lactobacillus in many women.
Factors that disrupt Lactobacillus colonization can
lead to a shift of the genital microbiota to bacterial vag-
inosis (BV) in which Lactobacillus is no longer the pre-
dominant type of bacteria [1, 5–7]. Epidemiologic
studies show that a genital microbiota dominated by
Lactobacillus is protective from the acquisition of
human immunodeficiency virus (HIV) when compared
Received 13 February 2014; accepted 4 April 2014; electronically published 15
April 2014.
Correspondence: Gregory T. Spear, PhD, Department of Immunology/Microbiology,
Rush University Medical Center, 1653 W Congress Parkway, Chicago, IL 60612
(gspear@rush.edu).
The Journal of Infectious Diseases 2014;210:1019–28
© The Author 2014. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/infdis/jiu231
to BV (reviewed in [8]). In HIV-infected women, a
Lactobacillus-dominated microbiota is also associated
with decreased shedding of virus, decreased risk of
heterosexual transmission to men, and vertical trans-
mission to the fetus during birth [9–14]. Further, a gen-
ital microbiota dominated by Lactobacillus is associated
with a lowered risk of acquiring other sexually trans-
mitted diseases (STDs), such as herpes simplex virus
type 2, chlamydia, and gonorrhea [15, 16]. Finally, Lac-
tobacillus also protects against developing BV, pelvic
inflammatory disease, and adverse fetal outcomes
[17–21]. Certain species of Lactobacillus have been
found to be more protective [4, 22].
Although Lactobacillus is recognized as important
for genital tract health, the factors responsible for its
colonization are not well understood. For example,
while BV can be treated with antibiotics, Lactobacillus
does not always become reestablished as the dominant
bacteria type, and BV recurs in 60%–80% of cases after
1 year [23, 24]. However, a relationship between vaginal
epithelial glycogen and Lactobacillus colonization in the
female lower genital tract has been recognized for
many decades. Thus, Cruickshank and others [25, 26]

Vaginal Amylase and Glycogen Degradation • JID 2014:210 (1 October) • 1019

observed that at puberty, glycogen becomes expressed in the
vaginal epithelium and at the same time colonization with Lac-
tobacillus becomes apparent. Those studies showed that both
glycogen expression and colonization by Lactobacillus persist
through life until at menopause, when both Lactobacillus colo-
nization and glycogen expression decline.
Despite the evidence that glycogen promotes colonization by
lactobacilli, most isolates of Lactobacillus do not grow in vitro
in media containing glycogen as the carbohydrate source.
Thus, Stewart-Tull [27] reported in 1964 that a number of isolates
of Lactobacillus did not ferment glycogen. Later in that decade,
Wylie and Henderson [28] showed that 39 of 42 isolates of Lac-
tobacillus did not ferment glycogen, even when the glycogen was
isolated from the genital tract of women. More recently, Martin
et al [29] showed 45 strains of vaginal Lactobacillus could not use
glycogen. The lack of Lactobacillus growth in vitro in the presence
of glycogen leaves open the question of how glycogen could be
utilized in vivo by Lactobacillus. The goal of this study was to in-
vestigate how glycogen could be utilized by genital lactobacilli in
the environment of the lower female genital tract.
MATERIALS AND METHODS
Subjects
Genital cervical-vaginal lavage (CVL) samples were obtained
from women who had provided written informed consent. All
procedures followed Department of Health and Human Service
guidelines. The study was approved by the Cook County Health
and Hospitals System Institutional Review Board. Samples were
collected weekly over a 1–3 month period. All women reported
not douching and not engaging in sex within the 48 hours be-
fore sample collection. Trichomonas, yeast, and BV were not
detected at the time of sample collection as determined by
wet mount, potassium hydroxide, and Amsel criteria [30].
The CVL samples were obtained by irrigation of the cervix
with 10 mL of nonbacteriostatic sterile saline, followed by aspi-
ration from the posterior fornix.
Bacterial Cultures
All reagents were purchased from Sigma (Sigma-Aldrich, St
Louis, MO) unless noted. A basal de Man, Rogosa, and Sharpe
(MRS) medium contained proteose peptone #3 (10.0 g, Remel,
Thermo Fisher Scientific, Lenexa, KS), beef extract (10.0 g,
Remel), yeast extract (5.0 g, Fisher), sorbitan monooleate
(1.0 g), ammonium citrate (2.0 g), sodium acetate (5.0 g),
MnSo4xH2O (0.05 g), and Na2HPO4 (2.0 g) per liter. Medium
was supplemented with 2% glucose or other carbohydrates, in-
cluding glycogen (from oyster), maltodextrins, maltose, malto-
triose, and maltopentaose. In some experiments, normal saliva
collected from 1 donor was immediately filtered (0.45 µm), and
aliquots frozen. Upon thawing, saliva was added to glycogen-
containing MRS medium immediately before the addition of
1020 • JID 2014:210 (1 October) • Spear et al
bacteria to give concentrations of 2.5%, or 0.25% of the culture
volume. Most cultures were in ambient air, except some run
under anaerobic conditions using the Anaero Pack System (Mit-
subishi Gas Chemical Co, Tokyo).
