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Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of forming biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main questions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-existence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp. DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneumoniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demonstrated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed pathogenic structure. In summary, our study is the first to show Borrelia–Chlamydia mixed biofilms in infected human skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship for their mutual survival.
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Borrelia and Chlamydia Can Form Mixed Biofilms in
Infected Human Skin Tissues
E. Sapi
1*
, K. Gupta
1
, K. Wawrzeniak
1
, G. Gaur
1
, J. Torres
1
, K. Filush
1
, A. Melillo
1
and B. Zelger
2
1
Department of Biology and Environmental Science, University of New Haven, West Haven, CT 06516, USA
2
Department of Dermatology and Venereology, Medical University Innsbruck, Innsbruck, Austria
Received: 15 January 2019; accepted: 04 March 2019
Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of form-
ing biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often
contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental
stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main ques-
tions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-exis-
tence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected
human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in
Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp.
DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneu-
moniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demon-
strated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed
that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed patho-
genic structure. In summary, our study is the first to show BorreliaChlamydia mixed biofilms in infected human
skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship
for their mutual survival.
Keywords: Lyme disease, biofilm, Borrelia lymphocytoma, alginate, chlamydia, confocal microscopy
Introduction
Lyme disease is a tick-borne illness that is caused by Borre-
lia burgdorferi sensu stricto and sensu lato in the United
States and Europe, respectively [15]. Lyme disease is esti-
mated to affect 300,000 people a year in the United States and
65,000 people a year in Europe [6]. The most common skin
manifestation is a red rash that is observed after a tick bite
called erythema migrans (EM) [7, 8]. The other well-studied
dermatological conditions of Lyme disease are Borrelia lym-
phocytoma (BL) that appears in the early phase of Borrelia in-
fection and acrodermatitis chronica atrophicans (ACA), which
is the late onset cutaneous manifestation [912]. However,
Lyme disease is a multi-systemic disease with manifestations
that may also include other several chronic conditions such as
Lyme carditis and neuroborreliosis [1318].
Recently, our research group provided evidence for both the
B. burgdorferi sensu stricto and the sensu lato groups of B.
burgdorferi to exist in biofilm form in vitro and in vivo in
Borrelia lymphocytoma [1921]. Like other bacterial biofilms,
Borrelia biofilms have shown increased resistance towards the
standard antibiotics that are used to treat Lyme disease [22].
Biofilms are an aggregation of planktonic bacteria that attach
on biotic and abiotic surfaces to form a three-dimensional ar-
chitecture to withstand various environmental stressors [23].
The presence of a protective surface matrix called extracellular
polymeric substance (EPS) and persister cells with low meta-
bolic activity helps the survival of community inside the bio-
film [2430]. Clinically, biofilm infections represent a very
significant problem due to the extraordinary resistance to both
antimicrobial drugs, as well as host immune systems, which
eventually could lead to persistent human infections [29, 3133].
According to the National Institute of Health (NIH), 80%
of all chronic infections have been linked to pathogenic
biofilms [33, 34]. Several biofilm-related chronic infections
have been reported such as Pseudomonas aeruginosa in cystic
fibrosis [35], Escherichia coli in urinary tract infections,
Staphylococcus aureus in osteomyelitis and endocarditis, and
Streptococcus pneumoniae in pulmonary infections [33, 3638].
Highly diverse in nature, biofilms have been reported to ex-
ist in a polymicrobial fashion, where several bacterial species
along with fungi, yeast, and viruses reside in a community
[39, 40]. The microbial community communicates through
quorum sensing, co-operates with each other by developing a
symbiotic relationship, protects and fights against antimicro-
bial treatments [39, 40]. The presence of mixed biofilms has
been suggested in oral plaques, gastrointestinal tract, chronic
wounds, and lungs, enhancing biofilm formation and increas-
ing the resistance against stress and the host immune re-
sponses [4144].
In Lyme disease, co-infections are common because ticks
are well known to carry and transmit several human patho-
genic microbes along Borrelia such as Bartonella,Ehrlichia,
Babesia,Anaplasma, and even Mycoplasma species [4549].
Recently, the presence of Chlamydia-like organisms was also
reported by several studies in a significant fraction of Ixodes
ricinus ticks [5052]. Furthermore, the presence of Chlamydia
DNAs in 68% of the skin biopsies obtained from patients with
a suspected tick bite history was found [52]. The follow up
*Author for correspondence: 300 Boston Post Road, West Haven CT 06516;
Tel.: +1-203-479-4552; E-mail: E-mail: esapi@newhaven.edu.
DOI: 10.1556/1886.2019.00003
© 2019 The Author(s)
European Journal of Microbiology and Immunology
Original Research Paper
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study from the same research group reported that all Borrelia
positive granuloma annulare skin conditions were also positive
for Chlamydia related bacteria [53]. Furthermore, a recent
Australian study also confirmed that DNA from ticks contains
DNA belonging to the chlamydial order genotype [54] sug-
gesting that Chlamydia can be a very frequent co-infection in
Borrelia infected tissues.
The bacterial order Chlamydiales includes intracellular
Gram-negative bacteria that follow a biphasic development cy-
cle and are dependent on the host organism for ATP synthesis
[55]. The bacteria primarily exist as elementary bodies capable
of invading the host cell [56]. Following infection in the host
cell, they fuse with the membrane-bound cytoplasmic vacuole,
termed inclusion bodies, where they are in a protective envi-
ronment [56, 57].
There are 3 main Chlamydia pathogens responsible for
causing human infections. Chlamydophila pneumoniae is a re-
spiratory pathogen whose infection leads to extra-pulmonary
symptoms such as myocarditis, atherosclerosis, reactive arthri-
tis, and nervous system disorders [58, 59]. Chlamydia tracho-
matis is a bacterium responsible for causing sexually
transmitted diseases such as urethritis, cervicitis, and some
other infections, such as Reiter's syndrome, reactive arthritis,
ocular infections, atypical pneumoniae, or pelvic inflammatory
diseases [60]. Chlamydia psittaci is a pathogen that affects
avians and is known to cause the human infection psittacosis
leading to severe pneumonia [61]. Erythema nodosum, an in-
flamed skin condition with painful, red deep-seated nodules
on lower legs, is also observed after chlamydial infection [62].
Several chlamydial infections have similar symptoms, as ob-
served in Lyme patients such as arthritis, atherosclerosis, neu-
rocognitive symptoms, and skin rashes [45, 63].
Chlamydia-related infections have been reported to be de-
veloping an emerging resistance to antibiotics in vitro and in
clinical samples [64, 65]. There is no direct evidence for the
existence of Chlamydia in biofilm form; however, studies
have reported the existence of chlamydial aggregates due to
stressful conditions such as calcium imbalance [66, 67].
Based on these findings, the goal of this study was to inves-
tigate the potential presence of Chlamydia spp. in BL skin bi-
opsies and their potential relationship to Borrelia biofilms
reported previously in BL skin biopsies.
Materials and Methods
Human Skin Sections. From the files of our
dermatohistopathologic laboratory, paraffin materials from 6
cases of clinically confirmed Borrelia lymphocytoma were
archived from January 1975 to December 2005. All six cases
had positive serology for Borrelia IgG and characteristic
features of Borrelia lymphocytoma with acralpredilection
were found. All six patients were female (average age = 33
years) from endemic areas of borreliosis in Austria with a rate
of positive serology in the population between 3060%.
Polymerase chain reaction (PCR) confirmation for all 6 cases
was performed independently in 2 different laboratories
located in Austria and the US. The archived hematoxylin-and-
eosin (H&E)-stained sections were reexamined, and the
previous diagnosis also confirmed. Institutional Review Board
exemption for this study was obtained from the University
of New Haven. The paraffin blocks were sectioned by
McClain Laboratories LLC [Smithtown NY] at 4 μmon
TRUBOND200 adhesive slides. The sections then were
deparaffinized by washing the sections three times in 100%
xylene for 5 min each, followed by rehydration in a series of
graded alcohols (100%, 90%, and 70%) and washed in 1×
phosphate buffered saline (PBS) of pH 7.4 for 5 min. For the
immunohistochemical experiments, the tissues were incubated
in 10 mM sodium citrate buffer for 45 min at 95 °C for
antibody retrieval.
DNA Extraction. DNA extraction from FFPE samples was
performed using the Qiagen Gene Read DNA FFPE Kit
(Qiagen, Germantown, MD) according to the manufacturer's
handbook with some modifications: 4-μm paraffin-embedded
tissue sections were deparaffinized by heating slides for
10 min at 45 °C followed by 3 xylene washes, 5 min each
wash. Tissues were then rehydrated in a series of alcohol
(100%, 100%, 90%, and 70%) washes for 3 min each. Slides
were run under a slow stream of tap water in a container with
70% alcohol for 30 min. Tissue sections were scraped into
1.5-mL tubes using a sterile razor blade; the 56 °C proteinase
K digestion step was performed for 72 h; the AW1 and AW2
wash steps were performed three times.
