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Evaluation of the antibacterial potential of Burkholderia cepacia complex
bacteria secretome and of BT-12, an antimicrobial produced by BioMimetx SA
Inês Raquel Carvalho Leonardo
Under supervision of Prof. Doctor Isabel Maria de Sá Correia Leite de Almeida and
Doctor Patrick de Oliveira Freire
This thesis work was developed in collaboration with BioMimetx start-up, dedicated to the production of biocides
to tackle problems caused by biofouling. BT-12, a sub-fraction of the secretome produced by a BioMimetx
proprietary Pseudomonas strain, composed by small peptides and quorum-sensing-related molecules and
exhibiting a strong antibacterial activity, was examined. BT-12 antimicrobial potential was tested against 11
sequential clonal variants of Burkholderia cenocepacia, isolated from a cystic fibrosis patient during a 3.5-years
of chronic infection, as well as against other clinical and environmental isolates of B. cenocepacia and B. dolosa.
BT-12 was proved to inhibit bacterial growth of all the clinical and environmental Burkholderia cepacia complex
(Bcc) isolates tested and to affect the size of the biofilm formed.
Bcc bacteria were also explored as producers of secretomes with antibacterial activity against Escherichia coli
ATCC 25922, Staphylococcus aureus ATCC 33591 and Enterococcus faecalis 20478. Bcc-supernatants, collected
from cultures of the clinical and environmental isolates tested for BT-12 activity and grown at different conditions,
were tested against the selected target bacteria. The majority of these supernatants inhibited bacterial growth of
both Gram-positive and Gram-negative bacteria, decreasing the maximum specific growth rate and the final
biomass obtained in a dose-dependent manner. B. cenocepacia IST01 gave rise to supernatants with the
strongest antibacterial activity but their efficacy was not identical for all the bacteria species tested.
This thesis work provided preliminary results to characterize the antibacterial potential of BT-12 against antibiotic
resistant Bcc bacteria as well as the antibacterial activity of Bcc isolates secretomes, raising relevant questions
and providing opportunities to be addressed and explored in the future.
Key-words: Burkholderia cepacia complex, antibacterial activity, bacterial secretome, antimicrobial peptides, quorum
sensing, biofilm
In the past few years, strains of the Burkholderia genus have
been extensively explored in research and in biotechnology
applications since these versatile bacteria occupy a wide
range of diverse ecological niches and can be used for
biocontrol, bioremediation among other applications. A
specific group of species within this genus, the Burkholderia
cepacia complex (Bcc), includes opportunistic pathogens
able to colonize plants and animals, including individuals
with specific pathogenicities, in particular Cystic Fibrosis
(CF) patients, and immunocompromised humans. Different
antimicrobial compounds are used in the treatment of Bcc
infections but, since these organisms are intrinsically multi-
resistant, a cocktail of antibiotics is often used, and still can’t
always eradicate the infection. [1], [2] This project was
based on a scientific collaboration between the group of
Prof. Isabel Sá Correia at iBB-Institute for Bioengineering
and Biosciences and BioMimetx that enabled the evaluation
of the antibacterial potential of one of the antimicrobial
products developed by this biotechnology start-up against
Bcc bacteria. In addition, the assessment of the
antibacterial capacity of compounds secreted by Bcc was
also performed, due to the established potential of this group
as bioactive compounds producer.[3]
BioMimetx SA is a Biotech start-up dedicated to the
production of innovative ecological biocides, able to tackle
big societal and environmental problems caused by
bioincrustration or biofouling. Besides the evident economic
impact, bioincrustration on hulls is a threat to marine
ecosystems through the introduction of foreign organisms.
The technology of BioMimetx translates in the development
of bio-additives that are incorporated in marine antifouling
paints. These additives are produced by a strain of
Pseudomonas sp. that was isolated from brackish waters,
directly from the environment in Portugal, when grown under
precise conditions optimized by BioMimetx Research &
Development team. Its secretome is purified using different
techniques, including lyophilization, defined by the
company. This mixture was described to have bactericide,
fungicide, algaecide and solvent properties. In this project a
sub-fraction of this mixture was used composed of
molecules of molecular mass < 2 kDa, named BT-12,
already described by BioMimetx to have strong antibacterial
activity. The composition of BT-12 is not entirely known yet,
but it is constituted at least by peptides and quorum sensing
(QS) – related molecules. The eradication of Bcc bacteria
from CF patients and immunocompromised humans is a
relevant goal and it is also possible that Bcc bacteria, due to
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their complex adaptation mechanisms, are potential
producers of compounds with interest for antibiofouling
applications that can be of interest to BioMimetx. This thesis
project has explored the potential antimicrobial activity of
BT-12 produced by BioMimetx against Bcc bacteria
resistant to many common antibiotics. Also, the antibacterial
potential of these bacteria secretome was assessed during
this work.
Burkholderia cepacia complex (Bcc) is a bacterial group of
20 closely related species known to be Gram-negative
chemoorganotrophs that have a respiratory type of
metabolism with oxygen as the terminal electron acceptor,
and some Bcc species have the ability to perform anaerobic
respiration with nitrate.[3]–[5] These bacteria are ubiquitous
and have the ability to occupy diverse niches, since they
have already been isolated from soil, water, fungus, plant
rhizospheres, various animal species, hospital
environments and from infected humans.[6] All of them are
opportunistic pathogens, either to plants or animals,
including immunocompromised humans and other patients
with specific pathogenicities, in particular Cystic fibrosis
(CF) patients. Bcc bacteria represent a major threat for CF
patients since they are able to colonize patients’ lungs and
establish a chronic infection of the respiratory tract that
cause a more rapid deterioration of respiratory function.[7]
Chronic infections can progress to a stage of lethal
necrotizing pneumonia which leads to septicemia known as
Cepacia Syndrome.[8] Although Bcc bacteria pose a
potential threat to particular humans, some species of this
bacterial group have been used for biological control and
plant growth promotion, in particular B. cepacia and B.
vietnamiensis that are able to fix nitrogen.[1] Therefore, Bcc
bacteria are considered relevant within the Burkholderia
genus and the mentioned above justifies the interest of the
study of these specific microorganisms to better understand
both the mechanisms involved in antimicrobial resistance
and their potential use in biotechnological applications.