Bacteria tested included Lactobacillus jensenii (ATCC
#25258), L. gasseri (ATCC #9857), and L. johnsonii (ATCC #
33200). Cultures (4 mL) were initiated from frozen stocks
with 50 µL phosphate-buffered saline–washed bacteria at time
0 and the optical density (OD) at 600 nm was taken at 24-
hour intervals.
Glycogen Degradation by Genital Fluids
Genital fluid samples collected by CVL were centrifuged at 4°C
in a microfuge for 10 minutes to pellet bacteria. The superna-
tants (25 µL) were added to oyster glycogen (75 µL of 5 mg/mL)
dissolved in phenol-red-free Roswell Park Memorial Institute
1640 medium. Aliquots were incubated at either 37°C or 4°C
for 90 minutes and the amount of glycogen then measured
using a colorimetric assay [31] by adding 50 µL of each of the
following solutions: 10% potassium iodide, 0.01 N potassium
iodate, and 1 N acetate. The mixture was incubated for 10 min-
utes and read at 565 nm. In some experiments, no extra glyco-
gen was added to genital fluid samples so that degradation of
endogenous glycogen could be observed.
Analysis of Oyster Glycogen Degradation Products
Oyster glycogen (20 mg/mL, weight per volume [w/v]) was hy-
drolyzed by adding saliva (see above) at 2.5% or 0.5% of the final
volume for 10, 30, and 120 minutes at 37°C . Hydrolyzed sam-
ples were diluted 10× with deionized water. Molecular weight
(MW) distributions of salivary α-amylase-treated glycogen sam-
ples were obtained by high-pressure size exclusion chromatog-
raphy (HPSEC) using a Varian model 9012 high-performance
liquid chromatography pump system connected to a Varian
Star 9040 refractive index detector (Agilent Technologies,
Santa Clara, CA) at room temperature. Injected samples
(100 µL, 0.45 µm filtered) were separated using Sephacryl TM
S-500 HR gel filtration (GE Healthcare, Piscataway, NJ). The el-
uent was purified water (18.2 MΩ) with 0.02% sodium azide
[32]. Pullulan standards (Polymer Laboratories Inc, Amherst,
MA) were used as a standard curve.
Oligosaccharide Analysis by High-Performance Anion-
Exchange Chromatography
Hydrolysis products from oyster glycogen treated with either
the collected saliva or the vaginal fluids were determined by a
high-performance anion-exchange chromatography (HPAEC)
system equipped with an electrochemical detector (Dionex,
Sunnyvale, CA). The filtered (0.45 µm) samples were separated
using a CarboPac PA-1 pellicular anion-exchange column (Di-
onex) with gradient elution from 100% eluent A (150 mM
NaOH) to 100% eluent B (600 mM NaOAc in 150 mM
NaOH) [33].
Bacterial Polymerase Chain Reactions
Bacteria specific quantitative polymerase chain reaction poly-
merase chain reaction (qPCR) assays were performed on isolat-
ed CVL genomic DNA [34]. Each 20 µL qPCR reaction
contained Supermix (Bio-Rad Hercules, CA), primers, probes
(IDT, Coralville, IA), and 1–10 ng template DNA. Primers
and probes for the bacteria were described previously [34, 35].
Known quantities of 16S rRNA plasmid targets were used as
standards [34, 35].
Enzyme Detection
Enzyme-linked immunosorbent assay (ELISA) was used to
quantify human pancreatic α-amylase (AbCam, Cambridge,
UK) and acidic α-glucosidase (MyBiosource, San Diego, CA).
RESULTS
Growth of Lactobacillus in Glucose, Glycogen, and Other
Glucose Polymers
We first performed experiments to confirm previous reports
that some genital Lactobacillus isolates do not grow in media
with glycogen as the source of carbohydrate [27–29]. L. gasseri
(ATCC #9857) was cultured in medium containing either 2%
glucose, 2% glycogen, or no added carbohydrate (Figure 1).
