REVIEW ARTICLE


Biofilm Targeting Strategy in the Eradication of Burkholderia Infections: A Mini-Review



Wani D. Gunardi1, *
iD
, Kris H. Timotius1
iD
, Agustine Natasha2
iD
, Paulina R. Evriarti2, 3
iD

1 Department of Microbiology, Faculty of Medicine, Krida Wacana Christian University (UKRIDA), Jakarta, Indonesia
2 Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
3 Department of Microbiolgy, Health Labortory of Makassar (BBLK Makassar), Makassar, Indonesia


Article Metrics

CrossRef Citations:
0
Total Statistics:

Full-Text HTML Views: 244
Abstract HTML Views: 67
PDF Downloads: 0
Total Views/Downloads: 311
Unique Statistics:

Full-Text HTML Views: 153
Abstract HTML Views: 40
PDF Downloads: 0
Total Views/Downloads: 193



Creative Commons License
© 2021 Gunardi et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at Department of Microbiology, Faculty of Medicine, Krida Wacana Christian University (UKRIDA), Jakarta 11510, Indonesia; E-mail: wani.gunardi@ukrida.ac.id


Abstract

Burkholderia are intracellular pathogenic bacteria which can produce biofilm. This biofilm protects the intracellular pathogenic bacteria from antibiotic treatment and the immunological system of the host. Therefore, this review aims to describe the capacity of Burkholderia to form a biofilm, the regulation of its biofilm formation, the efficacy of antibiotics to eradicate biofilm, and the novel therapy which targets its biofilm. Burkholderia's biofilm is characterized by its lipopolysaccharides, exopolysaccharides (EPSs), biofilm-associated proteins, and eDNA. Its regulation is made by quorum sensing, c-di-AMP, sRNA, and two component systems. Many antibiotics have been used as sole or mixture agents; however, they are not always effective in eradicating the biofilm-forming Burkholderia. Inhibitors of quorum sensing and other non-conventional antibiotic approaches are promising to discover effective treatment of Burkholderia infections.

Keywords: Antibiotic, Biofilm, Burkholderia, Exopolysaccharide, Lipid A, Quorum sensing.



1. INTRODUCTION

Burkholderia is a gram-negative bacilli group and living as saprophyte primarily in soil and water [1]. However, several Burkholderia species can cause infectious diseases in human beings, such as Burkholderia pseudomallei, which cause Melioidosis and B. cepacia complex, which cause pneumonia, bacteremia, UTIs, and septic arthritis in patients with cystic fibrosis [2, 3]. Melioidosis is endemic in Southeast Asia and Northern Australia. From 2014 - 2017, a study reported that there were increasing Melioidosis cases (145 cases) in Indonesia, with a mortality percentage that reached 43% more than the previous year [4]. The virulence factors such as hydroxytetradecanoic acid, biofilm formation, flagella expression, and ultrastructure adapt to the host tissue and probably impact it by causing Melioidosis [2].

Biofilm formation by Burkholderia has an important role in its pathogenesis process. As a virulence factor, Biofilm is defined as sessile microbial communities that attached irreversibly to organic or inorganic surfaces of each other. Burkholderia produces matrixes of extracellular polymeric substances (EPS), which is embedded in them, and exhibit phenotype changes in line with the growth rate and gene transcription [5]. Biofilm increases the ability of B. pseudomallei to survive in the host cell and escape from the immune system [6]. Compared to other bacterial infections, Burkholderia infections cause a very high relapse rate associated with biofilm formation [7]. Moreover, previous studies reported that Burkholderia is more resistant to antibiotics when growing as a biofilm than their free-living [planktonic] counterparts [6, 8].

Resistance to many antimicrobial agents, including first and second generations of cephalosporins penicillins, macrolides, colistin, rifamycins, and aminoglycosides have been found in Burkholderia [9]. Several novel antibiotic combination therapies for Burkholderia infections have been reported [10]. However, biofilm-targeted therapies among Burkholderia have not been reported. Therefore, this review aims to describe the capacity of Burkholderia to form biofilm, the regulation of Burkholderia biofilm formation, antibiotic efficacy in the eradication of biofilm, and the biofilm targeting therapy in Burkholderia infections. The Burkholderia discussed in this review are the human pathogens, namely B. pseudomalei, B. mallei, and Bcc (Burkholderiacepaia complex), including B. cepacia, B. multivorans, B. cenocepacia, and B. stabilis.