Bcc bacteria are intrinsically multi-resistant and capable of
adapting to different environments due to their complex
adaptation mechanisms. The cellular envelope of any Gram-
negative bacteria is important to the antimicrobial resistance
phenotype since the outer and the inner membranes
represent a barrier against amphipathic and hydrophilic
compounds, respectively, which are properties of most
antibiotics currently used.[9] The lipopolysaccharide (LPS)
of Bcc bacteria is another determinant of their resistance
ability since it has a unique structure specific of this bacterial
group: the core oligosaccharide contains less phosphate
groups and lipid A backbone has 4-amino-4-
deoxyarabinose moieties attached to its phosphate
residues.[10], [11] These modifications reduce the negative
charge of LPS which increases resistance against cationic
antimicrobial peptides and polymyxins.[12], [13] As with
other Gram-negative bacteria, efflux pumps present in the
outer membrane of Bcc bacteria were described to play a
significant role in their antimicrobial resistance, in particular
efflux pumps from resistance nodulation cell division (RND)
family described to have chloramphenicol, tobramycin and
fluoroquinolones as major substrates. [14] Besides the
properties of cell envelope of Bcc bacteria, the production of
different molecules that are secreted to the extracellular
space also contribute to their resistant phenotype. An
exhaustive inventory of Burkholderia secretome was
reported in which are included diverse enzymes (lipases,
proteases, phospholipase C, between others),
siderophores, exotoxins and quorum sensing (QS)
molecules.[6] Biofilm formation is a very common adaptive
response among Bcc species and, besides being a
protection over host immune responses, it is one of the most
efficient resistance mechanisms against antimicrobials.
However, some antimicrobials like meropenem and
ceftazidime, between others, are able to affect biofilms,
even though it is always necessary to use higher
concentrations in comparison with the ones applied to
planktonic cells.[15], [16]
Various antimicrobials, from different classes, have been
tested against Bcc bacteria but only a few were found to be
able to have an antibacterial effect. Bcc bacteria were
described to be more susceptible in the presence of
semisynthetic penicillins, carbapenems and ceftazidime that
affect cell wall synthesis, fluoroquinolones and co-
trimoxazole that restrict DNA replication.[17], [18]
Nevertheless, the treatment strategies for Cystic fibrosis
patients whose lungs were colonized by Bcc bacteria are
often based on a cocktail of antibiotics that can’t always
inhibit infection evolution to the fatal Cepacia Syndrome. As
the development of new antimicrobials is urgent,
antimicrobial peptides (AMPs) and quorum sensing
inhibitors (QSI) have been explored in this context and
already showed to be potentially low cost and effective
antimicrobial agents that can be used as a source of new
pharmaceuticals for the treatment of different types of
infections such as antibiotic-resistant bacterial infections or
septic shock.[19]–[22]
AMPs are oligopeptides with 100 amino acids maximum that
have the ability to affect a wide variety of target organisms
from viruses to parasites. Antibacterial AMPs are the most
studied until now and most of them are amphipathic cationic
peptides that can interact with bacterial membranes by
binding to lipid components or phospholipid groups and
forming membrane pores causing their disruption.[23]
However, some of them, when present in low
concentrations, can diffuse trough the cell membrane and
affect essential pathways inside the cell like DNA replication
or protein biosynthesis. In similarity, anionic peptides, that
are active against both Gram-positive and Gram-negative
bacteria, are known to diffuse trough cell membrane and
cause the flocculation of intracellular contents.[19], [24] The
synthetic structurally nano-engineered antimicrobial peptide
polymers (SNAPPs) have a distinct multimodal mechanism
in which they are able to disrupt both outer and cytoplasmic
membranes, deregulate ion influx and efflux and to induce
apoptotic-like death pathway.[21] Most of the studies
published regarding the evaluation of AMPs activity against
Bcc bacteria were performed with cationic peptides and the
majority showed no significant effect. [25] However, a
derivative of LL-37 was able to inhibit biofilm formation of B.
cenocepacia with a reduction of biomass of over 50%, but
had no effect in planktonic cells.[26]
Quorum sensing (QS) is a cell density dependent
phenomenon and it is maybe one of the most relevant
mechanisms of bacterial adaptation, since it can modulate
the expression of genes involved in virulence, being an
important regulator of pathogenicity.[27] Bcc bacteria
commonly have more than one QS system. CepIR is a QS
system present in all of them but other systems have been
described within this complex.[28], [29] CepIR regulates the
expression of several virulence factors like the production of
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extracellular proteases, swarming motility, ornibactin
synthesis, a protein involved on iron uptake, and biofilm
maturation.[31] Taking into account all cell functions that
are regulated by QS system, it is to expect that when it is
defective, susceptibility of Bcc bacterial cells to
antimicrobials can increase. QS systems’ inhibition restricts
the biosynthesis of virulence factors without directly kill
bacteria, which makes the development of drug resistance
less likely.[22] Therefore, the inhibition of Bcc bacteria’s QS
system has been explored as a possible approach to treat
these type of bacterial infections. QS system can be
inhibited by the interruption of distinct steps of the
mechanism, so it is possible to reduce the activity of N-acyl
homoserine lactones (AHLs) receptor or synthase, to inhibit
AHLs production, to degrade the signal molecules or to
mimic AHLs using synthetic analogues of these
molecules.[32] Known QS inhibitors (QSI) can be synthetic
or natural and showed to be effective against a diverse
panel of organisms, including Bcc species. Some strains of
B. cenocepacia and B. multivorans appeared to be affected
by a QSI developed by Riedel et al. since it was observed a
decrease of biofilm biomass (~60% reduction) and virulence
factors’ production.[33], [34] In the past few years, several
in vitro and in vivo studies were performed where QS anti-
virulence drugs were tested in combination with established
antimicrobials and it was observed that QSI can improve the
currently available therapies increasing host survival.[35]
Additionally, some of these molecules were already tested
in human cells and showed low toxicity.[22] Therefore, the
use of QSI against Bcc bacteria represent an effective
approach to use as clinical therapy and efforts are being
made with that ultimate goal, but further studies are
necessary to be possible to use it in human patients.
Over the past 30 years, Bcc bacteria have been also
explored as antimicrobial producers since some of the
molecules present in their secretomes were described to
have antimicrobial potential.[3] Studies regarding this
subject were primarily focused on antifungals compounds
produced by Bcc, like pyrrolnitrin, xylocandins,
cepafungins/glidobactins, among others.[36]–[38]
Nowadays, further studies on the secretome of Bcc bacteria
have demonstrated that they also produce a range of other
potent antibacterial compounds.[3] Barthi et al. evaluated
the antimicrobial activity of B. gladioli OR1 supernatants
collected after 24h of bacterial growth against clinical
isolates of different species, including Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli, among
others, using a disc diffusion approach and the results
appear to indicate that the secretome of this Bcc bacteria
has, in fact, the ability to affect the growth of a broad
spectrum of organisms.[39] Unfortunately, the use of
antimicrobials produced by Bcc bacteria is currently not
permitted in most countries because they are considered a
risk to human health. However, it has been discussed if the
current control measures should be as rigid as they are or
more specific, taking into account all differences of
epidemiology and pathogenicity among Bcc species and
strains.[1], [40], [41] Nowadays, there are just a few
exceptions, as the case of B. cenocepacia M36 and B.