Over a 72-hour period, a relatively large increase in absorbance
was observed for glucose cultures, while the ODs of cultures
with either no carbohydrate or glycogen were similar. A similar
Figure 1. Lack of L. gasseri growth in glycogen. L. gasseri was cultured
in MRS medium that contained either no added carbohydrates (No Carb),
2% glucose, 2% glycogen, or 2% starch for 72 hours. The OD at 600 was
read each day. Each culture condition was run in duplicate and the dupli-
cates averaged. Each culture experiment was performed at least 3 times
with similar results. Abbreviations: MRS, de Man, Rogosa, and Sharpe;
OD, optical density.
Vaginal Amylase and Glycogen Degradation
lack of growth in glycogen when compared with no carbohy-
drate was observed with L. jensenii (ATCC #25258) and L. john-
sonii (ATCC # 33200) (Figure 2). The lack of growth in
glycogen was seen under both aerobic and anaerobic conditions
(data not shown). Soluble starch (2% w/v) was also tested be-
cause its structure is similar to that of glycogen. No growth
was observed using starch as the sole carbohydrate (Figure 1).
The lactobacilli tested above were able to utilize glucose for
growth, but not glycogen, a high-molecular-weight polymer of
glucose. Smaller polymers of glucose were also tested for their
ability to support growth of lactobacilli. All 3 isolates of Lacto-
bacillus were able to use 2% maltose for growth (Figure 2A–C).
L. jensenii grew to relatively high levels by 48 hours in both mal-
totriose ( polymer of 3 glucose units) and maltopentaose (5 glu-
cose units) (Figure 2D).
Maltodextrins are derived from the partial hydrolysis of
starch, and maltodextrins that are the least hydrolyzed have
the lowest dextrose equivalent (DE) values. Growth of L. gasseri,
L. jensenii, and L. johnsonii was also assessed in media contain-
ing 2% of 1 of 3 different maltodextrin preparations; DE 4–7,
13–17, and 16–19. All 3 of the species grew in the maltodextrins
as a source of carbohydrates (Figure 2A–C), although the level
of growth was generally less than that for glucose.
Growth of Lactobacilli in α-Amylase-Treated Glycogen
The above experiments indicated that some genital lactobacilli
cannot utilize glycogen or starch for growth but can utilize
smaller glucose polymers (dimers, trimers, pentamers). There-
fore, the effect of glycogen breakdown by α-amylase on Lacto-
bacillus growth over 72 hours was tested by adding a small
amount of filtered saliva (2.5% or 0.25%) to the culture medium
as a source of α-amylase. While L. gasseri did not grow in gly-
cogen alone, it grew at a rate similar to cultures with glucose
when cultured in glycogen with 2.5% filtered saliva added (Fig-
ure 3A). No growth above the control was seen with 2.5% saliva
only. Similar rapid growth of L. gasseri was seen when 0.25%
saliva was added to glycogen in cultures (data not shown).
Growth of L. johnsonii in glycogen with 2.5% saliva was also
similar to growth in glucose (Figure 3B). In contrast, L. jensenii
grew slowly in glycogen with saliva for the first 48 hours when
compared to glucose, but growth increased by 72 hours to a
level similar to glucose (Figure 3C).
The characteristics of the saliva-treated and control-treated
glycogen were analyzed (Figure 4). HPSEC showed a peak at ap-
proximately 45 minutes that corresponded to undegraded gly-
cogen. As little as 10 minutes of incubation with 2.5% saliva
resulted in the appearance of a peak at 69 minutes, which was
not present in untreated glycogen (Figure 4A). Longer incuba-
tion times (30 minutes, 120 minutes) led to an increase in the
size of this peak. Analysis of the smaller carbohydrates in the
120-minute tube by HPAEC (Figure 4B) showed the appearance
of peaks corresponding to maltose (G2) and maltotriose (G3)
• JID 2014:210 (1 October) • 1021
Figure 2. Growth of lactobacilli in glucose polymers. L. gasseri (A), L. jensenii (B and D), and L. johnsonii (C ) were cultured in MRS medium
that contained either no added carbohydrates (No Carb) or 2% of either glucose, maltose, maltodextrin 4–7, maltodextrin 13–17, maltodextrin 16–19,
maltotriose, or maltopentaose for 72 hours. The OD at 600 was read each day. Abbreviations: MD, maltodextrin; MRS, de Man, Rogosa, and Sharpe;
OD, optical density.

with some maltotetraose (G4), which are known major prod-
ucts of the α-amylase reaction [36, 37].