Polymerase Chain Reaction. PCR reactions for Borrelia
burgdorferi sensu lato were performed on all BL biopsy
samples in previous studies by 2 independent laboratories [8,
21], and positive Borrelia afzelii DNAs were found on all 6
samples. To detect the specific Chlamydia spp. in the skin
tissue samples, 2 different previously published PCR methods
were used to maximize the probability to amplify Chlamydia
spp. [68, 69]. Both PCR protocols were designed to detect the
Outer Membrane Protein A (OmpA) gene, which was proven
to be specific enough to identify the different Chlamydia
species [68, 69]. Positive control reactions consisted of
commercially available DNA samples (not live cultures) from
Chlamydophila pneumoniae strain CM-1 [ATCC
W
VR-1360]
and Chlamydia trachomatis, both obtained from American
Type Culture Collection (ATCC) (Serovar E Chlamydiaceae
VR 348BD BOUR strain). As negative controls, reactions with
no template DNA and normal healthy human DNA samples
were used. The first PCR protocol was slightly modified and
included an additional pre-amplification of the OpmA DNA in
the BL tissues in a nested PCR reaction. In the first round,
primers specific to the outer membrane protein A (OmpA)
gene were used: forward 5-CGCATTTGCTGGTTCTGTT-3
and reverse 5-CCAACGAGATTGAACGCTGT-3primer
sequences (Integrated DNA Technologies). In a 25 μL reaction,
1× PCR buffer (Promega), 1.5 mM MgCl
2
, 0.2 mM dNTPs,
0.2 μM forward primer, 0.2 μM reverse primer, 1.25 U of
DNA polymerase, and 50 ng of DNA template were added.
Reaction conditions were defined by an initial denaturing time
of 95 °C for 5 min, followed by 35 cycles of 95 °C/45 s,
53 °C/15 s, 55.4 °C/15 s, 72 °C/45 s, and a final extension of at
72 °C/5 min. Primers for the nested reaction were as follows:
forward 5-CTCCTTACAAGCCTTGCCTGTAGGG-3, reverse
5-GCGATCCCAAATGTTTAAGGC-3. [68]. A 50 μL nested
PCR reaction was prepared by adding 1× Buffer B (Promega,
Madison WI), 1.5 mM MgCl
2
, 0.2 mM dNTPs, 0.2 μM
forward primer, 0.2 μM reverse primer, 1.25 U of DNA
polymerase, and 1 μL of a 1:100 dilution of the first reaction
product. Reaction conditions were defined by an initial
denaturing time of 95 °C for 5 min, followed by 35 cycles of
95 °C/60 s, 53.4 °C/30 s, 72 °C/60 s, and a final extension of at
72 °C/5 min. The 337 bp PCR products were analyzed by
standard agarose gel electrophoresis, and the PCR products
were purified using a QIAquick PCR purification kit
(Qiagen, Germantown, MD) according to the manufacturer's
instructions. Samples were eluted twice in 30 μL, and the
eluates from each sample were pooled and sequenced in both
directions twice (4× coverage) using the same primers that
generated the products. All DNA sequencing was performed by
Eurofins Genomics (Louisville, KY).
In the second PCR protocol, a different published primer
pair spanning the major outer membrane protein (OmpA)
2
Borrelia and Chlamydia Can Form Mixed Biofilms
region of the Chlamydia species was used (69). Primers were
forward 5-CCTGTGGGGAATCCTGCTGAA-3and reverse
5-GTCGAAAACAAAGTCACCATAGTA-3flanking a
144 bp region of the gene. For the PCR conditions, a final
reaction volume of 50 μL was set with 0.2 mM dNTPs, 2.5 U
of Taq DNA polymerase (Invitrogen, Carlsbad CA), 0.2 μM
of each forward and reverse primer, 1.5 mM of MgCl
2
, and
1× Buffer B (Promega, Madison WI). The temperature profile
was set for initial denaturation at 94 °C for 4 min, followed
by 40 cycles of denaturation at 94 °C for 1 min, annealing at
53 °C for 1 min, and extension at 72 °C for 2 min, followed
by a final extension at 72 °C for 5 min. The PCR products
were analyzed by standard agarose gel electrophoresis, and
the PCR products were purified using the QIAquick PCR
purification kit (Qiagen, Germantown, MD) and sequenced as
described above.
All resulting sequences were first analyzed using the Basic
Local Alignment Search Tool on the NCBI website (BLAST,
https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were
aligned to reference sequences using the CLUSTEL OMEGA
multiple sequence alignment tool (EMBL-EBI, http://www.
ebi.ac.uk/Tools/msa/clustalo/).
Immunohistochemistry. Before proceeding with
immunostaining, the deparaffinized slides were rinsed with 1×
phosphate buffered saline (PBS, Sigma, St. Louise MO) and
distilled water for 2 min each. Slides were pre-incubated with
10% normal goat serum (Thermo Scientific) in PBS0.5%
bovine serum albumin (BSA, Sigma) for 30 min at room
temperature (RT) to block the nonspecific binding of the
secondary antibody. Slides were then rinsed twice with 1×
PBS and distilled water for 2 min each at RT. The slides were
then treated with a dilution of 1:200 (dilution buffer: PBS pH
7.4 + 0.5% BSA) of monoclonal antibody for Chlamydia spp.
(Cat# C65815M, Meridian Life Sciences, USA) and incubated
overnight in a humidified chamber at 4 °C. The slides then
were washed in 1× PBS and distilled water five times for
2 min each at RT. The tissue sections were then incubated
with a 1:200 dilution of the secondary anti-mouse antibody
(dilution buffer: PBS pH 7.4 + 0.5% BSA) with a fluorescent
red tag (goat anti-mouse IgG (H+L), DyLight 594 conjugated
for an hour at RT in a humidified chamber. The excess
solution around the tissue was gently wiped, washed five
times as mentioned above with 1× PBS, and then, the
polyclonal rabbit anti-alginate antibody (generously provided
by Dr. Gerald Pier, Harvard Medical School) was diluted in a
1:500 ratio (dilution buffer: PBS pH 7.4 + 0.5% BSA) and
added to the slides. The slides were incubated at RT overnight
in a humidified chamber. The next day the slides were washed
five times with 1× PBS for 2 min each. The tissue sections
were then treated with a 1:200 dilution (dilution buffer: PBS
pH 7.4 + 0.5% BSA) of the secondary anti-rabbit antibody
with a fluorescent blue tag (goat anti-rabbit IgG (H+L),
DyLight 405 conjugated) and incubated for an hour. This step
was followed by the abovementioned washes and then
treatment with a 1:50 dilution of a fluorescein isothiocyanate
(FITC)-labeled polyclonal rabbit anti-Borrelia burgdorferi
antibody (PA-1-73005, Thermo scientific), for an hour in a
humidified chamber at room temperature. The slide sections
were then washed and processed as mentioned above and then
counterstained with 0.1% Sudan black (Sigma) for 20 min,
washed again, and then mounted with PermaFluor (Thermo
Scientific). Images were taken and processed using a Leica
DM2500 fluorescence microscope at 200× and 400×
magnification.
As negative controls, commercially available human new-
born foreskin tissue sections and healthy human skin sections
(Biomax, HuFPT136) were stained following the same
procedure as mentioned above. Additional negative controls
such as omitting the primary antibody and the use of non-spe-
cific isotype IgG controls (IgG1 Isotype Control, Invitrogen,
MA1-10406) were also utilized to confirm the specificity of
the antibodies.
Fluorescent in situ Hybridization (FISH). The paraffin-
embedded tissue sections were deparaffinized and hydrated in
a series of alcohol washes as mentioned above. The tissue
sections were then placed in a solution of sodium borohydride
for 20 min on ice. Tissues were fixed with 4%
paraformaldehyde (PFA, J.T Baker) for 15 min at RT. Next,
the sections were washed with 2× saline sodium citrate (SSC)
buffer for 5 min and digested with 100 ug/mL of proteinase K
(Sigma) at RT for 15 min. The slides were then treated with
denaturing solution (70% v/vformamide and 2× SSC) and
incubated for 5 min at 95 °C and at RT. The slides were fixed
again with 4% PFA for 10 min at RT and washed with 2×
SSC before being again placed in denaturing solution at 60 °C
for 2 min. The salmon sperm DNA (2.5 ng, Thermo Fisher
Scientific) was prewarmed at 95 °C for 5 min and added to
the slides for blocking for an hour at 48 °C. The slides were
then incubated with previously validated fluorescent in situ
DNA probes [21, 70]: Borrelia-specific 16S rDNA probe
(FAM-5-GGATATAGTTAGAGATAATTATTCCCCGTTTG-
3) and Chlamydia-specific 16S rDNA probe (Alexa 568 5-
CCTCCGTATTACCGCAGC-3) after denaturing the probes at
95 °C for 10 min. A coverslip was placed on the slide to
ensure that the tissue did not dry out, and the tissue sections
were incubated for 16 h overnight at 48 °C. After overnight
incubation, the coverslip was removed by placing the slides in
2× SSC for 5 min, followed by five-time washes of 0.2× SSC
buffer for 5 min each at RT in the dark. Sections were then
counterstained with 0.1% Sudan black dye (Sigma) for 20 min
in the dark at RT. The slides were washed five times with 2×
SSC for 5 min before mounting the slides with PermaFluor
mounting media (Thermo Scientific) and stored at 4 °C.