ambifaria M54, which are registered as biopesticides for use
in fungi biocontrol in the United States of America. The
function of antimicrobials produced by Bcc in the natural
environment is still unknown and more studies about their
ecological relevance are needed to fully explore their
apparent multifunctionality. For instance, these natural
compounds could be used as antibiofouling agents which
consists in the purge of the accumulation of unwanted
biomass on surfaces, with biofilms created by
microorganisms and macrofouling created by their
association with higher organisms.[42] This phenomenon is
responsible for significant financial losses in marine and
industrial fields and for health risks related to medical
biofouling that occurs in prosthetic implants, biosensors,
catheters and medical equipment that can cause implants
rejection, malfunction of biosensors and spread of infectious
diseases.[43] Bcc has the potential to produce novel active
compounds able to attenuate this threat.[42]
This thesis project has two main goals: the evaluation of
antimicrobial activity of the sub-fraction BT-12, provided by
BioMimetx, against Bcc isolates and the assessment of
antibacterial activity of Burkholderia supernatants from
isolates of the Bcc against Gram-positive and Gram-
negative bacteria. The first Bcc isolates selected to work
with were clinical isolates retrieved from a Cystic Fibrosis
(CF) patient - patient J – already thoroughly examined by
the Biological Sciences Research Group of iBB-Institute for
Bioengineering and Biosciences.[44]–[48] This patient was
chronically infected with the same B. cenocepacia strain
(recA lineage III-A) for 3.5 years, until the patient’s death
with Cepacia syndrome. Patient J was followed at Hospital
de Santa Maria (HSM) in Lisbon where 11 sequential B.
cenocepacia clonal isolates were obtained.[44] Proteomic
studies in which genome-wide expression patterns of three
of these isolates (IST439, IST4113 and IST4134) were
compared revealed that relevant phenotypes in the context
of bacterial pathogenesis vary according to the isolate
studied. [47]–[49] It was already described that B.
cenocepacia IST439 is the only clonal variant that has the
O-antigen subunit in the lipopolysaccharide and it was found
to have less virulence potential in comparison with late
isolates (IST4113 and IST4134) which suggests that the
virulence potential of these B. cenocepacia clonal variants
increase through time of chronic infection.[49] B.
cenocepacia IST439 was found to be the most susceptible
isolate to clinically used antibiotics between all the
remaining clonal variants, in the opposite of B. cenocepacia
IST4113 which was described as the most resistant.[45] B.
cenocepacia IST4129 was described to be the only isolate
that do not have the third replicon which contains the
majority of the virulence genes. The loss of this replicon was
shown to be an extremely rare event that is responsible for
a highly attenuated virulence of this B. cenocepacia isolate
in different infection models (Galleria mellonella larvae and
Caenorhabditis elegans). The fact that these clonal variants
are isolates with practically the same genome sequence but
with already described distinct phenotypes, justify the
interest on study them. Therefore, they were the starting
point for the studies performed, exploring them either as
targets for the BioMimetx antimicrobial or as producers of
antimicrobials, and based on the first screening results the
project strategies were defined.
MATERIALS AND METHODS
Strains and growth media. In this project, 11 clinical isolates
of Burkholderia cenocepacia isolated at Hospital de Santa Maria
(HSM) from respiratory secretions of the same chronically
infected Cystic fibrosis patient during 3.5 years of chronic
infection. Additional Burkholderia cepacia complex (Bcc)
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isolates of B. cenocepacia and B. dolosa, retrieved from other
patients in HSM were also tested (B. cenocepacia IST432,
IST4484, IST4136, IST4197 and B. dolosa IST4208 and
IST4616) as well as two environmental isolates, obtained from
a culture collection (BCCM/LMG, Belgium): B. cenocepacia
LMG 19238 and B. dolosa 21443. Additionally, Enterococcus
faecalis 20478, Escherichia coli ATCC 25922, Pseudomonas
aeruginosa 12 and F117 and Staphylococcus aureus ATCC
33591 were used. Bacterial cells were usually cultured at 37ºC
but 26 and 30ºC were also used. Bacterial cells were cultured in
orbital incubators at 250 rpm, using two different media. One of
the media was a rich medium Luria-Bertani (LB) (Nzytech)
composed, per litre, by 10 g of Peptone, 5 g of Yeast extract and
5 g of NaCl. The other was a chemically defined mineral medium
(CDM) that contains, per liter, 0.54 g of KCl (Merck), 0.36 g of
NaCl (VWR), 6.34 g of (NH4)2SO4 (Scharlau), 0.124 g of
MgSO4.7H2O (Merck), 1.40 g of K2HPO4 (Merck), 1 g of
Casaminoacids (Difco) and 12.56 g of 3-Morpholinopropane-1-
sulfonic acid (MOPS) (Serva) with pH adjusted to 7.2 and
supplemented with 0.8 or 1.6% of Glucose (Merck) and with 1
ml of FeCl3 100 mM solution. Mueller-Hinton (MH) (Sigma-
Aldrich) medium is composed by, per litre, 17.5 g of Casein
hydrosylate, 2 g of beef infusion solids and 1.5 g of starch and
it was also used. All media were autoclaved for 15 min at 121ºC.
Effect of BT-12 in Bcc bacteria growth. Liquid cultures of
patient J isolates were cultivated until medium exponential
phase, at 37ºC, 250 rpm, in a final volume of 50 ml of LB
inoculated with an initial OD640nm of 0.05. Cells were washed
twice with NaCl 0.9% (5 min centrifugation, 8220g, 23ºC) and
re-suspended in Mueller-Hinton (MH) medium, with OD640nm
adjusted to 0.21. In a 96-well microplate, BT-12 was tested by
adding 10 µl of this mixture, in a range of final concentrations of
8 to 1024 µg ml-1, in 190 µl of culture. Two additional higher
concentrations of BT-12 were also tested: 1500 and 2000 µg ml-
1. The positive control was prepared using 200 µl of culture. The
negative controls were prepared with 200 µl of MH medium and
10 µl of diluted fraction added to 190 µl of MH medium. The
microplate was incubated without agitation at 37ºC during 24 h,
the liquid present in each well was resuspended manually
before OD was measured in SPECTROstarNano (BMG
LABTECH) microplate reader after that time (OD640nm). In the
antibacterial assays performed with the remaining Bcc isolates
were used final concentrations in a range of 300 – 1000 µg ml-1
and the 96-well microplate was incubated at 37ºC in the
FilterMaxF5 Multi-Mode Microplate Reader, Molecular
Devices® which provides a mechanical agitation before each
measurement (one measurement per hour (OD620nm). The
microplates prepared from liquid cultures of Bcc isolates were
incubated during 24 h and the ones prepared with liquid cultures
of P. aeruginosa 12 and E. coli ATCC 25922 were incubated
during 48 h.
Effect of BT-12 in biofilm size. For each isolate to be tested
with BT-12, a liquid culture was cultivated until medium
exponential phase, at 37ºC, 250 rpm, in a final volume of 50 ml
of LB inoculated with an initial OD640nm of 0.05. The culture was
diluted in LB to an OD640nm of 0.05 and 190 µl of this culture were
added to 10 µl of a BT-12 solution, in each well of a 96-well
microplate. BT-12 was tested using final concentrations in a
range of 300 – 1000 µg ml-1. Two additional higher
concentrations of BT-12 were also tested: 1500 and 2000 µg ml-
1. The positive controls were prepared using 200 µl of bacterial
culture in LB. The negative controls were prepared using 200 µl
of sterilized LB medium. The microplates were incubated at
37ºC for 24 h without shaking. Each well was washed two times
with H2O, 25 µl of 1% Crystal violet (Merck) solution were added
to the empty wells and after 15 min at room temperature, each
well was washed twice with H2O. 200 µl of 96% ethanol were
added to the empty wells and the OD590nm of this solution was
measured using SPECTROstarNano (BMG LABTECH)
microplate reader.