Vaginal Fluid From Normal Women Contains an Activity That
Degrades Glycogen
The above experiments showed that salivary α-amylase could
process glycogen into products that could be used by vaginal
lactobacilli for growth. Experiments were next performed to de-
termine if any α-amylase-like activity was present in the genital
fluid of women [31]. Samples were obtained from women that
were all premenopausal with ages ranging from 34 to 48. Ten
were African American and the race of 1 classified as “other.”
Aliquots of 2 genital mucosal fluid samples (collected by lavage)
with detectable glycogen were incubated at either 4°C or 37°C
for 90 minutes, and a colorimetric iodine assay was then used to
detect glycogen [31]. Sample A had more detected glycogen
than sample B after incubation at 4°C (Figure 5A). For both
of the samples, less glycogen was detected in the aliquot incu-
bated at 37°C than in the portion incubated at 4°C, indicating
temperature-dependent degradation of glycogen.
Several other genital-fluid samples were tested (not shown)
for glycogen breakdown in a similar manner. However, many
of the samples had very low or no detectable glycogen and so
glycogen degradation could not be assessed. Therefore, in fur-
ther experiments, exogenous glycogen was added to genital flu-
ids collected from women to ensure that degradation could be
detected. Figure 5B shows the results from incubating genital
fluid from 4 different individuals with exogenous glycogen at

1022 • JID 2014:210 (1 October) • Spear et al

Figure 3. Growth of lactobacilli in salivary α-amylase-treated glycogen. L. gasseri (A), L. jensenii (B), or L. johnsonii (C) were cultured in MRS broth that
contained either no added carbohydrates (No Carb), 2% glucose, 2% glycogen, 2.5% saliva alone, or glycogen plus saliva for 72 hours. The OD at 600 was
read each day. Abbreviations: MRS, de Man, Rogosa, and Sharpe; OD, optical density.

Figure 4. Degradation of glycogen by salivary α-amylase. Glycogen (2%
oyster) was incubated for 10 minutes with no saliva (Oyster) or for 10, 30,
or 120 minutes with 2.5% saliva. The size of the degraded glycogen was
determined by HPSEC (A) and the products analyzed by HPAEC (B). Abbre-
viations: HPAEC, high-performance anion-exchange chromatography;
HPSEC, high-pressure size exclusion chromatography.
4°C and 37°C. Less glycogen was detected for all 4 samples
after incubation at 37°C compared to incubation at 4°C, with
sample F exhibiting the most glycogen degradation and C
having the least. Twenty-one samples from a total of 11
women were tested in this manner and the percent degradation
was calculated using the following formula: 1 – glycogen 37/gly-
cogen 4 × 100. The median degradation for the 21 samples was
73%, with only 3 samples having values less than 20%
(Figure 5C).
To investigate whether the source of the glycogen-degrading
activity could be bacterial, PCR was used to quantify several
types of bacteria in the same samples. No significant relation-
ships were observed between either L. crispatus, L. iners, or
Gardnerella vaginalis and the glycogen degradation activity in
women (P > .05, Spearman rank correlation, Figure 6A–C).
Similarly, no relationships were observed between either L. jen-
senii, Mycoplasma hominis, or Sneathia (not shown). Further,
the vaginal pH that was measured for diagnosing bacterial vag-
inosis was not significantly associated with α-amylase activity
(P > .05, Figure 6D). These experiments therefore showed no
obvious relationship between the types of microbiota in genital
tract samples and the levels of the glycogen degradation activity.
To investigate a possible human source of the glycogen deg-
radation activity, the genital fluid samples were tested for
human α-amylase using an ELISA for pancreatic α-amylase.
All tested samples had ELISA-detectable α-amylase, and the
median α-amylase detected in genital fluids was 1.86 mU/mL
(range, 0.02–12.7 mU/mL). The pancreatic α-amylase levels
were strongly associated (Spearman r = 0.73; P = .0002) with
the amount of glycogen degradation (Figure 6E ). Acidic
α-glucosidase was also detected in some of the samples
(Figure 6F ). However, its presence was not significantly associ-
ated with glycogen degradation (P > .05).

Vaginal Amylase and Glycogen Degradation • JID 2014:210 (1 October) • 1023

Figure 5. Temperature-dependent degradation of glycogen in genital fluid. A, Aliquots of genital fluid collected from 2 subjects (A and B) were incubated
at either 37°C or 4°C for 90 minutes and the level of endogenous glycogen was measured with a colorimetric iodine assay. B, Glycogen was added to
genital fluids collected from 4 other women (C–F) or saline, and aliquots were incubated at either 4°C or 37°C for 90 minutes. At the end of the incubation,
the amount of glycogen in the samples was measured with the colorimetric iodine assay. C, Degradation in 21 genital fluid samples was measured as in
B and the percent degradation was calculated by the following formula: 1 – glycogen 37/glycogen 4 × 100.

