Eur J Clin Microbiol Infect Dis
DOI 10.1007/s10096-015-2564-x
ORIGINAL ARTICLE
Mycoplasma hominis and Gardnerella vaginalis display
a significant synergistic relationship in bacterial vaginosis
C. Cox 1,2 & A. P. Watt 1 & J. P. McKenna 1 & P. V. Coyle 1
Received: 21 August 2015 / Accepted: 21 December 2015
# Springer-Verlag Berlin Heidelberg 2016
Abstract Gardnerella vaginalis plays an important role in
bacterial vaginosis (BV,) while the role of genital Mollicutes
is less obvious. The diagnosis of BV by use of the current
Gram stain Nugent score is also suboptimal for defining the
role of Mollicutes that lack a cell wall. Since bacterial load and
diversity is an important prerequisite for BV, real-time quan-
titative polymerase chain reaction (qPCR) assays enable these
to be assessed. The purpose of this study was to define the role
of genital Mollicutes and potential patterns of synergy with
G. vaginalis in women with BV. Vaginal swabs from 130
women categorised by Nugent score as BV (n = 28), interme-
diate (n = 22) and non-BV (n = 80) were tested against four
qPCR TaqMan assays targeting G. vaginalis, Mycoplasma
hominis, M. genitalium, Ureaplasma urealyticum and
U. parvum. Statistical analyses were used to compare bacterial
prevalence and load between the three groups of women.
Mycoplasma hominis and G. vaginalis co-infection was sig-
nificantly more common in BV (60.7 %) compared to inter-
mediate (36.4 %) and non-BV (8.8 %) Nugent scores
(p < 0.001). Significantly higher loads of M. hominis
(p = 0.001) and G. vaginalis (p < 0.001) were detected in
women with BV and the respective loads in M. hominis and
G. vaginalis co-infections displayed a significant positive cor-
relation (p < 0.001; r = 0.60). No significant associations were
seen with the other Mollicutes. The findings strengthen the
* C. Cox
ciara.cox@belfasttrust.hscni.net
1
Regional Virus Laboratory, Belfast Health and Social Care Trust,
Belfast, Northern Ireland, UK
2
Centre for Infection and Immunity, Queen’s University Belfast,
Belfast, Northern Ireland, UK
evidence of a role for M. hominis in BV and a potential syn-
ergy with G. vaginalis. This synergy could be an important
trigger of the condition and sexual contact the conduit for the
transmission of an otherwise commensal bacterium that could
initiate it.
Introduction
Bacterial vaginosis (BV) is a complex polymicrobial disorder
commonly recognised between menarche and menopause in
women of all ethnicities. It is associated with adverse out-
comes relating to both pregnancy and fertility [1–3]. The con-
dition is caused by a reduction in protective lactobacilli spe-
cies, accompanied by an overgrowth of strict or facultative
anaerobic bacteria, predominantly Gardnerella vaginalis;
quantitative molecular assays demonstrate increased loads of
G. vaginalis and a prevalence of 100 % in the condition [4–7].
The increase of other highly fastidious bacteria results in the
polymicrobial dysbiosis that defines BV [8–10]. Women can
be colonised with both cohesive and dispersive strains of
G. vaginalis. Cohesive G. vaginalis strains dominate the bac-
terial biofilm seen in BV, which is thought to be transmissible
between sexual partners [11–15]. The biofilm confers resis-
tance to effective antimicrobial treatment and is linked to re-
currence [11, 12, 14–17].
Current laboratory diagnosis of BV uses the Gram staining
technique to establish a numeric score from 1 to 10, the
Nugent score, which estimates the quantity of different bacte-
rial morphotypes in a stained vaginal smear [18]. It is simple
to perform but lacks sensitivity, is subjective and is operator
dependent [19]. The score also accommodates an indetermi-
nate score (6–8), which does not confirm or exclude BV. Also,
by definition, the Gram stain cannot detect bacteria lacking a
cell wall, such as the genital Mycoplasma and Ureaplasma