Images were taken using a Leica DM2500 fluorescence
microscope at 200× and 400× magnification.
All FISH steps were repeated with several negative controls
such as the following: 1) 100 ng random oligonucleotide, (5-
FAM-GCATAGCTCTATGACTCTATACTGGTACGTAG-3),
2) 200 ng of unlabeled competing oligonucleotide added
before the hybridization step [competing Borrelia
(5-CAAACGGGGAATAATTATCTCTAACTATATCC-3) and
competing Chlamydia (5-CCTCCGTATTACCGCGGC-3)],
and 3) a DNase treatment of the sections before the hybridiza-
tion step to digest all genomic DNA (100 μg/mL for 60 min
at 37 °C).
A combination of immuno and in situ protocols was per-
formed in a similar fashion as described earlier [21]. Briefly,
after the 0.2× SSC wash in the FISH protocol the sections
were blocked with a 1:200 dilution of goat serum (Thermo
Scientific) for an hour at RT in a humidified chamber. The
slides were washed five times with PBS followed by adding
the primary polyclonal anti-alginate antibody for overnight in-
cubation at RT. The next day the slides were tagged with a
1:200 dilution of the secondary anti-rabbit antibody with a
fluorescent blue tag (goat anti-rabbit IgG (H+L), DyLight 405
conjugated) and incubated for an hour at RT. This step was
then followed by a counterstaining step with 0.1% Sudan
black for 20 min, followed by several washes in 0.2× SSC
and mounting with PermaFluor mounting media (Thermo Sci-
entific) and storing at 4 °C. Images were taken using Leica
DM2500 fluorescent microscope at 200× and 400×
magnification.
Confocal Microscopy. The tissue sections were visualized
and scanned with a confocal scanning laser microscope (Leica
3
E. Sapi et al.
DMI6000) for generating z-axis stacks for visualization of the
dual species biofilm in a three-dimensional view. ImageJ
software was used to process the generated zstacks in order
to receive a detailed analysis of the spatial distribution of the
multi-species biofilms (Plugins: Interactive 3D Surface Plot
and Volume Viewer).
Ethics. The study used archived paraffin embedded
sections which was sent to University of New Haven without
any identification. The Institutional Review Board at the
University of New Haven approved the study under 45 CFR
46.101(b)(4): Research involving the collection or study of
existing data, documents, records, pathological specimens, or
diagnostic specimens, if these sources are publicly available
or if the information is recorded by the investigator in such a
manner that subjects cannot be identified, directly or through
identifiers linked to the subjects.
Results
PCR Analyses of Borrelia-Positive BL Skin Tissues for
Chlamydia spp.. The first aim of the study was to evaluate
the potential presence of Chlamydia spp. in the Borrelia-
positive biopsy tissues of BL patients. We used archived skin
biopsies from our previous studies, in which we proved that
the BL tissues are positive for Borrelia afzelii DNA using PCR
methods performed by two independent research laboratories
previously [8, 21]. To amplify Chlamydia spp. DNA, several
previously published PCR protocols were utilized which were
designed to amplify the major outer membrane protein A
(OmpA) gene and were able to identify the species [68, 69].
The Chlamydia OmpA-specific PCR protocols resulted in
positive bands in the BL tissues studied. Interestingly, when
the DNAs were sequenced and analyzed by Basic Local
Alignment Search Tool (BLAST, bioinformatics tool on NCBI
website), the results revealed that multiple Chlamydia species
were present in the Borrelia-infected BL skin tissues. In some
of the BL tissues (4 out 6 samples), we were able to identify a
common sequence with 99% identity to C. pneumoniae
(KC512913; 98% coverage with E value: 7e-151), 86%
identity to C. psittaci (KM247620; in 64% coverage with E
value: 1e-61), and 76% to C. trachomatis (EU040365, in 71%
of coverage and E value: 1e-40). The sequences were further
analyzed by Clustal Omega multiple sequence alignment tool
on the EMBL-EBI server (https://www.ebi.ac.uk/Tools/msa/
clustalo/). Figure 1A shows a representative multiple sequence
alignment of the BL Chlamydia OmpA sequence to the
pathogenic Chlamydia sequences.
Using another published OmpA PCR protocol [69], we am-
plified a significantly different common sequence in some of
the BL tissue samples (2 out 6 samples), showing 97% iden-
tity to C. trachomatis (JX559522; 93% coverage with E value
9e-50), 81% to C. psittaci (HM214490, in 67% of coverage
Figure 1. (A) A multiple sequence alignment obtained from Clustal Omega analyses representing BL Chlamydia OmpA DNA sequence mapped
against different Chlamydia strains of Chlamydia psittaci (KM247620), Chlamydia trachomatis (EU040365), and Chlamydia pneumoniae
(KC512913). (B) Clustel Omega multiple sequence alignment of OmpA gene DNA sequences obtained from BL tissues against different Chla-
mydia strains such as Chlamydia trachomatis (JX559522), Chlamydia psittaci (HM214490), and Chlamydia pneumoniae (DQ358972). Asterisks
represent identical nucleotide sequence in all four Chlamydia sequences
4
Borrelia and Chlamydia Can Form Mixed Biofilms
and E value 2e-19), and 79% identity to C. pneumoniae
(DQ358972; in 80% coverage with E value 2e-20).
Figure 1B shows a multiple sequence alignment obtained
from Clustal Omega EMBI/EBI server representing DNA se-
quences from BL tissue OmpA DNA samples mapped against
3strainsofChlamydia trachomatis (JX559522), Chlamydia
psittaci (HM214490), and Chlamydia pneumoniae (DQ358972).
Immunohistochemical (IHC) Staining of Human Biopsy
Skin Tissues for Borrelia and Chlamydia.To further prove
the presence of Chlamydia species and to determine whether
there is a potential co-existence of the previously identified
Borrelia biofilms found in these BL biopsy tissues [21], IHC
staining techniques were used which were specific for Borre-
lia,Chlamydia, and alginate (biofilm marker) antigens.
Figure 2 shows that positive immunostaining for Borrelia
(Figure 2, panels A, F, and K, green arrows,) and for the bio-
film-specific marker alginate (Figure 2, panels C, H, and M,
blue arrows) is present in all 6 BL biopsy tissues. For some
tissue sections, the Borreliaalginate positive aggregates also
showed positive co-staining for Chlamydia spp. (Figure 2,
Panels B and G, red arrows); however, some of the tissue sec-
tions only stained positive for Borrelia and alginate (Figure 2,
panels K and M) but not for Chlamydia (Figure 2,panel L).
Crucially, there was no immunostaining for Chlamydia spp. in
the biofilm-free regions of tissues; however, Borrelia spiro-
chetes were found frequently in the vicinity of the biofilm
structures (Figure 2, panels A, F, and K green arrowheads).
As reported previously [21, 22], those spirochetes were all
negative for alginate antibody [Figure 2, panels C, H, and M].
All of the IHC experiments included 2 independent nega-
tive controls to prove the specificity of antibodies: non-spe-
cific IgG antibody and normal human skin samples. No signal
was observed in the BL skin tissues when non-specific anti-
body was used in the IHC procedure (Figure 2, panels D, I,
N, S, and Y). Furthermore, there were no immunostaining on
the 20 commercially purchased human foreskin (Figure 2,
panels P, Q, R, S, and T) and 20 healthy skin tissue sections
(Figure 2, panels V, W, X, Y. and Z) for Borrelia and Chla-
mydia, as well as alginate antigens. To demonstrate the struc-
ture of the biofilm and how it is embedded in the tissue, the
morphology of the BL skin tissues was visualized using a
differential interference contrast microscopy method (DIC;
Figure 2, panels E, J, O, T, and Z).
All images are taken with relatively low magnification
(200×) to demonstrate the biofilm surrounding tissues and to
show the background signals of the IHC methods.