Glycogen Breakdown Products Generated by Genital Fluids
Because the above experiments suggested that glycogen pro-
duced by the genital epithelium could be degraded into smaller
glucose polymers such as maltose and maltotriose in genital flu-
ids, genital fluids from several women were analyzed by HPAEC
for small carbohydrates. However, only trace amounts of small
sugars were detected, and these levels were too low to determine
the characteristics of these sugars. Therefore, in further experi-
ments, exogenous glycogen was added to genital fluids and the
types of carbohydrates that were generated were analyzed by
HPAEC. The glycogen used for this experiment had only
trace peaks corresponding to glucose, maltose, maltotriose,
and maltotetraose (Figure 7A). After incubation for 48 hours
in buffer with no added genital fluids, there was essentially no
change in these peaks (Figure 7B). However, incubation for 48
hours with genital fluid from 2 different subjects resulted in
substantial increases in peaks corresponding to maltose (G2),
maltotriose (G3), and maltotetraose (G4), the major known
α-amylase digestion products (Figure 7C and 7D). Trace
peaks corresponding to glucose and maltopentaose were
also observed. Similar results were observed after 24 hours of
incubation, although the size of each of the peaks was reduced
when compared with the 48-hour peaks (not shown). No peaks
were detected in genital fluids with no added glycogen (not
shown).
DISCUSSION
Genital colonization with Lactobacillus has been associated with
protection from STDs, BV, and adverse pregnancy outcomes [1,
5, 6, 8–21]. However, the factors that promote and maintain Lac-
tobacillus colonization are not well understood. Glycogen is

1024 • JID 2014:210 (1 October) • Spear et al

Figure 6. Relationship of glycogen degradation with genital bacteria, vaginal pH, α-amylase and α-glucosidase. The percent of glycogen degradation
(Figure 5C) in 21 samples was plotted along with either genital-tract levels of L. iners (A), L. crispatus (B), G. vaginalis (C), vaginal pH (D), pancreatic
α-amylase (E ), or α-glucosidase (F ).

synthesized in the vaginal epithelium and has long been
postulated to provide an energy source for genital Lactobacillus
[25, 26]. However, it has not been clear how Lactobacillus uses
glycogen because most genital isolates do not grow on glyco-
gen in vitro [27, 28]. This study shows that (1) isolates of
Lactobacillus that cannot use glycogen can grow in glycogen
breakdown products, including maltose and maltotriose; (2)
an activity that breaks down glycogen to maltose and malto-
triose is present in the lower-genital-tract fluid of women; (3)
human α-amylase is present in the genital fluids of women;
(4) the glycogen breakdown activity in genital fluid correlates
with genital levels of α-amylase; and (5) incubation of genital
fluids with glycogen generated products such as maltose,
maltotriose, and maltotetraose. This study therefore supports
a model where α-amylase present in the genital tract breaks
down glycogen into smaller polymers, such as maltose and mal-
totriose, that are then utilized by Lactobacillus to aid its
colonization.
Glycogen is composed mainly of α1,4-linked chains of glu-
cose but also has α1,6-linked branches. Human α-amylase
(both salivary and pancreatic) preferentially processes glycogen
to α1,4-linked multimers, such as maltose and maltotriose, but
also results in production of α-dextrins that contain α1,6
branches and α1,4 linkages [36, 37]. This study shows that gen-
ital lactobacilli can grow well in the small unbranched glucose
polymers. It is not clear, however, if lactobacilli can utilize
branched carbohydrates.
There are some previous studies of utilization of maltose by
Lactobacillus in the food and beverage industries, such as L.
casei, and L. reuteri [38, 39]. However, those Lactobacillus spe-
cies are different than the ones found in the genital tract, includ-
ing L. crispatus, L. jensenii, L. iners, and L. gasseri [4, 20, 40, 41].
There is a paucity of studies on the utilization of small glucose
polymers by genital tract lactobacilli. This study suggests that
the small glucose polymers can support growth by genital Lac-
tobacillus in vivo, and the consequent acid production suppress-
es growth of nonlactobacilli. The low genital pH found may vary
somewhat depending on the species of Lactobacillus that is
dominant [4]. The pH can be neutralized by factors such as sex-
ual activity due to semen or by douching [42–44]. During peri-
ods of increased pH, other bacteria may utilize the glycogen
breakdown products, and due to competition for this food
source and production of pH buffering amines [45], prevent
Lactobacillus from acidifying the genital environment.