2. BIOFILM FORMATION IN Burkholderia

Clinical signs of Melioidosis vary from acute septicemia to chronic inflammation or subclinical infections. These signs are determined by the capacity of B. pseudomallei to produce biofilm. B. pseudomallei biofilm plays a role during bacterial-host interaction. Biofilm determines the adhesion to epithelial cells of the human lungs, interactive intercellular processes, apoptosis/necrosis, proinflammatory responses, and cellular pathogenesis [11]. Biofilm formation begins with biofilm surface attachment by flagella or pili, then makes cell-to-cell interactions or micro-colony formations, and finishes with exopolysaccharide secretion or a biofilm matrix [12]. A biofilm matrix glues cells together on a surface and eventually releases them to make new colonization of other surfaces [13]. The capacity of Burkholderiato form biofilm is affected by several factors, such as the cell wall structure (lipopolysaccharides), biofilm proteins, and extracellular DNA (eDNA).

2.1. Lipopolysaccharides (LPS)

Lipopolysaccharides (LPS) are common structural components in a Gram-negative bacteria cell wall, including Burkholderia. Generally, LPS has a role as endotoxin and triggers the defense-related immune responses of the host [14, 15]. Moreover, LPS also able to determine the resistance to antimicrobial peptides and also have the other function that was listed in Table 1. Structurally, the LPS component of Burkholderia species is varied, but mostly it has three regions such as Lipid A, core oligosaccharide, and O-polysaccharides (OPS) [16].

Table 1. Several functions of LPS [57-60].
Type of LPS Function
BP-LPS Endotoxin, tumour necrosis factor, interleukin-6, and nitric oxide
LPS
B.cenocepacia
cytokine induction
LPS
B.cenosepacia
Determine resistance to antimicrobial peptides
Prevent binding of the peptide to the bacterial cell envelope
LPS
Bcc
Induce a strong immune response that can contribute to host cell damage.
Lower the anionic charge of the Bcc cell surface, which inhibits the binding and subsequent effects of cationic antibiotics
LPS
B.mallei
Play a significant role in the pathogenesis of human disease.
Produce high levels of TNF-alpha, IL-6 and RANTES
CPS, B. mallei and B.pseudomallei Antigen

Lipid A is composed of hexa-acylated diglucosamine that binds LPS to the outer membrane [15, 16]. Lipid A is potentially immunogenic and can interact with receptor proteins of the innate immune system such as Toll-like receptor 4; such interactions are the first line of defense against bacterial infections and can trigger caspase-11 mediated cell death [17]. O-polysaccharides (OPS) are attached to the core of oligosaccharides and are comprised of repeating units of three to five sugars, each of which may bear a range of post-glycosylation modifications [16, 17].

LPS in different species of Burkholderia is varied. LPS of B. multivorans has two O-polysaccharide chains. The B. cenocepacia LPS inner core oligosaccharide determines the in vitro resistance to antimicrobial peptides and B. pseudomallei. LPS has three types that are serologically different [type A, type rough, and type B smooth] [18]. A study conducted by Narisara et al. [18] showed that the type A LPS of B. pseudomallei produced the lowest amount of biofilm. Therefore, it means that the different types of LPS impact the capacity of Burkholderiato from a biofilm.

2.2. Exopolysaccharides (EPSs)

Burkholderia can produce extracellular polysaccharides or exopolysaccharides (EPSs), the high-molecular-weight sugar-based polymers that are synthesized and secreted by multi microorganisms.EPSs have the function as a scaffold of biofilms to cross-link the bacterial cells together and make bacterial adaptation to different stress conditions [19, 20]. Pellizzoni et al.reported that B. cenocepacia in a non-mucoid state could form biofilm containing EPSs [21].

2.3. Biofilm-Associated Proteins

Biofilm-associated proteins in Burkholderia are unclearly identified. However, previous studies reported that some proteins and encoded genes had a role in biofilm formation.

Pinweha reported the bpsl1039-1040 ATP-binding cassette transporter's inactivation reduced the biofilm formation of B. pseudomallei [22], and BcpA protein in Burkholderia plays an important role in biofilm development. [23] However, the BcpA activity is an independent contact-dependent growth inhibition (CDI) system. CDI is the toxic C-terminus of a surface exoprotein used to inhibit the growth of susceptible bacteria on cell contact. CDI proteins are important in cooperative behaviors to build biofilm communities and prevent non-self-bacteria from entering the community [23]. Encoded genes for surface proteins involved in the biogenesis and maintenance of an integral outer membrane. Encoded genes for regulatory factors are required for biofilm maturation [24].

2.4. Extracellular DNA

Extracellular DNA (eDNA) is an essential component of biofilm. eDNA is released from the autolysis processes. The lysis of a bacterial subpopulation generates eDNA under the control of the quorum-sensing system or proceeded in a fratricide mode/suicide similar to eukaryotic cells' necrosis. The eDNA is an important substance in biofilm formation, especially in DNA damage repair, gene transfer, and nutrient source. Other functions of eDNA are to stabilize biofilm, bind and shield biofilm from antibiotics and aminoglycosides, as well as to induce antimicrobial peptide resistance. The acidification of biofilm by eDNA increases aminoglycoside resistance [25]. Pakkulnan et al. (2019) reported that eDNA is important during bacterial cell attachment and biofilm formation [26].