Preparation of Bcc bacteria supernatants. Liquid cultures of
Bcc bacteria isolates in a final volume of 50 ml of medium
inoculated with an initial OD640nm of 0.05 were cultivated with
orbital agitation until early-stationary phase at 250 rpm, at the
different conditions indicated below (Table 3). Cultures were
centrifuged at 4ºC and 8200g during 5 min and supernatants
were filtered with 0.2 µm sterile filters and conserved until used
at -80ºC
Table 1 – Supernatants collected from Burkholderia cultures. The different growth parameters are: medium (LB or CDM with 0.8 or 1.6% of Glucose), and temperature (37, 30 and 26ºC). The 13 selected isolates were: 11 sequential B. cenocepacia clonal isolates from patient J and two environmental isolates obtained from LMG collection. Only
marked [ ] conditions correspond to supernatants extractions. [ _ ] correspond to tests that were not performed.
Burkholderia supernatants susceptibility assay. The
different supernatants to be tested for susceptibility assays were
lyophilized using Thermo ScientificTM Heto PowerDry PL9000
Freeze Dryer and re-suspended in H2O to a given concentration.
Considering Table 4, in the assays I and II the supernatants
were used at final concentrations of 5X and 1X concentrated in
comparison with the original supernatant and in the assay III the
supernatants were used at a maximum final concentration of
300 mg ml-1. Strains to be tested as targets of Bcc bacteria
supernatants (Escherichia coli ATCC 25922, Staphylococcus
aureus ATCC 33951 and Enterococcus faecalis 20478) were
grown at 37ºC for 5 hours and OD600nm was adjusted to 0.08 in
LB medium. In a 96-well microplate, supernatants were tested
by adding 50 µl of this fraction to 50 µl of culture. The positive
controls were prepared with 50 µl of bacterial culture in 50 µl of
LB or CDM medium, according to the supernatants tested. The
negative controls were prepared with 100 µl of sterilized LB
medium and 50 µl of supernatants in 50 µl of sterilized LB
medium. The 96-well microplates were incubated following the
same method previously described for BT-12 antimicrobial
assays against Bcc isolates during 16 h or 48 h and the OD
values were measured at 595 nm in the Biochrom® Zenyth 200
microplate reader or in the BioScreenTM C MBR system.
Table 2 – Target bacteria used in susceptibility assays related with Burkholderia supernatants performed during 16 h. Three antimicrobial assays performed with different sets of Burkholderia supernatants indicated in the Table 3. Three bacterial isolates were used as target organisms E. coli ATCC 25922, S. aureus ATCC 33591 and E.
faecalis 20478 on the correspondent marked [ ] assays. [ _ ] correspond to tests that were not performed.
5
N-Acyl-Homoserine lactone quantification. To evaluate if
quorum sensing signaling molecules could be indicators of
antibacterial activity of the collected supernatants for being
related to high cell densities, B. cenocepacia IST01 isolate was
grown in liquid cultures, at different conditions used to collect
supernatants (Table 3). Quantification of the N-acyl-homoserine
lactones (AHLs) produced was performed following the protocol
described by Riedel et al., 2001 with few alterations.[50]
Pseudomonas putida F117, used as biosensor, was grown in
liquid culture, in an orbital incubator at 250 rpm, 30ºC, in a final
volume of 50 ml of LB supplemented with 20 µg ml-1 of
Gentamicin inoculated with an initial OD640nm of 0.05 until mid-
exponential phase. A 96-well solid black polystyrene microplate
was prepared with 100µl of liquid culture of P. putida F117 and
the same volume of the selected supernatant to test in each
well. The negative controls were prepared with 200µl of
sterilized LB medium and 100 µl of P. putida F117 liquid culture
added to the same volume of sterilized LB medium. After 6h of
incubation at 30ºC without agitation in FilterMax F5 Multi-Mode
Microplate Reader, Molecular Devices®, fluorescence was
measured. Induction of fluorescence was measured with an
excitation and emission wavelengths of 485 nm and 535 nm,
respectively.
Table 3 – Conditions of growth to obtain the supernatants used for N-acyl-homoserine lactones quantification. Conditions used for B. cenocepacia IST01 isolate growth with respective time points used
for supernatants extraction. Only marked [ ] conditions correspond to supernatants extractions. [ _] correspond to tests that were not performed.
RESULTS
Inhibitory effect of BT-12 in Burkholderia cepacia
complex isolates growth. The antimicrobial activity of BT-
12 was tested using the broth microdilution method and ten
different BT-12 concentrations. BT-12 was used at a stock
solution of 40 mg ml-1 that were tested directly as the
maximum BT-12 concentration used in the assay,
corresponding to a final concentration of 2000 µg ml-1. BT-
12 was tested using three internal replicates against each
Bcc isolate, using cells from three independent bacterial
cultivations and the results presented are the average of
these independent assays. The eleven sequential clonal
variants collected from patient J during 3.5 years of chronic
infection were the first to be tested. After 24 h of microplate
incubation at 37ºC without agitation, liquid culture of each
well was resuspended manually under aseptic conditions.
The OD values were measured in SPECTROstarNano (BMG
LABTECH) microplate reader. It was observed that, even
using the maximum BT-12 concentration tested, the OD
values obtained were always higher than zero and it was
first thought that the initial OD of the pre-inoculum could be
interfering with the measurements. Therefore, the initial OD
value (average value of 0.42) was subtracted to the average
for each experimental point and the results obtained are
represented in Figure 1.
Figure 1 – Susceptibility of B. cenocepacia sequential clonal variants isolated from patient J to the BioMimetx mixture BT-12. This growth susceptibility test was based on the broth microdilution
method, using Mueller-Hinton medium. The ODs were measured 24h after microplate inoculation at 37ºC without agitation. The OD values represented are the average of three independent assays performed from different bacteria growth experiments for each Bcc isolate tested. To each average value the initial OD value corresponding to the pre-inoculum (0.42) was subtracted. All target bacteria were grown at the same selected standard conditions.
Although the pre-inocula were adjusted to have similar initial
OD values in all the growth assays performed, the initial OD
achieved after growth in the absence of BT-12 were not the
same for all the B. cenocepacia isolates tested. This result
is possibly due to the intrinsic growth characteristics of each
isolate, under the specific growth conditions used, 37ºC and
Mueller-Hinton medium. The Mueller-Hinton medium used
has low thymidine content and different strains of B.
cenocepacia were already described to be slow growers in this
medium in comparison with other media.[51] In all cases
tested, a decrease of the final biomass reached at 24 h in
the presence of increasing concentrations of BT-12 revealed
the impact of BT-12 on Burkholderia cenopacia growth.