To further analyze and understand the frequency of co-exis-
tence of Chlamydia spp. in Borrelia biofilms, an additional
150 sections were stained using IHC staining procedures de-
scribed above. Figure 3 shows representative images of Borre-
lia/alginate and Chlamydia-positive staining biofilms; a higher
magnification (400×) is used than that used in Figure 2 for a
better visualization of their structures. Borrelia-positive aggre-
gates were seen in all BL skin tissue samples (Figure 3, panels
A, E, I, M, and R, green arrows, ref). Those structures were
also stained positive for alginate, showing that they are indeed
biofilms (Figure 3, panels C, G, K, O, and T, blue arrows).
Most Borrelia and alginate positive structures stained positive
for Chlamydia (Figure 3, panels B and F, red arrows). How-
ever, not all of those biofilm structures were positive for
Chlamydia which further shows the specificity of our IHC
protocol, (Figure 3, panels J, N, and S). The differential inter-
ference images depict the tissue morphology and the structure
of the biofilm (Figure 3,panels D, H, L, P, and V).
Figure 2. Representative IHC images of Borrelia,Chlamydia, and alginate staining in Borrelia-infected BL skin tissues. Panels A, F, K, P, and V
show IHC staining results of skin tissues using a FITC labeled anti-Borrelia antibody (green arrows and arrowheads). Panels B, G, L, Q, and W
show staining results with anti-chlamydia antibody (red arrows). Panels C, H, M, R, and X show staining of anti-alginate antibody (blue arrows).
Panels D, I, N, S, and Y show results of staining with non-specific IgG antibody. Panels E, J, O, T, and Z are the differential interference contrast
(DIC) images that show the morphology of the tissues. Panels AO corresponds to BL skin tissues while panels PT include negative controls cor-
responding to skin tissues from healthy human foreskin, and panels VZ include negative controls corresponding to healthy skin tissues. All im-
ages were taken at 200× magnification. Scale bar: 200 μm
5
E. Sapi et al.
Quantitative analysis of a total of 150 IHC stained slides
was carried out to categorize the size and frequency of the co-
localization of Borrelia biofilms with Chlamydia spp. in BL
skin tissues by direct counting of the positive structures. Each
slide contained 24 biofilms and each biofilm size varied from
a range of 2080 μm.
Approximately 84% of Borrelia positive biofilms were pos-
itive for co-existence with Chlamydia spp. (Figure 4).
FISH Staining of Human Biopsy Skin Tissues for Borre-
lia and Chlamydia.To further confirm the results obtained
by IHC staining, fluorescent in situ hybridization (FISH)
methods were utilized. FISH probes specific for 16S rDNA of
Borrelia and Chlamydia were chosen from previously vali-
dated studies [21, 70]. For each slide containing structures,
IHC-positive for Borrelia (green staining, Figure 5, panel A)
that co-stained with Chlamydia spp. antibody (red staining,
Figure 5, panel B) and with the biofilm marker alginate
(blue staining, Figure 5, panel C), the next consecutive slide
was stained using a combined IHC and FISH technique. The
Borrelia-species-specific 16S rDNA probe (green staining,
Figure 5, Panel E) was co-localized with the Chlamydia-
DNA-specific 16S rDNA probe (red staining, Figure 5, panel
F). The Borrelia/Chlamydia-positive structures also stained
positive for IHC staining using anti-alginate antibody which
confirmed the co-localization of Borrelia biofilms with Chla-
mydia spp. in the BL skin tissues. Several negative controls
were included in the study to confirm the specificity of the
chosen FISH probes with our target organisms. Competing ol-
igonucleotide probes showed no significant staining for both
Borrelia (Figure 5, panel I) and Chlamydia (Figure 5,panel
J). As additional negative controls, a random DNA probe
(Figure 5, panel K) and a DNAse I pre-treated sample (Fig-
ure 5, panel L) were used which resulted in no significant
staining. The tissue morphology was visualized using the DIC
images, which show how the biofilm is embedded in the tissue
(Figure 5, panels D and H).
Confocal Imaging of Borrelia and Chlamydia Positive
Tissues. A tissue section that was IHC positive for co-exis-
tence of Borrelia and Chlamydia and for the biofilm marker
alginate (Figure 6,panels A, B, and C) were scanned with a
confocal scanning laser microscope (Leica DMI6000) to fur-
ther analyze the structure of the biofilm in the BL skin tissues
Figure 3. Representative images of IHC staining of BL biopsy skin tissues with Borrelia,Chlamydia, and alginate-specific antibodies. Panels A,
E, I, M, and R show IHC positive staining for Borrelia (green arrows), and panels B and F show positive staining of Chlamydia (red arrows),
while panels J, N, and S show negative staining for Chlamydia spp. Panel C, G, K, O, and T depict positive staining for alginate (blue arrows).
Panel D, H, L, P, and V show DIC images. All images were taken at 400× magnification. Scale Bar: 200 μm
Figure 4. Quantitative analysis of Borrelia biofilms for positive Bor-
relia and Chlamydia IHC staining
6
Borrelia and Chlamydia Can Form Mixed Biofilms
in a three-dimensional view. The obtained image shows the
spatial distribution and the integrity of the biofilm along with
the individual Z stacks further providing evidence for Borrelia
and Chlamydia co-existence in the Borrelia/alginate positive
structure (Figure 6, panel E, F, and G). The individual Z
stacks show aggregates of Chlamydia enclosed within the cen-
ter of Borrelia biofilm (Figure 6,panel F, red arrows). The in-
dividual Z stacks of Borrelia and alginate show how alginate,
a component of the EPS layer, surrounds the Borrelia biofilm
(Figure 6,panel G, blue arrow).
Discussion
Previous studies have shown that Borrelia burgdorferi
sensu stricto and the sensu lato group are capable of forming
biofilms in vitro [19, 20]. Recently, we also provided in vivo
evidence for the presence of Borrelia burgdorferi biofilms in
Figure 5. Representative images of the IHC staining on BL skin tissue section and the images of the consecutive slides stained with combined
fluorescent in situ hybridization (FISH) and IHC techniques. Panels A, B, and C are the results of skin tissues stained with antibodies against Bor-
relia (green arrow), Chlamydia (red arrow), and alginate (blue arrow), respectively. Panels E and I show the staining results of skin tissues with
16S rDNA probe for Borrelia burgdorferi (green arrow). Panels F and J show the staining results of skin tissues with 16S rDNA probe for Chla-
mydia spp. (red arrow). Panel G is stained with antibodies for alginate (blue arrow). Panels D and H depict the morphology of the skin tissues
using DIC microscopy methods. As comprehensive negative controls, a competing oligonucleotide (panels I and J for Borrelia and Chlamydia, re-
spectively), a random DNA probe (panel K), and a DNase-treated samples (panel L) were used on consecutive tissue sections to further show the
specific city of the 16S rDNA probe (for further details of the experimental conditions can be in Materials and Methods). All images were taken at
400× magnification. Scale bar: 100 μm
Figure 6. Three-dimensional (3D) analyses of Borrelia and Chlamydia mixed biofilm in human BL skin biopsy tissue using confocal microscopy.
Panels A, B, and C are the results of skin tissues positively immunostained with antibodies against Borrelia (green arrow), Chlamydia (red arrow),
and alginate (blue arrow), respectively. Panel D shows the DIC image to depict the morphology of the tissue. Confocal microscopy shows the 3D
distribution of mixed biofilms and the individual Z stacks focus on the biofilms showing Borrelia and Chlamydia (panel F) and Borrelia and algi-
nate (panel G) spatial distribution. Scale bar: 100 μm
7
E. Sapi et al.
Borrelia-infected skin lesions called Borrelia lymphocytoma
(BL) [21]. However, the question of co-existence of Borrelia
biofilms in the multi-species form is yet to be answered. This
study investigated the presence of potential co-infections of
Borrelia biofilms with Chlamydia spp. It is among the first to
document the co-existence of Borrelia biofilms with the intra-
cellular pathogen Chlamydia spp. in infected human skin tis-
sues, and to the best of our knowledge, this is the first study
to show Chlamydia within the biofilm.
Our PCR and sequencing analyses showed that Borrelia
positive BL tissue samples are also positive for Chlamydia
DNA, and the obtained sequencing was mapped to several
chlamydial strains and was found to have the best match to 2
human pathogens, C. pneumoniae and C. trachomatis strains.
Studies conducted in Finland and Australia reported Chla-
mydia-like DNA in skin biopsies of patients suspected to have
a tick bite and who were PCR-positive for Borrelia DNA as
well [5052]. A very recent study provided evidence that IgM
and IgG antibodies for both C. pneumoniae and C. trachoma-
tis can be detected in 2030% of patients with tick bite history
[71]. Those studies strongly indicated that co-infection of Bor-
relia with Chlamydia spp. is possible.
After finding chlamydial DNA in BL skin biopsies, the
question became whether they exist in biofilm form. To exam-
ine BL skin lesions for co-existence of Borrelia and Chla-
mydia in biofilm form, IHC staining and FISH techniques
were used.