Vaginal Amylase and Glycogen Degradation • JID 2014:210 (1 October) • 1025

Figure 7. Breakdown of glycogen by genital fluids. Glycogen was incubated for 0 hours (A) or 48 hours (B) with saline or 48 hours with genital fluid
collected in saline from 2 different subjects (C and D). After incubation the size of small carbohydrates was determined by HPAEC. Abbreviation: HPAEC,
high-performance anion-exchange chromatography.
Starch and glycogen have similar structures and there are re-
ports of nongenital lactobacilli, such as L. fermentum and L.
plantarum, degrading starch [46]. While there have been
some reports of genital Lactobacillus isolates breaking down gly-
cogen, this was later discovered in some cases to have been due
to serum added to the culture medium that likely was a source
of α-amylase [27, 47]. However, some genital Lactobacillus iso-
lates can directly utilize glycogen [28].
We observed in this study that α-amylase and Lactobacillus
levels were not correlated (Figure 6). However, we recently ob-
served that there are variable levels of glycogen in the genital
fluids of different women and that the levels of glycogen corre-
late strongly with the level of colonization by Lactobacillus
(manuscript in preparation). Based on that observation, we pos-
tulate that women must have 2 conditions met to be dominantly
colonized by Lactobacillus: (1) an adequate amount of glycogen
in genital fluid; and (2) α-amylase to cleave the glycogen into
smaller carbohydrates. If glycogen is low, then Lactobacillus lev-
els will also be low. Potentially, if α-amylase levels were low but
glycogen high, this could also lead to low Lactobacillus. Our
studies do not rule out the possibility that other enzymes, in-
cluding bacteria-derived enzymes, could process glycogen in
1026 • JID 2014:210 (1 October) • Spear et al
some women. Also, because these experiments were conducted
with women consisting mostly of 1 ethnic group, it is possible
that women from other groups could have differing genital am-
ylase activity.
This study has implications for the measurement of glycogen
in genital secretions because α-amylase acts on glycogen, result-
ing in a mixture of glycogen and smaller glucose polymers. Gly-
cogen is made in the epithelium, but it has not been clearly
established how glycogen in epithelial cells becomes available
for Lactobacillus utilization. Factors such as the amount of gly-
cogen in cells, the rate of cell release, and the rate of glycogen
breakdown are unknown. In our study, samples were collected
on ice so that glycogen breakdown was arrested relatively quick-
ly, but even so, many women had glycogen levels too low to
measure with the iodine method. Further, glycogen breakdown
products were too low to measure, possibly due to rapid uptake
by bacteria.
While this study showed that human α-amylase was in gen-
ital fluid, the source for it was not established. As mentioned
above, α-amylase is produced in both the pancreas and salivary
glands, and essentially all of the extracellular α-amylase activity
in the body is produced by those 2 glands [47, 48]. Both
pancreatic and salivary α-amylase are in serum and urine at rel-
atively high levels that can become elevated during certain clin-
ical conditions such as alcoholism or trauma. The salivary and
pancreatic enzymes are closely related, with only about 3% dif-
ference in the protein sequence [47]. Because the protein se-
quences are so similar, the ELISA for pancreatic α-amylase
may have cross-reactivity with salivary α-amylase. Therefore,
it is possible that the α-amylase in genital fluids is from 1 or
both of these glands.
In conclusion, this study provides evidence that human α-
amylase in the genital tract processes epithelial glycogen.
This, in turn, suggests a mechanism for how glycogen can sup-
port genital colonization by strains of Lactobacillus that cannot
directly use glycogen. Further studies are needed to clarify the
source of the enzyme and whether too much or too little
α-amylase could affect levels of Lactobacillus.
Notes
Financial support. This work was supported by National Institutes of
Health (NIH) grants P01 AI08297 and P30 AI 082151.
Potential conflicts of interest. All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
References
1. Lamont RF, Sobel JD, Akins RA, et al. The vaginal microbiome: new
information about genital tract flora using molecular based techniques.
BJOG 2011; 118:533–49.
2. O’Hanlon DE, Moench TR, Cone RA. Vaginal pH and microbicidal lac-
tic acid when lactobacilli dominate the microbiota. PLOS ONE 2013; 8:
e80074.
3. Aldunate M, Tyssen D, Johnson A, et al. Vaginal concentrations of lactic
acid potently inactivate HIV. J Antimicrob Chemother 2013; 68:
2015–25.
4. Ravel J, Gajer P, Abdo Z, et al. Microbes and Health Sackler Colloqui-
um: vaginal microbiome of reproductive-age women. Proc Natl Acad
Sci USA 2010; 108 (Suppl 1):4680–7.