However, lower concentrations of this mixture apparently
potentiate the clonal isolates growth, suggesting that in the
presence of lower concentrations of BT-12, the isolates of
B. cenocepacia may use it as a nutrient source, thus
reaching a higher biomass concentration at the stationary
phase of growth. With the exception of B. cenocepacia
IST4112 and IST4113, the most significant OD reduction
was detected between BT-12 concentrations in the range of
512 to 1024 µg ml-1. Additionally, it was observed that for
higher BT-12 concentrations the OD obtained maintains its
value in a baseline, reaching minimal stable values in all
cases above zero, rendering difficult the definition of a MIC
value. Nonetheless, BT-12 definitely does have an inhibitory
effect against the growth of all these B. cenocepacia
isolates, decreasing the final biomass obtained at stationary
phase. To confirm these results it would be necessary to
6
perform a cell viability test because, in fact, the minimal OD
values obtained do not mean growth or even the presence
of viable cells, it can be, for instance, the presence of some
colored compound whose production is potentiated by BT-
12 presence or aggregates of bacterial cells that are already
unviable but that did not lysed. Similar results were obtained
for Bcc isolates from other CF patients and the
environmental isolates from LMG collection (data not
shown).
Inhibitory effect of BT-12 in the size of biofilm produced
by Bcc isolates. BT-12 was also tested to determine its
effect in B. cenocepacia biofilm formation. In this assay, only
four bacterial isolates were tested: B. cenocepacia IST439,
IST4103, IST4113 and IST4131 from patient J, in the
presence of higher BT-12 concentrations (from 300 to 2000
µg ml-1). These isolates were selected since each
corresponds to different phases of a chronic infection of a
CF lung whose biofilm formation ability was already
examined by the lab group. Biofilms were quantified
following the previously described O’Toole and Kolter
method.[71] The following analysis (Figure 2) takes in
consideration only biofilm formation in absence of BT-12
and in presence of its maximum concentration tested (2000
µg ml-1).
Is o la te
OD
59
0n
m
IST
439
IST
4103
IST
4113
IST
4131
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
Figure 2 – Effect of BT-12 in the biofilm size produced by patient J isolates of B. cenocepacia IST439, IST4103, IST4113 and IST4131. Quantification of biofilms produced by B. cenocepacia IST439,
IST4103, IST4113 and IST4131 isolates in presence and absence of BT-12. This analysis was based on O’Toole and Kolter method. The OD values represented are the average of three independent assays performed from different bacterial cultivations of each isolate tested. All target bacteria were grown at the same selected standard conditions.
The acquired results indicate that BT-12 affects the size of
the biofilm produced by the B. cenocepacia isolates
IST4103, IST4113 and IST4131, but no significant
differences were detected in the size of the biofilm produced
by IST439 in presence of BT-12.The isolates IST4113 and
IST4131 revealed to be more affected by the compound
than the first two isolates since they have shown a reduction
of 87.9 and 94.7% of biofilm size produced under presence
of high amounts of BT-12, respectively, in comparison with
B. cenocepacia IST439 (2.6%) and IST4103 (24.5%). The
effect of BT-12 in biofilm size produced by the isolates tested
can be due to its inhibitory effect in bacterial growth but the
ability of this mixture to affect biofilm formation cannot be
discarded. Nonetheless, BT-12 seems to be more effective
against IST4113 in comparison with IST439 which is an
unexpected result, since the first was described to be one of
the most resistant to known antibiotics and, in the opposite,
IST439 was the more susceptible.[52] Taking this into
account, it is to expect that BT-12 interacts with isolates in a
different way of all the antibiotics previously tested.
Antibacterial activity of Bcc bacteria supernatants. In
order to explore the potential of Burkholderia cepacia
complex bacteria to produce extracellular compounds that
can be of interest to BioMimetx, in particular compounds
with antimicrobial ability, supernatants from liquid cultures of
different Bcc isolates were prepared and their antimicrobial
activity was assessed against different pathogenic bacterial
strains. In the first screening performed (assay I from Table
2), supernatants were collected from cultures of all the B.
cenocepacia clonal isolates from patient J and from the two
environmental bacteria used before to test the antibacterial
effect of BT-12. The antimicrobial activity of the
supernatants was tested against two bacterial strains, the
Gram-negative E. coli ATCC 25922 and the Gram-positive
S. aureus ATCC 33591. The lyophilized selected
supernatants were resuspended in water to be 10X
concentrated in comparison with the volume firstly collected
and were tested in a final concentration of 1X and 5X
concentrated. As the selected type of growth medium and
temperature can influence bacterial growth and metabolism,
it is to expect that these parameters also have an impact on
their secretome. Taking this into account, Burkholderia
supernatants were collected at the end of exponential phase
from cultures grown in two different media (LB or CDM), at
two different temperatures (30 and 37ºC), to know in which
way growth conditions affect supernatants properties.
Figure 3 - Susceptibility of E. coli ATCC 25922 and S. aureus ATCC 33591 to supernatants of Burkholderia isolates grown in 0.8% Glucose CDM at 30 and 37ºC. Antimicrobial activity was analyzed following broth microdilution method using Burkholderia cepacia complex supernatants. The OD values represented were measured 16h after microplate incubation at 37ºC without agitation. The
OD values represented are the average of two independent assays performed from different bacterial cultivations of each bacterial isolate tested.
Figure 4 - Susceptibility of E. coli ATCC 25922 and S. aureus ATCC 33591 to supernatants of Burkholderia isolates grown in LB at 30 and 37ºC. Antimicrobial activity was analyzed following broth microdilution method using Burkholderia cepacia complex supernatants. The OD values represented were measured 16h after microplate incubation at 37ºC without agitation. The OD values represented are the average of two independent assays performed from different bacterial cultivations of each bacterial isolate tested.
7
The results obtained (Figure 3 and 4) indicate that, in
general, E. coli ATCC 25922 is more susceptible to Bcc
bacteria supernatants than S. aureus ATCC 33591 whose
growth appears to be, in some cases, potentiated by their
presence, even when they were present in relatively high
concentrations. S. aureus ATCC 33591 final biomass
obtained was found to be not significantly different in
presence of all the tested supernatants, with exception for
the one collected from B. cenocepacia IST01 culture in LB
medium at 37ºC and from the environmental isolates B.
dolosa LMG 21443 and B. cenocepacia 19238 in LB
medium at 30 or 37ºC. These supernatants appeared to be
the only ones able to significantly affect bacterial growth if
present in a concentration of 5X, in the case of B.