species, which may play a role in BV. These problems suggest
the need for a new diagnostic approach [7, 20].
Detection of Mycoplasma and Ureaplasma species not on-
ly in healthy women but in women with vaginal discharge and
other conditions has led to uncertainty as to their role in BV
[21]. The inability of the Nugent score to register these bacte-
ria through Gram stain makes it suboptimal and deficient in
recognising significant synergies between bacterial species in
BV. Since increased diversity and loads are both important in
the establishment of BV, quantitative real-time polymerase
chain reaction (qPCR) assays are well placed to address these
elements. While BV is associated with vaginal discharge, a
significant number are asymptomatic and require laboratory
confirmation [22]. The use of qPCR in women with and with-
out a disturbed vaginal microflora would allow the role of
Mollicutes in BV and in synergy to be assessed and could be
used for studies of biofilm transmission [23–30].
This present study consisted of vaginal swabs from women
attending a local genitourinary medicine (GUM) clinic who
had been co-tested for BV, with scores across the full Nugent
score range of confirmed, intermediate and normal. The swabs
were tested against qPCR assays targeting genes in
G. vaginalis, Mycoplasma hominis, M. genitalium,
U. urealyticum and U. parvum. Establishing the respective
prevalence of each Mollicute and G. vaginalis and their loads
was performed between the three groups of women to identify
associations with BV and if patterns of synergy were present.
Materials and methods
Ethical approval
All anonymised residual specimens in the study were collect-
ed and processed under research ethics approval from
Northern Ireland REC Committee 1, reference number 10/
NIR01/20.
Study samples
Patients included in the study were women with a vaginal
discharge attending a GUM clinic over a consecutive 12-
month period. A vaginal smear was submitted for Gram stain-
ing for the detection of BV in accordance to the Nugent score
technique [18]. The residual extract from a vaginal swab sub-
mitted concurrently for chlamydia testing was used by qPCR.
The respective Nugent score was recorded. All samples were
anonymised before testing.
Sample collection and processing
Vaginal swabs were collected using the Cobas® PCR swab
sample kit (Roche Molecular Diagnostics, Mannheim,
Eur J Clin Microbiol Infect Dis
Germany). Swabs were transported in Cobas® PCR media
(Roche Molecular Diagnostics, Mannheim, Germany) to the
laboratory and stored at 4 °C until further processing. Cobas®
PCR media tubes containing vaginal swabs were subjected to
Cobas® 4800 (Roche Molecular Diagnostics, Mannheim,
Germany) for nucleic acid extraction and dual testing of
Chlamydia trachomatis and Neisseria gonorrhoeae (CT/
NG) in accordance to the manufacturer’s instructions. A final
volume of 100 μl of nucleic acid extract was output in the
Cobas® 4800 extraction procedure (Roche Molecular
Diagnostics, Mannheim, Germany).
Molecular testing
Nucleic acid extracts were tested against previously described
U. urealyticum, U. parvum [31], M. genitalium [32],
M. hominis [33] and G. vaginalis [7] real-time qPCR assays
using the LightCycler® 480 instrumentation (Roche
Molecular Diagnostics, Mannheim, Germany). Clinical and
analytical validation has been completed in our laboratory
for each of the real-time qPCR assays. Logarithmic dilutions
of gene-specific plasmid standards tested against each qPCR
assay demonstrated limits of detection of less than 54 gene
copies per 2 μl and no cross-reactivity was observed when the
five qPCR assays were tested against a panel of nucleic acid
extracts from 40 different organisms. All assays were carried
out using 1X Platinum® Quantitative PCR SuperMix-UDG
(Life Technologies, Paisley, UK). The final working concen-
trations of reagents in the qPCR assays were as follows:
0.4 μM forward and reverse primer, 0.1 μM TaqMan® probe,
0.2 μg ml−1 BSA (Thermo Scientific, Epsom, UK) and 4 mM
MgCl2 (Life Technologies, Paisley, UK). Final reaction vol-
umes of 10 μl comprising 2 μl nucleic acid extract and 8 μl
mastermix were used with the following PCR cycling condi-
tions: 95 °C for 5 min, followed by 45 cycles of 95 °C for 15 s
and 59 °C for 45 s. MS2 internal control was included in each
real-time PCR run to monitor PCR inhibition and DNA ex-
traction. The results were analysed using LightCycler® 480
software, version 1.5 (Roche Molecular Diagnostics,
Mannheim, Germany) and recorded as the cycle threshold
(CT) value. Any reaction which failed to produce a CT value
after 45 cycles was recorded as negative.
Target quantification
Individual plasmids containing each qPCR amplicon were
cloned and the DNA content was measured using a
NanoDrop 2000 Spectrophotometer (Thermo Scientific,
Epsom, UK). With reference to the size of the plasmid and
the genome of each target organism, the gene copy number
per ml was calculated for each plasmid standard. Logarithmic
serial dilutions of appropriate plasmid were tested against each
specific qPCR assay and by inputting the known copy number
Eur J Clin Microbiol Infect Dis