5. Marrazzo JM. Interpreting the epidemiology and natural history of bac-
terial vaginosis: are we still confused? Anaerobe 2011; 17:186–90.
6. Li J, McCormick J, Bocking A, et al. Importance of vaginal microbes in
reproductive health. Reprod Sci 2012; 19:235–42.
7. Macklaim JM, Cohen CR, Donders G, et al. Exploring a road map to
counter misconceptions about the cervicovaginal microbiome and dis-
ease. Reprod Sci 2012; 19:1154–62.
8. Atashili J, Poole C, Ndumbe PM, et al. Bacterial vaginosis and HIV ac-
quisition: a meta-analysis of published studies. AIDS 2008; 22:1493–501.
9. Coleman JS, Hitti J, Bukusi EA, et al. Infectious correlates of HIV-1
shedding in the female upper and lower genital tracts. Aids 2007;
21:755–9.
10. Sha BE, Zariffard MR, Wang QJ, et al. Female genital-tract HIV load
correlates inversely with Lactobacillus species but positively with bacte-
rial vaginosis and Mycoplasma hominis. J Infect Dis 2005; 191:25–32.
11. Cu-Uvin S, Hogan JW, Caliendo AM, et al. Association between bacte-
rial vaginosis and expression of human immunodeficiency virus type 1
RNA in the female genital tract. Clin Infect Dis 2001; 33:894–6.
12. Mitchell C, Balkus JE, Fredricks D, et al. Interaction between lactobacil-
li, bacterial vaginosis–associated bacteria, and HIV type 1 RNA and
DNA genital shedding in U.S. and Kenyan women. AIDS Res Hum Ret-
roviruses 2013; 29:13–9.
Vaginal Amylase and Glycogen Degradation
13. Cohen CR, Lingappa JR, Baeten JM, et al. Bacterial vaginosis associated
with increased risk of female-to-male HIV-1 transmission: a prospective
cohort analysis among African couples. PLOS Med 2012; 9:e1001251.
14. Watts DH. Mother to child transmission of HIV—another complication
of bacterial vaginosis? J Acquir Immune Defic Syndr 2012; 60:221–4.
15. Cherpes TL, Meyn LA, Krohn MA, et al. Association between acquisi-
tion of herpes simplex virus type 2 in women and bacterial vaginosis.
Clin Infect Dis 2003; 37:319–25.
16. Wiesenfeld HC, Hillier SL, Krohn MA, et al. Bacterial vaginosis is a
strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis
infection. Clin Infect Dis 2003; 36:663–8.
17. Wiesenfeld HC, Hillier SL, Krohn MA, et al. Lower genital tract infec-
tion and endometritis: insight into subclinical pelvic inflammatory dis-
ease. Obstet Gynecol 2002; 100:456–63.
18. Hillier SL, Nugent RP, Eschenbach DA, et al. Association between bac-
terial vaginosis and preterm delivery of a low-birth-weight infant. The
Vaginal Infections and Prematurity Study Group [see comments]. N
Engl J Med 1995; 333:1737–42.
19. Goldenberg RL, Klebanoff MA, Nugent R, et al. Bacterial colonization
of the vagina during pregnancy in four ethnic groups. Vaginal Infec-
tions and Prematurity Study Group. Am J Obstet Gynecol 1996;
174:1618–21.
20. Antonio MA, Hawes SE, Hillier SL. The identification of vaginal Lacto-
bacillus species and the demographic and microbiologic characteristics
of women colonized by these species. J Infect Dis 1999; 180:1950–6.
21. Eschenbach DA, Davick PR, Williams BL, et al. Prevalence of hydrogen
peroxide–producing Lactobacillus species in normal women and
women with bacterial vaginosis. J Clin Microbiol 1989; 27:251–6.
22. Verstraelen H, Verhelst R, Claeys G, et al. Longitudinal analysis of the
vaginal microflora in pregnancy suggests that L. crispatus promotes the
stability of the normal vaginal microflora and that L. gasseri and/or L.
iners are more conducive to the occurrence of abnormal vaginal micro-
flora. BMC Microbiol 2009; 9:116.
23. Larsson PG, Forsum U. Bacterial vaginosis—a disturbed bacterial flora
and treatment enigma. APMIS 2005; 113:305–16.
24. Mehta SD. Systematic review of randomized trials of treatment of male
sexual partners for improved bacteria vaginosis outcomes in women.
Sex Transm Dis 2012; 39:822–30.
25. Rogosa M, Sharpe ME. Species differentiation of human vaginal lacto-
bacilli. J Gen Microbiol 1960; 23:197–201.