cenocepacia IST01, and in a concentration of 1X in the case
of the environmental isolates. Differently, E. coli ATCC
25922 was affected by all the supernatants tested at
relatively high concentrations and even at lower
concentrations in the case of supernatants collected in CDM
medium. However, in these cases there was no significant
differences between the antibacterial activity of
supernatants collected in CDM 1X or 5X concentrated. The
more marked growth decrease of E. coli ATCC 25922 was
observed in the presence of supernatants collected from
liquid cultures of B. cenocepacia IST01 (in LB at 30ºC) and
of B. cenocepacia LMG 19238 (in LB at 37ºC). Based on the
first evaluation of supernatants that showed to have a
significant effect on bacterial growth inhibition, five isolates
out of the initial thirteen were selected to be further explored
as antimicrobial producers – B. cenocepacia IST01, IST02,
IST05, IST10 and B. cenocepacia LMG 19238. In general,
these isolates appeared to produce supernatants with
higher antibacterial activity when collected from liquid
cultures grown at 30ºC compared with the ones collected at
37ºC. The results obtained for the selected supernatants
collected at 30ºC in LB were confirmed in continuum assays
in which OD values were measured at numerous time points
during 48 h (data not shown). Specific growth rates (µ),
defined as the increase of biomass per time, were
determined from the exponential growth phase of E. coli
ATCC 25922 and S. aureus ATCC 33591 growth curve in
absence of Bcc supernatants and in presence of the
maximum concentration tested (5X). The acquired results
confirmed that E. coli ATCC 25922 is more susceptible to
the selected Bcc bacteria supernatants in comparison with
S. aureus ATCC 33591. E. coli ATCC 25922 was more
affected in the presence of high concentrations of the
supernatants collected from liquid cultures of B.
cenocepacia IST01 and IST02 since it was observed the
total absence of bacterial growth. The remaining tested
supernatants were able to affect bacterial growth when they
were present at high concentrations since they were able to
induce a significant decrease of the specific growth rate (µ)
of E. coli ATCC 25922. The same phenomenon was
observed in S. aureus ATCC 33591 growth curve in
presence of high concentrations of supernatants collected
from cultures of B. cenocepacia IST01, IST02 and IST05,
which are results inconsistent with the previously obtained
in antimicrobial activity assays performed during 16 h.
Collectively, these results are consistent with the previously
obtained in assays performed during 16 h for all E. coli
ATCC 25922 assays, but not for all the selected
supernatants tested against S. aureus ATCC 33591.
Since the selected Bcc isolates appeared to produce
supernatants with higher antibacterial activity when
collected from liquid cultures grown at 30ºC compared with
the ones collected at 37ºC, another assay was performed
(assay II from Table 2) with the supernatants extracted in
new conditions using a lower growth temperature (growth in
LB medium at 26ºC). The selected supernatants were tested
against E. coli ATCC 25922 and a new bacterial target:
Enterococcus faecalis 20478. This Gram-positive bacteria
was selected as new bacterial target because former results
obtained for S. aureus ATCC 33591 were not always
consistent between the two methodologies tested.
Figure 5 – Comparison of the final biomass concentration determined on the culture OD at 595 nm of E. faecalis 20478 and E. coli ATCC 25922 when different concentrations of the supernatants of B. cenocepacia isolates IST01, IST02, IST05 and IST10 and B. cenocepacia LMG 19238, collected from growth in LB at 26ºC, were added. The OD values were measured
after 16 h of microplate incubation at 37ºC without agitation. The OD values represented are the average of two independent assays performed from distinct bacterial growths of each bacterial isolate tested. To each average point value was subtracted the initial OD value of the pre-inoculum (0.08). All target bacteria were grown at the same selected standard conditions.
E. faecalis 20478 showed to be more susceptible to Bcc
supernatants in comparison with E. coli ATCC 25922 with
exception for the supernatant collected from B. cenocepacia
IST02 culture. The results obtained for E. coli ATCC 25922
obtained in these antibacterial assays performed with
supernatants collected at 26ºC (Figure 5) showed no
significant differences in comparison to the antimicrobial
activity of supernatants collected at 30ºC. The supernatant
that showed a more efficient ability to inhibit bacterial growth
was the collected from the liquid culture of B. cenocepacia
IST01 isolate. Once again, results were validated in assays
performed during 48 h and specific growth rates were
determined for bacterial growth curves in absence of Bcc
supernatants and in presence of the maximum
concentration tested (5X). (data not shown)
All the supernatants tested against E. coli ATCC 25922 were
able to affect bacterial growth being responsible for a
significant decrease of the specific growth rate when
supernatants were present at high concentrations of 5X
concentrated. The maximum concentration tested of the
supernatant collected from B. cenocepacia IST01 culture
had a more marked inhibitory effect on E. coli ATCC 25922
growth because its presence lead to a significantly
prolonged lag phase. Once again, the supernatant produced
by this isolate stood out as the most effective one against E.
faecalis 20478, since it was observed the total absence of
bacterial growth in presence of high concentrations of B.
cenocepacia IST01 supernatant. With the exception of the
supernatant collected from B. cenocepacia IST02 culture, all
the remaining supernatants tested against E. faecalis 20478
were able to significantly decrease the specific growth rate
when supernatants were present at high concentrations of
5X. Additionally, relatively high concentrations (5X and 2.5X
concentrated) of the supernatant collected from B.
8
cenocepacia IST05 were able to decrease the final biomass
concentration obtained of E. faecalis 20478. This inhibitory
effect was also observed for the supernatant collected from
B. cenocepacia IST02 culture only when it was present 5X
concentrated.
Collectively, this information indicates that the antimicrobial
properties of the supernatants do fluctuate according to the
media and temperature used for the selected Bcc isolates
growth. It was postulated that supernatants’ antimicrobial
activity could be related to cell density of the culture. In an
attempt to reach higher antimicrobial activity, growth of the
isolate that showed to be more consistently active through
all the tests – B. cenocepacia IST01 – was optimized to
obtain higher cell densities.The higher values of cell density
were achieved when B. cenocepacia IST01 was grown in
CDM supplemented with 1.6% of Glucose at 37ºC.
Therefore, new supernatants were collected at these
conditions and tested for antimicrobial activity against the
three organisms studied before: E. coli ATCC 25922, S.
aureus ATCC 33591 and E. faecalis 20478 (assay III from
Table 2). All assays were performed twice with bacterial
cells from two distinct growths and the average points are
presented in the following figure (Figure 6). Since basal OD
value of medium used (LB) and of the supernatants tested,
that are yellowish, could influence the final results, the OD
value corresponding to that mixture (0.08) was subtracted
from all average points obtained.
Figure 6 – Susceptibility of E. coli ATCC 25922, E. faecalis 20478 and S. aureus ATCC 33591 to different supernatants of B. cenocepacia IST01 isolate, grown at 37ºC in CDM with 1.6% Glucose. Antimicrobial activity was analyzed following the broth
microdilution method. The inhibitory effect of four different supernatants, which just differ in extraction time-point (16, 24, 36 and 50h of growth), was analyzed against the isolates (A) E. coli ATCC 25922, (B) E. faecalis
20478 and (C) S. aureus ATCC 33591. The OD values represented were measured 16h after microplate incubation at 37ºC without agitation. The OD values represented are the average of two independent assays performed from distinct bacterial growths of each bacterial isolate tested. To each average point was subtracted the OD value referent to the medium and supernatant used (0.08). All target bacteria were grown at the same selected standard conditions.