of dilution into the LightCycler® 480 instrumentation (Roche
Molecular Diagnostics, Mannheim, Germany), a calibration
curve was constructed and stored. For subsequent qPCR test-
ing, which consisted of clinical samples, a gene-specific plas-
mid standard equivalent to 105 gene copies ml−1 was included
for quantification, where the generated CT value for each
sample was converted to the gene copy number per ml
by extrapolation against the internally stored calibration
curve.
Statistical analysis
All statistical analyses were performed using the SPSS 19.0
statistical package (SPSS Science, Chicago, IL, USA).
Women were categorised into three groups as BV, intermedi-
ate and non-BV based on the Nugent score results. Chi-
squared analysis was used to compare the prevalence of (a)
Mollicute single infections and (b) Mollicutes and
G. vaginalis dual infections between the three groups of wom-
en. Mollicute and G. vaginalis loads were log10 transformed to
fit a normal distribution and a one-way analysis of variance
(ANOVA) was performed for comparison of the mean load
between the three groups of women. Correlation of
M. hominis and G. vaginalis loads in dual infections were
assessed using Pearson’s product-moment correlation, which
measures the strength and direction of association which
exists between two variables. An R-value close to 1
represents a strong positive correlation. p-Values <0.05
were regarded as significant.
Results
Study population
A total of 130 women with a mean age of 26.85 years [stan-
dard deviation (SD) ± 8.45 years] were eligible for the study,
each having two routine vaginal swabs submitted, one for dual
C. trachomatis/N. gonorrhoeae molecular testing and one for
a Nugent score by Gram stain. Twenty-eight (21.5 %) women
had BV confirmed, 22 (16.9 %) women had an intermediate
flora and 80 (61.5 %) women were reported as non-BV.
Mollicute and G. vaginalis prevalence
Using individual qPCRs, the respective detection rates in the
cohort of 130 women for G. vaginalis, U. parvum,
M. hominis, U. urealyticum and M. genitalium were: 104
(77.6 %), 83 (63.8 %), 34 (26.2 %), 26 (20 %) and 6
(4.6 %). A comparison of the prevalence of each Mollicute
in BV, intermediate and non-BV women are summarised in
Table 1. Mycoplasma hominis had a significantly higher prev-
alence (p = 9.6 × 10−7) across the three groups of women and
had a significant trend (p = 1.6 × 10−7) increasing from non-
BV (11.3 %), intermediate (36.4 %) and confirmed BV
(60.7 %). The other Mollicutes lacked a statistically signifi-
cant association, as outlined in Fig. 1.
Mollicute and G. vaginalis co-infection
Comparisons of G. vaginalis and Mollicute co-infections are
summarised in Table 2. The association of M. hominis co-
infection with G. vaginalis was significantly higher in women
with BV (60.7 %) compared to intermediate (36.4 %) and
women diagnosed as non-BV (8.8 %) (p = 1.0 × 10−7); this
demonstrated a significant linear trend (p = 1.6 × 10 −8 ).
Ureaplasma parvum and G. vaginalis co-infections were
higher in women with BV (78.6 %) compared to intermediate
(50 %) and women diagnosed as non-BV (46.3 %); this asso-
ciation approached statistical significance (p = 0.06). There
were no significant associations across the three clinical
groups between G. vaginalis, U. urealyticum and
M. genitalium.