As previously reported, alginate is successfully being
adapted as a biofilm marker and was used to confirm the co-
existence of both bacterial species in the biofilm form [19].
Alginate has been reported to be a key component of the EPS
layer in Borrelia burgdorferi sensu stricto and sensu lato bio-
films [1921]. Although no direct evidence suggests the exis-
tence of Chlamydia individually in a biofilm form, they could
be a part of a microbial community with other bacterial bio-
films. Biofilm forming bacteria can promote the participation
of strains of non-biofilm forming bacteria in a community as
is observed in dental plaques with Actinomycetes spp. [72].
In fact, our IHC and FISH data suggest that Chlamydia spp.
can exist in aggregate forms as suggested on other systems
[60, 66]. Furthermore, environmental stressors are known to
push Chlamydia into a state of persistence, in which they are
viable but non-infectious [73]. Persistent like morphological
characteristics of Chlamydia have been identified in vivo [74]
and several studies have shown resistance of chlamydial infec-
tion to antibiotics both in vitro and in vivo [75].
Our confocal microscopy data suggest a very specific spa-
tial distribution of Chlamydia in the Borrelia biofilm. Previous
studies suggested that the different bacteria in multi-species
biofilms could have specific spatial distribution which sup-
ports our confocal image findings showing that Chlamydia is
localized in the middle of Borrelia biofilm rather than ran-
domly distributed [42, 77, 78]. Our confocal analyses also
demonstrated that the Borrelia/Chlamydia-positive biofilm
structure is surrounded by alginate. The observation raises the
question about which organism secretes alginate rich protec-
tive matrix. While studies show that Chlamydiae-infected cell
cultures express a glycolipid that is similar to alginate in its
polysaccharide content and molecular weight [76], our data
suggests that the alginate being expressed is probably secreted
by Borrelia burgdorferi and not by Chlamydia because all
Chlamydia negative biofilms are positive for alginate.
Ticks are capable of inoculating and harvesting several dif-
ferent pathogens upon infection to the host organism. A study
conducted in Switzerland and Algeria evaluated ticks and fleas
for the presence of Chlamydiales DNA and found ticks to be
a possible vector for transmission of Chlamydia spp. [50]. The
same group in 2015 then reported a higher prevalence and di-
versity of Chlamydiales DNA in ticks [51]. Another study
supported and confirmed the presence of Chlamydia-related
organism in ticks [52], and they also found sequences similar
to Chlamydia DNA in human skin biopsies. The study
screened skin biopsies of patients with suspected history of
tick bite and reported Chlamydia DNA in 85% Borrelia PCR
positive biopsies and 71% positive for Chlamydia DNA in
Borrelia PCR negative skin biopsies [52].
Mono-species biofilms alone have proven to be 100 to
1000 times more resistant to antibiotics, leading to persistent
infections [79]. Our research group has demonstrated the ex-
traordinary resistance of Borrelia biofilms to several antibi-
otics in vitro [22, 80], which may explain the persisting
symptoms observed in Lyme patients. Multi-species biofilms
are being studied extensively in relation to several chronic in-
fections. Chronic wound infections in a porcine model showed
increased resistance to antimicrobial activity upon infection
with Staphylococcus aureus in a multi-species biofilm form
[39, 81, 82]. Pulmonary infections in cystic fibrosis patients
have been suggested to contain several different airway patho-
gens making them more complex and resistant to treatments
[40]. Studies have identified Dolosigranulum pigrum and
Pseudomonas aeruginosa in biofilm form in pulmonary infec-
tions and have shown increased resistance to antimicrobial
treatments [40]. Diabetic foot ulcers show polymicrobial infec-
tion involving S. aureus,P. aeruginosa, and E. coli at the site
of infection, slowing the healing process and, in some cases,
leading to antimicrobial resistance [83]. These findings
strongly suggest that microbial communities behave synergis-
tically with each other in a mixed biofilm form.
The symptoms observed during Lyme infection are very
similar to those of chlamydial infection [62, 63]. Arthritis is
one of the major symptoms observed in both of these bacterial
infections, and a study suggested the intra-articular co-infec-
tion of Chlamydia trachomatis and Borrelia burgdorferi in pa-
tients with oligoarthritis [63]. Furthermore, Chlamydia and
Borrelia DNAs were found in the synovial fluid of patients
with undifferentiated oligoarthritis [84].
Another example for skin infections, which can be caused
by Borrelia or Chlamydia, is erythema nodosum, a condition
leading to skin inflammation with painful, red deep-seated
nodules [62, 85]. The skin condition erythema multiforme has
been also associated with Borrelia and C. pneumoniae infec-
tions [86, 87].
Furthermore, C. pneumonia infections have been linked to
atherosclerosis and well characterized in atherosclerotic pla-
ques [58]. Lyme carditis is one of the chronic infections of
Lyme disease, and an independent study reported seropositiv-
ity results for anti-Borrelia IgG antibodies in carotid athero-
sclerosis [88]. In addition, a recent study observed biofilm
formation in atherosclerotic plaques, which indeed suggests
that biofilms could be present in cardiac tissues and be a part
of the biofilm community with several other species [89, 90].
The obvious question is whether multi-species biofilms
could have even higher antibiotic resistance for antibiotics
than mono-species biofilm. In a synergistic relationship, both
biofilm partners should provide advantage for the whole com-
munity [42]. The obvious question is why Borrelia and Chla-
mydia can be found together so frequently and how they can
build symbiotic relationships. Chlamydia, for example, cannot
produce the ATP molecule for its energetic processes [5861].
Therefore, it is possible that Borrelia must provide ATP inside
the biofilm structure. Furthermore, Borrelia biofilm is known
to have a very organized structure that confers high resistance
to environmental stressors [19, 20, 21]; therefore, Borrelia
could also provide the necessary shelter for Chlamydia.
8
Borrelia and Chlamydia Can Form Mixed Biofilms
Conversely, Chlamydia could supply iron necessary for Borre-
lia. Several studies have reported that Borrelia uses manga-
nese instead of iron for its own biological processes [91, 92].
Yet, iron appears to play a crucial role in biofilm formation
by stabilizing the polysaccharide matrix, as was shown in
S. aureus and P. aeruginosa biofilms [9395]. Moreover, in a
multi-species biofilm of Candida albicans and P. aeruginosa,
iron triggers virulence of the bacterial pathogens and can
cause significant damage to the host [96]. Relating the role of
Chlamydia in the biofilm form with Borrelia could suggest
that they have a symbiotic relation.
In summary, our data provides strong evidence for the co-
existence of Chlamydia spp. with Borrelia biofilms in human
skin biopsies of BL lesions with their involvement in Borrelia
biofilms. This study warrants further research to understand
the physiological role of mixed biofilms in chronic Lyme
disease.
Abbreviations
ATCC - American Type Culture Collection
BL - Borrelial lymphocytoma
BSA - bovine serum albumin
BSK-H - Barbour-Stoner-Kelly H
DIC - differential interference contract microscopy
EM - erythema migrans
EDTA - ethylenediaminetetraacetic acid
FAM - 6-fluorescein amidite
FFPE - formalin-fixed, paraffin-embedded
FISH - fluorescent in situ hybridization
FITC - fluorescein isothiocyanate
H&E - hematoxylin and eosin
IHC - immunohistochemistry
OmpA - major outer membrane protein A
PBS - phosphate-buffered saline
PCR - polymerase chain reaction
RT - room temperature
SSC - saline sodium citrate
Funding Sources
This work was supported by grants from the University of
New Haven, National Philanthropic Trust, LivLyme Founda-
tion, Lyme Warriors and CT Lyme Riders scholarships to KG
and KW. We also thank the Schwartz Research foundation for
the donation of the Leica DM2500 microscope, as well as the
Hamamatsu ORCA Digital Camera.
Authors' Contribution
ES conceptualized, designed, and supervised the study, ana-
lyzed and interpreted data, obtained funding, and wrote the
manuscript. KG designed and performed experiments, ana-
lyzed and interpreted data, and wrote manuscript. KW, JT, and
GG designed and performed experiments. AM analyzed and
interpreted data. BZ conceptualized and designed the study.
All authors had full access to all data in the study and take re-
sponsibility for the integrity of the data and the accuracy of
the data analysis.
Conflict of Interest
The authors have declared that no competing interest exists.
Acknowledgements. The authors would like to thank Dr.
Gerald B. Pier (Harvard University) for the anti-alginate
antibody. We also thank Dr. Dougles Brash and Dr. Joseph J.
Burrascano for their helpful suggestions for the final manuscript.
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10
Borrelia and Chlamydia Can Form Mixed Biofilms
... Those conditions are well known to be the most difficult infections to clear [66,67]. Multispecies biofilms were also recently discovered for B. burgdorferi [68,69]. In two different dermatological human diseases (Borrelial lymphocytoma and Morgellons diseases), Chlamydia spp. ...