In all target organisms analyzed were observed cases in
which bacterial growth was potentiated by the presence of
B. cenocepacia IST01 supernatants. This behavior was
more frequent in the tested Gram-positive isolates than in E.
coli ATCC 25922 that showed to have this kind of response
in one case only (supernatants collected at 50h, present at
18.75 mg ml-1). These results are similar to the ones
obtained for BT-12 antimicrobial activity assays against Bcc
isolates, suggesting that, in some cases, E. coli ATCC
25922, S. aureus ATCC 33591 and E. faecalis 20478 are
able to use B. cenocepacia IST01 supernatants as a nutrient
source. With exception for the case already mentioned, E.
coli ATCC 25922 growth was affected by all supernatants
tested, in all concentrations used. The supernatants that
appeared to inhibit its bacterial growth more efficiently were
the ones collected at 36 and 50 h if present at the maximum
concentration used in this assay (300 mg ml-1). E. faecalis
20478 was confirmed to be more susceptible than E. coli
ATCC 25922 in the presence of the maximum concentration
of the supernatant collected at 24 h of cultivation of B.
cenocepacia IST01 and most concentrations of
supernatants collected at 36 h (37.5 to 300 mg ml-1) and 50
h (75 to 300 mg ml-1). E. faecalis 20478 growth was
potentiated in the presence of the remaining B. cenocepacia
IST01 supernatants tested. For the first time, it was possible
to completely inhibit S. aureus ATCC 33591 growth in
presence of high concentrations (75 to 300 mg ml-1) of B.
cenocepacia IST01 supernatants collected at 36 and 50 h.
In the other cases, growth appeared to not be significantly
affected (150 and 300 mg ml-1 of 16 and 24h supernatants)
or it was even potentiated by the presence of supernatants
tested.
All the acquired results indicate that depending on the
selected growth parameters for the production of
supernatants, each supernatant is able to inhibit bacterial
growth of each microorganism tested with different
efficiency. E. coli ATCC 25922 appeared to be more
susceptible to supernatants collected from liquid cultures of
B. cenocepacia IST01 and IST02 in LB at 30ºC, the growth
curve of E. faecalis 20478 was more affected by
supernatants collected from B. cenocepacia IST01 grown in
LB at 26ºC and S. aureus ATCC 33591 were more
susceptible to supernatants of this Bcc isolate grown in CDM
supplemented with 1.6% of Glucose at 37ºC. This
information seem to indicate that the active compound that
defines the antibacterial activity of the Bcc bacteria
supernatants is different for each microorganism tested.
Content of N-Acyl-homoserine lactones in Bcc bacteria
supernatants. In an attempt to correlate N-acyl-homoserine
lactones (AHLs) present in supernatants with their
antibacterial potential, AHLs were quantified from
supernatants collected in different growth conditions. In this
experiment, only B. cenocepacia IST01 isolate was used
and, based on the behavior of its growth in different
conditions, the parameters used for growth and time points
for the supernatants extraction were defined (Table 3). This
assay was performed three times with supernatants
collected from three distinct B. cenocepacia IST01
cultivations at each growth condition selected. The results
obtained (Figure 7) showed that B. cenocepacia IST01
isolate grown in LB reached comparable ODs (~5)
independently of the temperature tested. Consequently, all
supernatants collected under these two different conditions
(30 and 37ºC) appear to have similar amounts of AHLs. In
contrast, the isolate tested reached higher ODs in CDM
supplemented with 0.8% of Glucose (~10) in comparison
with growth in LB and the extracted supernatants had, in
similarity, higher amounts of homoserine lactones.
Supernatants collected in the three already mentioned
conditions showed a comparable pattern of AHLs amount
along growth time, since the maximum quantity was
obtained around 12h followed by a decrease until stationary
phase. B. cenocepacia IST01 isolate appeared to have a
completely different behavior when grown in CDM
supplemented with 1.6% of Glucose at 37ºC. In this case, it
9
was detected a decrease of AHLs amount in the supernatant
extracted at 24 h followed by an unexpected increase until
50 h. The media used in this situation had the double
concentration of carbon source than the usual CDM
(supplemented with 0.8% Glucose) but all other nutrients
were unaltered. Thus, it is possible that, around 20 h,
nitrogen depletion obligate cells to use AHLs as a new
source of this element and afterwards, when bacteria are
again able to grow, AHLs start to accumulate on
supernatants as in the beginning. The ability to use AHLs as
nitrogen source was already described in different
microorganisms and would explain the fluctuations
observed of AHLs amount in the extracted
supernatants.[72], [73] These results indicate that there is
no direct relation between supernatants’ amount of AHLs
and their antimicrobial activity because, in some cases, two
different supernatants with similar antimicrobial activity can
have different amounts of AHLs. For instance, supernatants
collected from liquid cultures of B. cenocepacia IST01 in
CDM supplemented with 1.6% of Glucose at 16 and 24 h
demonstrated to have similar antimicrobial activities against
S. aureus ATCC 33591. However, the one collected at 16 h
had a significant higher amount of AHLs in comparison with
the collected at 24 h.
Figure 7- N-acyl-homoserine lactones quantification of the supernatants of B. cenocepacia IST01 collected during growth in different conditions. Representation of the concentration of N-
acyl-homoserine lactones in arbitrary units (a.u) present in supernatants collected at different time points of the growth curve of B. cenocepacia IST01 in (A) LB at 37ºC, (B) LB at 30ºC, (C) CDM supplemented with
0.8% of Glucose at 37ºC and in (D) CDM supplemented with 1.6% of Glucose at 37ºC. The N-acyl-homoserine lactones arbitrary units values represented were measured after 6h of microplate incubation at 30ºC without agitation. The arbitrary units and OD values represented are the average of three independent assays performed from independent B. cenocepacia IST01 growth curves at the growth conditions tested. The OD values are represented in a semi-logarithmic scale.
DISCUSSION
Burkholderia cepacia complex (Bcc) bacteria are not only a
threat to immunocompromised individuals and patients with
cystic fibrosis, but they also have biotechnological potential.
This thesis project has explored the potential antimicrobial
activity of BT-12 produced by BioMimetx against Bcc
bacteria resistant to many common antibiotics. Also, the
antibacterial potential of these bacteria secretome was
assessed during this work.