Table 1 Comparison of
Mollicute prevalence between Target organism Total number of positive women (%) p-Valuea p-Valueb
women with bacterial vaginosis
(BV), intermediate and non-BV BV Intermediate Non-BV
flora
Total 28 (21.5 %) 22 (16.9 %) 80 (61.5 %) – –
G. vaginalis 28 (100 %) 18 (81.8 %) 54 (67.5 %) 0.002 3.9 × 10−4*
U. urealyticum 5 (17.6 %) 3 (13.6 %) 18 (22.5 %) 0.62 0.49
U. parvum 22 (78.6 %) 12 (54.5 %) 49 (61.3 %) 0.16 0.17
M. genitalium 1 (3.6 %) 2 (9.1 %) 3 (3.8 %) 0.55 0.84
M. hominis 17 (60.7 %) 8 (36.4 %) 9 (11.3 %) 9.6 × 10−7 1.6 × 10−7*
a
Chi-square
b
Linear-by-linear association
*Significant at level <0.05
 Eur J Clin Microbiol Infect Dis

120
across the full range of Nugent scores on vaginal specimens
100 ****
from women attending a local GUM clinic. A total of 130
80 women were included in the study, with each submitting one
**** BV vaginal swab for C. trachomatis PCR and one for Nugent
%

60
intermediate
40 non-BV
20
0 C. trachomatis/N. gonorrhoeae test was assayed against five
GV MH MG UU UP
Fig. 1 The prevalence of Gardnerella vaginalis (GV), Mycoplasma.
hominis (MH), M. genitalium (MG), Ureaplasma urealyticum (UU) and
U. parvum (UP) in 130 women categorised as BV (n = 28), intermediate
(n = 22) and non-BV (n = 80) by Nugent score
Bacterial load
The mean load of each Mollicute in women with BV, inter-
mediate and non-BV floras were log10 transformed and com-
pared using a one-way ANOVA test. Comparisons of individ-
ual Mollicute and G. vaginalis loads are summarised in
Table 3. Gardnerella vaginalis loads significantly increased
from non-BV (6.31) to intermediate (7.75) and confirmed BV
(8.60) (p = 2.3 × 10−9). Similarly, M. hominis loads significant-
ly (p = 0.001) increased between the three groups of women
from non-BV (5.88) to intermediate (7.55) and confirmed BV
(7.45). Ureaplasma urealyticum, U. parvum and
M. genitalium loads did not display a significant difference
between the three groups of women, as shown in Fig. 1. The
respective loads of M. hominis and G. vaginalis in dual infec-
tions displayed a significant positive correlation (r = 0.60;
p = 1.3 × 10−4), as shown in Fig. 2. The other Mollicute loads
showed no correlation with G. vaginalis.
Discussion
This study described the investigation of the prevalence, loads
and synergy between genital Mollicutes and G. vaginalis
score by Gram stain; samples were categorised by Nugent
score as BV 28 (21.5 %), intermediate flora 22 (16.9 %) and
non-BV 80 (61.5). Anonymised residual DNA from the dual
real-time qPCR assays targeting U. urealyticum, U. parvum,
M. hominis, M. genitalium and G. vaginalis. An increased
load and prevalence of G. vaginalis and M. hominis in women
with BV was noted.
The four genital Mollicutes were detected in the study.
Ureaplasma parvum was the most common (63.8 %), follow-
ed by: M. hominis (26.2 %), U. urealyticum (20 %) and
M. genitalium (4.6 %). There was no significant difference
in the prevalence of U. urealyticum, U. parvum and
M. genitalium or in their respective loads between women
with and without BV. The high prevalence of U. parvum in
women with BV (78.6 %) compared to women with interme-
diate (54.5 %) and non-BV (61.3 %) almost reached statistical
significance (p = 0.17), making an association with BV plau-
sible. The evidence for a causal role for M. hominis was,
however, more convincing. A significantly (p = 9.6 × 10−7)
higher M. hominis prevalence was detected in women with
BV (60.7 %) compared to women with intermediate
(36.4 %) and non-BV (11.4 %) flora. The detection of a sig-
nificantly higher M. hominis load in BV women in this study
further corroborates a potential link between M. hominis and
BV (p = 0.001).
Interestingly, there was also a significantly (p = 1.0 × 10−7)
higher co-infection rate of M. hominis and G. vaginalis in BV
(60.7 %) compared to intermediate (36.4 %) and non-BV
(8.8 %) women. These co-infections in women with an inter-
mediate (n = 8) and BV flora (n = 17) all had G. vaginalis loads
≥107 gene copies per ml, a threshold which was shown to have
an independent potential for defining BV on its own, with a
negative predictive value for BV of 100 % [7]. These co-