... In two different dermatological human diseases (Borrelial lymphocytoma and Morgellons diseases), Chlamydia spp. and Helicobacter pylori species were found respectively inside of the B. burgdorferi biofilm structures [68,69]. These findings strongly suggest that we need to establish multi-pathogen infection model systems for the further understanding of those complex infections. ...
Article
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Borrelia burgdorferi, the causative agent of Lyme disease, has been recently shown to form biofilm structures in vitro and in vivo. Biofilms are tightly clustered microbes characterized as resistant aggregations that allow bacteria to withstand harsh environmental conditions, including the administration of antibiotics. Novel antibiotic combinations have recently been identified for B. burgdorferi in vitro, however, due to prohibiting costs, those agents have not been tested in an environment that can mimic the host tissue. Therefore, researchers cannot evaluate their true effectiveness against B. burgdorferi, especially its biofilm form. A skin ex vivo model system could be ideal for these types of experiments due to its cost effectiveness, reproducibility, and ability to investigate host–microbial interactions. Therefore, the main goal of this study was the establishment of a novel ex vivo murine skin biopsy model for B. burgdorferi biofilm research. Murine skin biopsies were inoculated with B. burgdorferi at various concentrations and cultured in different culture media. Two weeks post-infection, murine skin biopsies were analyzed utilizing immunohistochemical (IHC), reverse transcription PCR (RT-PCR), and various microscopy methods to determine B. burgdorferi presence and forms adopted as well as whether it remained live in the skin tissue explants. Our results showed that murine skin biopsies inoculated with 1 × 107 cells of B. burgdorferi and cultured in BSK-H + 6% rabbit serum media for two weeks yielded not just significant amounts of live B. burgdorferi spirochetes but biofilm forms as well. IHC combined with confocal and atomic force microscopy techniques identified specific biofilm markers and spatial distribution of B. burgdorferi aggregates in the infected skin tissues, confirming that they are indeed biofilms. In the future, this ex vivo skin model can be used to study development and antibiotic susceptibility of B. burgdorferi biofilms in efforts to treat Lyme disease effectively.
... As mentioned above, Borrelia have also been observed in clusters and aggregates of various sizes in vitro [325,326,339,340] and in vivo [323,341]. These clusters are capable of forming on a number of biotic and abiotic surfaces [325], and have been reported to contain extracellular DNA (eDNA), alginate, and calcium-all known components of the extra-polymeric substance (EPS) matrix characteristic of bacterial biofilms [325,340,342]. ...
... A recent study has identified polymicrobial biofilms featuring Borrelia spp. and Chlamydia spp. in human tissue [341]. Interestingly, the high presence of round bodies is closely associated with biofilm formation [325], although the exact relationship between these morphological variants has not been well studied. ...
Article
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Lyme disease is a complex tick-borne zoonosis that poses an escalating public health threat in several parts of the world, despite sophisticated healthcare infrastructure and decades of effort to address the problem. Concepts like the true burden of the illness, from incidence rates to longstanding consequences of infection, and optimal case management, also remain shrouded in controversy. At the heart of this multidisciplinary issue are the causative spirochetal pathogens belonging to the Borrelia Lyme complex. Their unusual physiology and versatile lifestyle have challenged microbiologists, and may also hold the key to unlocking mysteries of the disease. The goal of this review is therefore to integrate established and emerging concepts of Borrelia biology and pathogenesis, and position them in the broader context of biomedical research and clinical practice. We begin by considering the conventions around diagnosing and characterizing Lyme disease that have served as a conceptual framework for the discipline. We then explore virulence from the perspective of both host (genetic and environmental predispositions) and pathogen (serotypes, dissemination, and immune modulation), as well as considering antimicrobial strategies (lab methodology, resistance, persistence, and clinical application), and borrelial adaptations of hypothesized medical significance (phenotypic plasticity or pleomorphy).
... All these various organisms may be found in AD brains because the biofilms made by one organism have attachment sites for other organisms and may easily incorporate them into a multiorganism community [19]. Thus, the other organisms, while they are not primary, can, and very likely do, join the community; this finding has recently been demonstrated [20]. The pathway of spirochetes leading to AD is sequentially documented in the Supplementary Material. ...
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Utilizing the pathology and microbiology found in tissue from patients with documented Alzheimer’s disease (AD), the pathogenesis of this fateful disorder has been made clear. Borrelia burgdorferi and Treponema denticola spirochetes enter the brain, mostly via neuronal pathways and the entorhinal circulation. These organisms easily pass through the blood-brain barrier and have an affinity for neural tissue. Once in the brain, the spirochetes make intra- and extracellular biofilms, and it is the biofilms that create the pathology. Specifically, it is the intracellular biofilms that are ultimately responsible for neurofibrillary tangles and dendritic disintegration. The extracellular biofilms are responsible for the inflammation that initially is generated by the first responder, Toll-like receptor 2. The hypothesis that arises from this work is two-pronged: one is related to prevention; the other to treatment. Regarding prevention, it is very likely possible that AD could be prevented by periodic administration of penicillin (PCN), which would kill the spirochetes before they made biofilms; this would prevent the disease and would not allow any of the above deleterious changes generated by the biofilms to occur. As regards treatment, it may be possible to slow or prevent further decline in early AD by administration of PCN together with a biofilm disperser. The disperser would disrupt the biofilm coating and enable the PCN to kill the spirochetes. This protocol could be administered in a trial with the control arm utilizing the current treatment. The progress of the treatment could be evaluated by one of the current blood tests that is semi-quantitative. The specific protocols are listed.
... Thus, the studies of skin microbiota reported here is mostly derived from cases involving skin diseases. Sapi et al. (2019) investigated the presence of mixed-species biofilms in Borrelia-infected human skin biopsies, and found that it was possible to detect Chlamydia antigens and DNA in 84% of the sampled Borrelia-biofilms. In another study recently published, they found that Borrelia burgdorferi and Helicobacter pylori was detected in mixed-species biofilms in dermatological skin-samples from people with Morgellons disease (Middelveen et al., 2019). ...
Article
Full-text available
Chronic infections present a serious economic burden to health-care systems. The severity and prevalence of chronic infections are continuously increasing due to an aging population and an elevated number of lifestyle related diseases such as diabetes. Treatment of chronic infections has proven difficult, mainly due to the presence of biofilms that render bacteria more tolerant toward antimicrobials and the host immune response. Chronic infections have been described to harbor several different bacterial species and it has been hypothesized that microscale interactions and mixed-species consortia are present as described for most natural occurring biofilms i.e., aquatic systems and industrial settings, but also for some commensal human biofilms i.e., the mouth microbiota. However, the presence of mixed-species biofilms in chronic infections is most often an assumption based on culture-based methods and/or by means of molecular approaches, such as PCR and sequencing performed from homogenized bulk tissue samples. These methods disregard the spatial organization of the bacterial community and thus valuable information on biofilm aggregate composition, spatial organization, and possible interactions between different species is lost. Hitherto, only few studies have made visual in situ presentations of mixed-species biofilms in chronic infections, which is pivotal for the description of bacterial composition, spatial distribution, and interspecies interaction on the microscale. In order for bacteria to interact (synergism, commensalism, mutualism, competition, etc.) they need to be in close proximity to each other on the scale where they can affect e.g., solute concentrations. We argue that visual proof of mixed species biofilms in chronic infections is scarce compared to what is seen in e.g., environmental biofilms and call for a debate on the importance of mixed-species biofilm in chronic infections.
... It is likely that the biofilms in the Alzheimer's brains are more similar to the dental biofilms rather than those in the skin. Chlamydia pneumoniae, herpes simplex, and porphyromonas gingivalis have all been shown to be present in Alzheimer's brains along with the spirochetes [28][29][30]. It is likely that the spirochetes play a dominant role in these brains for many reasons. ...
Article
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Herein we present the findings related to biofilms in Alzheimer's disease and compare them to known findings related to biofilms in other chronic diseases. Similarities include microbes making the biofilms both intra and extracellularly, the interaction of the innate immune system in many instances, the devastating impact of the adaptive immune system, and the devastating impact resulting from the various genes involved. Differences include location, the production of beta amyloid, neurofibrillary tangles, and hyperphosphorylated tau protein. The diseases compared include atopic dermatitis, psoriasis, tinea versicolor, leprosy, gout, rheumatoid arthritis and other arthritides.
... The pathology of LD appears to be caused primarily by host immune response, as Borrelia is not known to produce toxins or proteases that directly damage tissues [9]. The formation of persister cells and biofilms harboring persisters and other microbes [10][11][12][13][14][15][16], as well as other immune evasion mechanisms, likely play roles in pathogenesis and tissue damage through the misdirection of host immune response. ...