The antimicrobial activity of BT-12 was evaluated using
broth microdilution assays against Bcc isolates. Final
biomass concentration obtained under the different
conditions was determined based on the culture optical
density (OD) measured. In these assays, minimum inhibitory
concentration (MIC) values cannot be defined as the lowest
concentration of antimicrobial agent that completely inhibits
growth in microdilution wells.[52], [53] The minimal OD
values obtained at high concentrations of BT-12, on the
different antimicrobial assays performed were above zero
which seems to indicate that other parameters besides initial
OD value of the pre-inoculum are interfering with the final
OD value. It is hypothesized that maybe the action of BT-12
may enable cell division during the first minutes of growth
until all the BT-12 targets are affected. The cells grown and
the turbidity remained, if the cells do not lyse even though
the cells may be not viable. Also, the secretion of a different
compound able to absorb light might be interfering with the
final OD value obtained. Independently of this, it is certain
that BT-12 has the ability to affect growth of Bcc bacteria,
decreasing the final OD obtained at the stationary phase in
a dose-dependent manner, no matter if the isolates tested
were clinical or environmental. The results obtained for BT-
12 inhibitory effect in the size of biofilms produced by clinical
isolates from patient J indicate a marked decrease of the
biofilm produced by B. cenocepacia IST4113 in presence of
high concentrations of BT-12 compared with B. cenocepacia
IST439, the first isolate retrieved from the patient and
thought to have initiated the infection. This is an unexpected
result since B. cenocepacia IST439 was described to be one
of the most susceptible isolates obtained from patient J to
known antibiotics while B. cenocepacia IST4113 was the
most resistant.[44] Taking this into account, it is likely that
the mechanisms behind the action of BT-12 on the isolates
are different from those behind all the commercial antibiotics
previously tested.
Since BT-12 contains small peptides, it was thought that
some of them have antimicrobial activity against Bcc
bacteria because antimicrobial peptides (AMPs) were
already described to interact with Gram-negative bacteria.
AMPs can be cationic or anionic being those of the first type
the best studied until now. Cationic AMPs are known to
interact with the outer membrane of Gram-negative bacteria,
specifically with negatively charged lipopolysaccharide
(LPS) groups, such as phosphate or carboxyl groups.[19] It
was proposed that as the ratio of AMPs/membrane lipids
increases, AMPs can assume a perpendicular orientation
across outer and inner membrane forming pores that
increase their permeability.[19] Taking this in consideration,
the outer membrane structure of the three bacterial species
under discussion were analyzed and compared, with special
attention given to the LPS structural composition. It is
already known that Bcc bacteria, in general, have less
phosphate or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) in
their LPS core oligosaccharide structure compared to other
Gram-negative bacteria.[10] In fact, Loutet et al.
demonstrated that these characteristics have main
relevance on the majority of B. cenocepacia resistance to
cationic peptides since they are related to the
impermeabilization of the outer membrane.[25] However, it
is possible that high concentrations of BT-12 may be able to
disrupt the outer membrane of all the target bacteria tested
but differences present in the inner membranes of Bcc
bacteria, E. coli and P. aeruginosa could be determinant for
the efficacy of BT-12 antimicrobial activity. Additionally,
there are AMPs that do not interact directly with cell
membranes, instead, they kill bacteria by inhibiting essential
pathways inside the cell like protein synthesis or DNA
replication.[20] Anionic peptides could be of major interest
in this case since they are able to attack bacterial cells in a
different mode, causing the flocculation of intracellular
content.[19] It was also thought that quorum sensing-related
10
molecules present in BT-12 could play an important role in
its antimicrobial activity. These molecules could be quorum
sensing inhibitors that are able to mimic Bcc bacteria N-acyl-
homoserine lactones, N-octanoyl-homoserine lactones (C8-
HSL) and N-hexanoyl-homoserine lactone (C6-HSL),
competing with them, or able to form a complex with them
inhibiting their binding to quorum sensing receptors.[28],
[54] These inhibitor molecules could have the ability to
reduce the function of this system which would implicate a
reduction of all functions that it regulates as the biosynthesis
of several virulence factors, including extracellular
proteases, and biofilm maturation, but would not necessarily
display growth defects.[31], [54]
Regarding the antimicrobial activity of Bcc bacteria
supernatants, it was observed that they have the ability to
affect growth of both Gram-positive and Gram-negative
bacteria decreasing their specific growth rate and/or the final
biomass obtained in the antimicrobial assays. Antimicrobial
activity of Bcc bacteria supernatants depended on the
conditions selected to grow the cells consistent with the role
on cell metabolism depends of the growth parameters (e.g.
culture medium and temperature), which affect the type and
amount of secreted molecules. B. cenocepacia IST01
isolate stood out as a producer of efficient antibacterial
supernatants since some of them were able to completely
inhibit bacterial growth under standardized conditions.
Considering that different growth parameters selected for
Bcc isolates produce supernatants with different
antimicrobial activity against each selected target bacteria
tested, it is likely that Bcc supernatants include a mixture of
different antimicrobial compounds whose percentage in the
supernatant may depend on the growth conditions selected.
Taking into account that supernatants were extracted at
different phases of bacterial growth and submitted to
lyophilization, it is likely that the molecules responsible for
the antibacterial activity are secondary metabolites or
extracellular enzymes.[55] However, as the identification of
the molecules present in different supernatants tested is too
time-consuming and expensive to be performed in the
context of this thesis, it was thought to quantify AHLs
present in different supernatants since they could be a
signal of antimicrobial activity once their presence and level
could mean that QS system is more or less active, modelling
cell metabolism and stimulating antimicrobials
production.[31] The results obtained demonstrated that, in
some cases, two different supernatants, with similar
antimicrobial activity can have different amounts of AHLs.
Therefore, no direct relation could be established between
the amount of homoserine lactones in the supernatants and
their antimicrobial activity. The fact that Bcc bacteria
supernatants are able to easily inhibit E. coli ATCC 25922
growth is one of the most relevant results obtained since it
is more efficient than BT-12 for this bacterial species. This
fact suggests that these supernatants, when fully optimized
and studied, could be of interest to be used at BioMimetx. If,
eventually, it is proved that Bcc bacteria supernatants may
have interest for BioMimetx, the major problem would be to
obtain autorization to use Bcc isolates or sub-products on
biotechnological applications because their use will always
have to be balanced against their potential as opportunistic
pathogens. Fortunately, genomic studies have been
relevant to this subject since it has recently been suggested
that one Bcc bacteria, B. contaminans MS14, could be used
because it was found to possess multiple antimicrobial
biosynthetic genes but not major genetic loci required for
pathogenesis.[56] Additionally, it was well recently accepted
the use of another human opportunistic pathogens, from
Staphylococcus genus (S. lugdunensis), as source of
antimicrobials to be used against Gram-positive
bacteria.[57] Thus, the idea that Bcc bacteria would be, in
the future, a potential producer of equivalent antibacterials
cannot be beforehand discarded.
ACKNOWLEDGMENTS
I want to thank my supervisors Professor Isabel Sá Correia
and Dr. Patrick Freire for giving me the opportunity to
integrate this project as well as Dr. Carla Coutinho, my co-
supervisor, for all the support and guidance. The present
work was developed at iBB-Institute for Bioengineering and
Biosciences and BioMimetx SA. Funding received by iBB-
Institute for Bioengineering and Biosciences from FCT
(UID/BIO/04565/2013) and from Programa Operacional
Regional de Lisboa 2020 (Project N. 007317) is
acknowledged.
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