26. Cruickshank R, Sharman A. The biology of the vagina in the human
subject. II. The bacterial flora and secretion of the vagina at various
age-periods and their relation to glycogen in the vagina epithelium. J
Obstet Gynaec Brit Emp 1934; 41:208.
27. Stewart-Tull DE. Evidence that vaginal lactobacilli do not ferment gly-
cogen. Am J Obstet Gynecol 1964; 88:676–9.
28. Wylie JG, Henderson A. Identity and glycogen-fermenting ability of
lactobacilli isolated from the vagina of pregnant women. J Med Micro-
biol 1969; 2:363–6.
29. Martin R, Soberon N, Vaneechoutte M, et al. Characterization of indig-
enous vaginal lactobacilli from healthy women as probiotic candidates.
Int Microbiol 2008; 11:261–6.
30. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis. Diagnostic
criteria and microbial and epidemiologic associations. Am J Med 1983;
74:14–22.
31. Morris DL. Colorimetric determination of glycogen; disadvantages of
the iodine method. J Biol Chem 1946; 166:199–203.
32. Ao Z, Quezada-Calvillo R, Sim L, et al. Evidence of native starch degra-
dation with human small intestinal maltase-glucoamylase (recombi-
nant). FEBS Lett 2007; 581:2381–8.
33. Lee CK, Le QT, Kim YH, et al. Enzymatic synthesis and properties of
highly branched rice starch amylose and amylopectin cluster. J Agric
Food Chem 2008; 56:126–31.
34. Fredricks DN, Fiedler TL, Thomas KK, et al. Changes in vaginal bacte-
rial concentrations with intravaginal metronidazole therapy for bacteri-
al vaginosis as assessed by quantitative PCR. J Clin Microbiol 2009;
47:721–6.
• JID 2014:210 (1 October) • 1027
35. Srinivasan S, Liu C, Mitchell CM, et al. Temporal variability of human
vaginal bacteria and relationship with bacterial vaginosis. PLOS ONE
2010; 5:e10197.
36. Gray GM. Starch digestion and absorption in nonruminants. J Nutr
1992; 122:172–7.
37. Larson SB, Day JS, McPherson A. X-ray crystallographic analyses of pig
pancreatic alpha-amylase with limit dextrin, oligosaccharide, and alpha-
cyclodextrin. Biochemistry 2010; 49:3101–15.
38. Ganzle MG, Zhang C, Monang BS, et al. Novel metabolites from cereal-
associated lactobacilli—novel functionalities for cereal products? Food
Microbiol 2009; 26:712–9.
39. Meroth CB, Walter J, Hertel C, et al. Monitoring the bacterial population
dynamics in sourdough fermentation processes by using PCR-denaturing
gradient gel electrophoresis. Appl Environ Microbiol 2003; 69:475–82.
40. Fredricks DN, Fiedler TL, Marrazzo JM. Molecular identification of
bacteria associated with bacterial vaginosis. N Engl J Med 2005;
353:1899–911.
41. Gajer P, Brotman RM, Bai G, et al. Temporal dynamics of the human
vaginal microbiota. Sci Transl Med 2012; 4:132ra52.
1028 • JID 2014:210 (1 October) • Spear et al
42. O’Hanlon DE, Lanier BR, Moench TR, et al. Cervicovaginal fluid and
semen block the microbicidal activity of hydrogen peroxide produced
by vaginal lactobacilli. BMC Infect Dis 2010; 10:120.
43. Boskey ER, Telsch KM, Whaley KJ, et al. Acid production by vaginal
flora in vitro is consistent with the rate and extent of vaginal acidifica-
tion. Infect Immun 1999; 67:5170–5.
44. Cherpes TL, Meyn LA, Krohn MA, et al. Risk factors for infection with
herpes simplex virus type 2: role of smoking, douching, uncircumcised
males, and vaginal flora. Sex Transm Dis 2003; 30:405–10.
45. Hillier SL. Diagnostic microbiology of bacterial vaginosis. Am J Obstet
Gynecol 1993; 169:455–9.
46. Ganzle MG, Follador R. Metabolism of oligosaccharides and starch in
lactobacilli: a review. Front Microbiol 2012; 3:340.
47. Pieper-Bigelow C, Strocchi A, Levitt MD. Where does serum amylase
come from and where does it go? Gastroenterol Clin North Am 1990;
19:793–810.
48. Vissers RJ, Abu-Laban RB, McHugh DF. Amylase and lipase in the
emergency department evaluation of acute pancreatitis. J Emerg Med
1999; 17:1027–37.