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Lyme disease (LD) is an increasingly prevalent, climate change-accelerated, vector-borne infectious disease with significant morbidity and cost in a proportion of patients who experience ongoing symptoms after antibiotic treatment, a condition known as post-treatment Lyme disease syndrome (PTLDS). Spirochetal bacteria of Borrelia species are the causative agents of LD. These obligate parasites have evolved sophisticated immune evasion mechanisms, including the ability to defeat the innate immune system’s complement cascade. Research on complement function and Borrelia evasion mechanisms, focusing on human disease, is reviewed, highlighting opportunities to build on existing knowledge. Implications for the development of new antibiotic therapies having the potential to prevent or cure PTLDS are discussed. It is noted that a therapy enabling the complement system to effectively counter Borrelia might have lower cost and fewer side-effects and risks than broad-spectrum antibiotic use and could avert the need to develop and administer a vaccine.
... Taken as a whole, our culture, histological and molecular findings are consistent with the modified Koch's postulates that support a biofilm-related infectious etiology of MD lesions [101,102]. Additional pathogens may also be involved in formation of these biofilms [103]. Further studies in experimental animal models are needed to confirm the role of biofilms in the pathology of MD. ...
Article
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Background: Morgellons disease (MD) is a dermopathy that is associated with tick-borne illness. It is characterized by spontaneously developing skin lesions containing embedded or projecting filaments, and patients may also experience symptoms resembling those of Lyme disease (LD) including musculoskeletal, neurological and cardiovascular manifestations. Various species of Borrelia and co-infecting pathogens have been detected in body fluids and tissue specimens from MD patients. We sought to investigate the coexistence of Borrelia burgdorferi (Bb) and Helicobacter pylori (Hp) in skin specimens from MD subjects, and to characterize their association with mixed amyloid biofilm development. Methods: Testing for Bb and Hp was performed on dermatological specimens from 14 MD patients using tissue culture, immunohistochemical (IHC) staining, polymerase chain reaction (PCR) testing, fluorescent in situ hybridization (FISH) and confocal microscopy. Markers for amyloid and biofilm formation were investigated using histochemical and IHC staining. Results: Bb and Hp were detected in dermatological tissue taken from MD lesions. Bb and Hp tended to co-localize in foci within the epithelial tissue. Skin sections exhibiting foci of co-infecting Bb and Hp contained amyloid markers including β-amyloid protein, thioflavin and phosphorylated tau. The biofilm marker alginate was also found in the sections. Conclusions: Mixed Bb and Hp biofilms containing β-amyloid and phosphorylated tau may play a role in the evolution of MD.
Thesis
La Borréliose de Lyme est la maladie vectorielle la plus importante dans l’hémisphère nord en termes de prévalence. Elle est causée par des bactéries du genre Borreliella qui sont transmises à l’Homme par l’intermédiaire de morsures de tiques. En Europe, B. afzelii et B. garinii sont deux espèces majeures qui sont fréquemment co-détectées au sein d’une tique, soulevant l’hypothèse de l’existence d’un système leur permettant d’établir des co-infections. Le Quorum Sensing est un système de communication bactérien qui joue un rôle crucial dans le cycle de vie et la virulence des bactéries. Le Quorum Sensing induit par l’autoinducteur-2 (AI-2) permet une communication à la fois intra et inter-espèces. L’un des objectifs de cette thèse a été d’investiguer la présence, la fonctionnalité et le rôle de ce type de communication chez B. afzelii et B. garinii. Les résultats ont montré que ces deux espèces possèdent un système de communication AI-2 dépendant fonctionnel. Le développement d’outils visant à bloquer cette communication a été initié et permettra d’étudier de manière plus précise le rôle du Quorum Sensing dans la virulence des Borreliella ainsi que dans l’établissement de co-infections. En parallèle, la présence de tiques infectées par des Borreliella en Poitou-Charentes a été étudiée pour la première fois. Pour cela, le microbiote bactérien interne de tiques Ixodes ricinus a été étudié par séquençage haut-débit d’amplicons du gène codant l’ARNr 16s. Cette méthode présente l’avantage de permettre la caractérisation du microbiote bactérien dans son ensemble, à la fois les pathogènes mais également les autres genres bactériens. Un microbiote conservé/partagé des tiques analysées a été défini et la présence de séquences assignées à Borreliella spp. a été mise en évidence. Au sein de plusieurs tiques, différentes espèces de Borreliella spp. ont été détectées, indiquant de possibles co-infections.
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During the past few years, Alzheimer’s disease (AD) has been shown to be a chronic infection originating with a spirochete. These spirochetes form biofilms like most other microbes; moreover, in large measure, the biofilms contribute to both the chronicity and the pathogenesis of the disease. Once in a biofilm, the microbes become undetectable and resistant to the immune system and to antibiotics. Stroke, diabetes, nicotine, haloperidol, diet soft drinks, and others have all been shown to cause worsening of Alzheimer’s disease (AD) by their impact on biofilms. Penicillin, administered before the spirochetes form biofilms, would very likely prevent the disease.
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Recently, it was shown that hydroxychloroquine (HCQ) had a positive effect in COVID 19 infection, and this was augmented by the addition of azithromycin (Z). A proposed mechanism of action was outlined as to how these repurposed medications caused this. This short review offers an alternative hypothesis as to how this occurs, namely that the virus creates biofilms that activate the immune system which causes tissue damage. This is similar to other diseases, e.g. psoriasis, in which microbes create biofilms that interact with the immune system, and that interaction leads to the disease. Many other examples of other microbes that do this have been presented. How and where these two medications (HCQ and Z) fit into this hypothetical pathway is discussed. Other repurposed medications known to impact COVID 19 are briefly discussed.
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There is insufficient evidence to support screening of various tick-borne diseases (TBD) related microbes alongside Borrelia in patients suffering from TBD. To evaluate the involvement of multiple microbial immune responses in patients experiencing TBD we utilized enzyme-linked immunosorbent assay. Four hundred and thirty-two human serum samples organized into seven categories followed Centers for Disease Control and Prevention two-tier Lyme disease (LD) diagnosis guidelines and Infectious Disease Society of America guidelines for post-treatment Lyme disease syndrome. All patient categories were tested for their immunoglobulin M (IgM) and G (IgG) responses against 20 microbes associated with TBD. Our findings recognize that microbial infections in patients suffering from TBDs do not follow the one microbe, one disease Germ Theory as 65% of the TBD patients produce immune responses to various microbes. We have established a causal association between TBD patients and TBD associated co-infections and essential opportunistic microbes following Bradford Hill’s criteria. This study indicated an 85% probability that a randomly selected TBD patient will respond to Borrelia and other related TBD microbes rather than to Borrelia alone. A paradigm shift is required in current healthcare policies to diagnose TBD so that patients can get tested and treated even for opportunistic infections.
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The emergence and spread of multidrug-resistant organisms (MDROs) across global healthcare networks poses a serious threat to hospitalized individuals. Strategies to limit the emergence and spread of MDROs include oversight to decrease selective pressure for MDROs by promoting appropriate antibiotic use via antibiotic stewardship programs. However, restricting the use of one antibiotic often requires a compensatory increase in the use of other antibiotics, which in turn selects for the emergence of different MDRO species. Further, the downstream effects of antibiotic treatment decisions may also be influenced by functional interactions among different MDRO species, with the potential clinical implications of such interactions remaining largely unexplored. Here, we attempt to decipher the influence network between antibiotic treatment, MDRO colonization, and infection by leveraging active surveillance and antibiotic treatment data for 234 nursing home residents. Our analysis revealed a complex network of interactions: antibiotic use was a risk factor for primary MDRO colonization, which in turn increased the likelihood of colonization and infection by other MDROs. When we focused on the risk of catheter-associated urinary tract infections (CAUTI) caused by Escherichia coli, Enterococcus, and Staphylococcus aureus we observed that cocolonization with specific pairs of MDROs increased the risk of CAUTI, signifying the involvement of microbial interactions in CAUTI pathogenesis. In summary, our work demonstrates the existence of an underappreciated healthcare-associated ecosystem and strongly suggests that effective control of overall MDRO burden will require stewardship interventions that take into account both primary and secondary impacts of antibiotic treatments.