Table 2 Comparison of Gardnerella vaginalis and Mollicute co-infection between women with BV, intermediate and non-BV flora

Mollicutes and G. vaginalis co-infection Total number of positive women p-Valuea p-Valueb

BV, n = 28 Intermediate, n = 22 Non-BV, n = 80

UU + GV 5 (17.1 %) 3 (13.6 %) 15 (18.7 %) 0.86 0.82

MG + GV 1 (3.6 %) 2 (9.0 %) 3 (3.8 %) 0.55 0.84
UP + GV 22 (78.6 %) 11 (50 %) 37 (46.3 %) 0.14 0.06
MH + GV 17 (60.7 %) 8 (36.4 %) 7 (8.8 %) 1.0 × 10−7 1.6 × 10−8*
a
Chi-square
b
Linear-by-linear association
*Significant at level <0.05
Eur J Clin Microbiol Infect Dis

Table 3 Comparison of
Mollicutes mean load (log10 Target organism Mean log10 gene copy number per ml p-Valuea
transformed) between women
with BV, intermediate and non- BV, n = 28 Intermediate, n = 22 Non-BV, n = 80
BV vaginal flora
G. vaginalis 8.60 7.75 6.31 2.3 × 10−9*
U. urealyticum 4.49 5.15 4.40 0.36
U. parvum 5.84 6.09 5.96 0.37
M. genitalium 5.29 4.20 5.16 0.17
M. hominis 7.45 7.55 5.88 0.001*
a
One-way analysis of variance (ANOVA) test
*Significant at level <0.05

infections indicate a synergy between M. hominis and
G. vaginalis in BV that may be clinically important. In addi-
tion, the positive correlation between M. hominis and
G. vaginalis loads in co-infections, with both demonstrating
synchronised and parallel changes in their respective loads,
indicates a potential quorum sensing-like interaction or co-
response to an environmental stimulus. If true, then transmis-
sion of one of these bacteria could trigger the outgrowth of the
other and start a process leading to BV.
BV is regarded as a sexually enhanced disease (SED) rather
than a sexually transmitted infection (STI), with increased
sexual activity and a higher number of lifetime sexual partners
increasing the risk [34]. This distinction is somewhat contra-
dictory and counter-intuitive, and suggests that we have more
to learn about the microbiology of the condition. An increas-
ing evidence base suggests the penis can harbour BV-
associated bacteria that may be transmitted between partners
and act as a source of re-introduction to a sexual partner post
treatment [12, 35–40]. Circumcision also reduces the rate of
BV in female partners [41, 42], in keeping with the penis
acting as a potential reservoir or conduit of infection. Strains
of G. vaginalis isolated from women with BV are also more
capable of displacing lactobacilli species from adhering to
vaginal epithelial cells. This favours the biofilm formation that
acts as a scaffold for the dysbiotic bacterial overgrowth that
characterise BV [43]. Whether SED or STI, the lack of mea-
sure of a time period or incubation period needed to establish
BV, which is often asymptomatic, will make it challenging to
confirm the condition along the lines of Koch’s postulates.
The current study strengthens the evidence for a synergistic
growth trigger between G. vaginalis and M. hominis, adding
to the complexity of the pathogenesis of BV, whilst providing
a possible explanation linking sexual contact and a causal
effect. In conclusion, the use of more sensitive and spe-
cific qPCR assays demonstrated a highly specific syner-
gy between G. vaginalis and M. hominis in women with
BV that could provide an explanation underlying the
pathogenesis of the condition.
Acknowledgements The authors would like to acknowledge the sup-
port of Randox Laboratories Ltd, who sponsored Ciara Cox and also
thank Innovate UK for co-funding this study.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.

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