Evaluation of the antibacterial potential of Burkholderia ... · Evaluation of the antibacterial potential of Burkholderia cepacia complex bacteria ... a cocktail of antibiotics ...

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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.

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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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