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The significance of polymicrobial infections is increasingly being recognized especially in a biofilm context wherein multiple bacterial species—including both potential pathogens and members of the commensal flora—communicate, cooperate, and compete with each other. Two important bacterial pathogens that have developed a complex network of evasion, counter-inhibition, and subjugation in their battle for space and nutrients are Pseudomonas aeruginosa and Staphylococcus aureus. Their strain- and environment-specific interactions, for instance in the cystic fibrosis lung or in wound infections, show severe competition that is generally linked to worse patient outcomes. For instance, the extracellular factors secreted by P. aeruginosa have been shown to subjugate S. aureus to persist as small colony variants (SCVs). On the other hand, data also exist where S. aureus inhibits biofilm formation by P. aeruginosa but also protects the pathogen by inhibiting its phagocytosis. Interestingly, such interspecies interactions differ between the planktonic and biofilm phenotype, with the extracellular matrix components of the latter likely being a key, and largely underexplored, influence. This review attempts to understand the complex relationship between P. aeruginosa and Staphylococcus spp., focusing on S. aureus, that not only is interesting from the bacterial evolution point of view, but also has important consequences for our understanding of the disease pathogenesis for better patient management.
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Bacteria use two strategies to avoid being killed by antibiotics: resistance and tolerance. Resistance mechanisms such as destruction of a drug or modification of its target allow bacteria to grow in the presence of antibiotics. Tolerance is a property of dormant, nongrowing bacterial cells in which antibiotic targets are inactive, allowing bacteria to survive. The two phenomena are mechanistically distinct and assumed to be unrelated. On page 826 of this issue, Levin-Reisman et al. ( 1 ) show that tolerance nevertheless leads to resistance.
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Identifying Borrelia burgdorferi as the causative agent of Lyme disease in 1981 was a watershed moment in understanding the major impact that tick-borne zoonoses can have on public health worldwide, particularly in Europe and the USA. The medical importance of tick-borne diseases has long since been acknowledged, yet little is known regarding the occurrence of emerging tick-borne pathogens such as Borrelia spp., Anaplasma phagocytophilum, Rickettsia spp., Bartonella spp., “Candidatus Neoehrlichia mikurensis”, and tick-borne encephalitis virus in questing ticks in Romania, a gateway into Europe. The objective of our study was to identify the infection and co-infection rates of different Borrelia genospecies along with other tick-borne pathogens in questing ticks collected from three geographically distinct areas in eastern Romania. We collected 557 questing adult and nymph ticks of three different species (534 Ixodes ricinus, 19 Haemaphysalis punctata, and 4 Dermacentor reticulatus) from three areas in Romania. We analyzed ticks individually for the presence of eight different Borrelia genospecies with high-throughput real-time PCR. Ticks with Borrelia were then tested for possible co-infections with A. phagocytophilum, Rickettsia spp., Bartonella spp., “Candidatus Neoehrlichia mikurensis”, and tick-borne encephalitis virus. Borrelia spp. was detected in I. ricinus ticks from all sampling areas, with global prevalence rates of 25.8%. All eight Borrelia genospecies were detected in I. ricinus ticks: Borrelia garinii (14.8%), B. afzelii (8.8%), B. valaisiana (5.1%), B. lusitaniae (4.9%), B. miyamotoi (0.9%), B. burgdorferi s.s (0.4%), and B. bissettii (0.2%). Regarding pathogen co-infection 64.5% of infected I. ricinus were positive for more than one pathogen. Associations between different Borrelia genospecies were detected in 9.7% of ticks, and 6.9% of I. ricinus ticks tested positive for co-infection of Borrelia spp. with other tick-borne pathogens. The most common association was between B. garinii and B. afzelii (4.3%), followed by B. garinii and B. lusitaniae (3.0%). The most frequent dual co-infections were between Borrelia spp. and Rickettsia spp., (1.3%), and between Borrelia spp. and “Candidatus Neoehrlichia mikurensis” (1.3%). The diversity of tick-borne pathogens detected in this study and the frequency of co-infections should influence all infection risk evaluations following a tick bite.
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Background Members of the order Chlamydiales are known for their potential as human and veterinary bacterial pathogens. Despite this recognition, epidemiological factors such as routes of transmission are yet to be fully defined. Ticks are well known vectors for many other infections with several reports recently describing the presence of bacteria in the order Chlamydiales in these arthropods. Australian wildlife are hosts to an extensive range of tick species. Evidence is also growing that the marsupial hosts these ticks parasitise can also be infected by a number of bacteria in the order Chlamydiales, with at least one species, Chlamydia pecorum, posing a significant conservation threat. In the current study, we investigated the presence and identity of Chlamydiales in 438 ixodid ticks parasitizing wildlife in Australia by screening with a pan-Chlamydiales specific targeting the 16S rRNA gene. Results Pan-Chlamydiales specific PCR assays confirmed the common presence of Chlamydiales in Australian ticks parasitising a range of native wildlife. Interestingly, we did not detect any Chlamydiaceae, including C. pecorum, the ubiquitous pathogen of the koala. Instead, the Chlamydiales diversity that could be resolved indicated that Australian ticks carry at least six novel Chlamydiales genotypes. Phylogenetic analysis of the 16S rRNA sequences (663 bp) of these novel Chlamydiales suggests that three of these genotypes are associated with the Simkaniaceae and putatively belong to three distinct novel strains of Fritschea spp. and three genotypes are related to the “Ca. Rhabdochlamydiaceae” and putatively belong to a novel genus, Rhabdochlamydia species and strain, respectively. Conclusions Sequence results suggest Australian wildlife ticks harbour a range of unique Chlamydiales bacteria that belong to families previously identified in a range of arthropod species. The results of this work also suggest that it is unlikely that arthropods act as vectors of pathogenic members of the family Chlamydiaceae, including C. pecorum, in Australian wildlife. The biology of novel Chlamydiales identified in arthropods remain unknown. The pathogenic role of the novel Chlamydiales identified in this study and the role that ticks may play in their transmission needs to be explored further. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-1994-y) contains supplementary material, which is available to authorized users.
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Urinary tract infections (UTIs) are one of the most important causes of morbidity and health care spending affecting persons of all ages. Bacterial biofilms play an important role in UTIs, responsible for persistent infections leading to recurrences and relapses. UTIs associated with microbial biofilms developed on catheters account for a high percentage of all nosocomial infections and are the most common source of Gram-negative bacteremia in hospitalized patients. The purpose of this mini-review is to present the role of microbial biofilms in the etiology of female UTI and different male prostatitis syndromes, their consequences, as well as the challenges for therapy.
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Atherosclerosis (AS) is a chronic disorder characterized by the formation and progression of plaques within arteries. Various microbes, most notably periodontal organisms, have been identified in plaques both epidemiologically and microbiologically, and have been deemed possible contributors to the disease. In this work, we have queried whether microbes acted similarly in AS, when compared to other chronic diseases such as atopic dermatitis, psoriasis, and Alzheimer's disease, by making biofilms to protect themselves and evade the immune system. In those diseases, the microbes created biofilms that activated the innate immune system reactant Toll-like receptor 2 (TLR2). We examined 12 endarterectomy specimens using probes similar to those used in our previous examinations of the above diseases. Specifically, we stained the pathology specimens with hematoxylin and eosin (H+E), and periodic acid Schiff (PAS); the PAS stain would reveal the extracellular polysaccharides that forms the mass of the biofilm. Congo red, which stains the amyloid that forms the infrastructure of biofilms, was also performed. Immunostaining with CD 282 was performed on each specimen for evaluation of TLR 2. Twelve of twelve atherosclerotic plaques showed the presence of biofilms and activation of TLR 2; this is entirely similar to our findings in atopic dermatitis, psoriasis and Alzheimer's disease. The TLR 2 seen in the specimens suggests that biofilms in atherosclerotic plaques may contribute to the progression of the disease as a result of their ability to contribute to chronic inflammation and continued immune system activation. Lipids have long been considered to be the major focus of atherosclerosis, but our recent work suggests that biofilms, due to their ability to induce a chronic inflammatory state, may be another determinant in the progression of atherosclerosis. In the future, we hope to characterize the microbes-with an initial focus on periodontal microbes because of the calcification in the plaques-that directly contribute to biofilm formation and propagation.
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A retrospective study of 109 skin biopsies with granuloma annulare (GA) or morphea histology from patients with suspected tick bite was performed. Biopsies were tested for cutaneous Borrelia burgdorferi DNA using PCR. The same biopsies were analysed for tick-borne novel agents, Chlamydia-related bacteria (members of the Chlamydiales order), using a PCR-based method. Borrelia DNA was detected in 7/73 (9.6%) biopsies with GA and in 1/36 (2.8 %) biopsies with morphea, while Chlamydiales DNA was found in 53/73 (72.6%) biopsies with GA and 25/34 (73.4%) biopsies with morphea. All Borrelia DNA-positive GA samples were also positive for Chlamydiales DNA. The Chlamydiales sequences detected in GA were heterogeneous and contained Waddliaceae and Rhabdochlamydiaceae bacteria, which are also present in Ixodes ricinus ticks, while the Chlamydiales sequences detected in morphea closely resembled those found in healthy skin. In conclusion, tick-mediated infections can trigger GA in some cases, while correlation of either Borrelia or Chlamydiales with morphea is unlikely.