3. REGULATION OF BIOFILM FORMATION

Biofilm formation is a complex process that needs multifactorial things to develop. The regulation of biofilm formation is controlled by a quorum-sensing system (QS), second messenger cyclic diguanosine-5′-monophosphate (c-di-GMP) as a global intracellular protein expression, small RNAs (sRNAs), and Two Component Systems (TCSs) [27, 28]. QS is responsible for regulating the eDNA release, biosurfactants production, and the expression of a large surface protein. c-di-GMP is important in the regulation of the production of EPS and surface proteins. sRNAs are able to regulate the production of EPS [27]. The last TCSs have functions in multiple mechanisms such as cross-regulate, integrate, and coordinate various input stimuli to control biofilm formation [28].

3.1. Quorum Sensing System of Burkholderia

Two main behavioral traits of Burkholderia are intracellular life and biofilm formation [29]. The expression of these two virulence factors determines antibiotic treatment failures and is associated with quorum sensing. Two recognized signaling systems of quorum sensing in Burkholderia are the diffusible signal factor cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactones (AHLs). Both control similar phenotypic traits [30].

Acyl-HSL [Acyl homoserine lactone] mediates the gene regulation that influences biofilm formation. Most of the synthesized acyl-HSL is octanoyl-HSL [31]. The synthesis of Acyl-HSL is associated with biofilm formation. Therefore, quorum sensing plays an important role in the pathogenesis of Burkholderia, even though it does not regulate biofilm formation under all growth conditions [31].

The diffusible signal factor (DSF) molecule involved in biofilm formation and pathogenesis is a medium-length chain of monounsaturated fatty acids with an unusual cis-2 double bond. Burkholderiahas DSF (named BDSF), which contains cis-2-dodecenoic acid that can inhibit the formation and cause the dispersion of biofilm. Moreover, BDSF can regulate the expression of many genes, such as chitinase, which is responsible for biofilm formation. The presence of chitinase affects the antibiofilm activity of BDSF [32].

3.2. Cyclic Diguanosine-5′-Monophosphate (c-di-GMP)

Cyclic diguanosine-5′-monophosphate (c-di-GMP) is a second messenger that plays a main role in regulating biofilm formation in many bacteria, including Burkholderia. [33] In general, high intracellular c-di-GMP levels induce the production of extracellular biofilm matrix components. In contrast, low intracellular c-di-GMP levels suppress the production of matrix components and promote single cell motility [34, 35]. To regulate these cellular functions, c-di-GMP binds to specific effectors, which could be proteins or RNA, and alters their structure [36]. The presence of higher intracellular c-di-GMP concentration correlates with the increasing cell-to-cell aggregation and EPS production, swimming motilityor absence of flagella, and abundance of biofilm [37].

3.3. Small Non-Coding RNAMolecules (sRNAs): Genetic Control for Bacterial Biofilms

Small non-coding RNA molecules (sRNAs) are an RNA fragment that is 50–500 nucleotides in size and can regulate gene expression by interacting with other RNAs or proteins [38, 39]. As a regulator, sRNAs play the main role in the regulatory network of the post-transcriptional level of biofilm formation.[40, 41] Sasset et al. reported that the biofilm matrix of B. cenocepacia had more sRNAs than planktonic cultures [42].

3.4. Two-Component Systems (TCSs)

Two-component systems (TCSs) are essential in signaling events. Adaptation to environments, cell-cell communication, and pathogenesis are associated with TCSs. TCSs are absent in humans and other mammals. Adaptive changes in cellular processes are regulated by TCSs to respond to changes in environmental conditions [42, 43]. A study reported a novel TCS in B. cenocepacia, namely RqpSR, which plays an important role in modulating QS and pathogenesis in B. cenocepacia. Mutations in rqpS and rqpR exert overlapping effects on B. cenocepacia transcriptomes and phenotypes, including motility, biofilm formation, and virulence [44].

Besides the rqpSR gene, another response regulator gene of TCS is bfmR (biofilm formation-associated regulator], which regulates B. pseudomallei biofilm formation. A mutant of the bfmR gene shows suppression of assembly of fimbriae on the cell surface. It reduces biofilm formation that is led by a decrease in the expression of fimbriae chaperone-usher assembly genes. The low-iron growth condition upregulates the bfmR gene expression. Bacteria in low-medium iron with mutant bfmR show retarded growth. Therefore, bfmR is considered an important positive regulator in controlling the assembly of fimbriae and biofilm formation and is upregulated under low-iron conditions [45]. Mangalea and Borlee also investigated the role of a two-component nitrate sensing system, NarX-NarL, in the biofilm formation of B. pseudomallei. They found that the deletion of narX and narL could decrease the biofilm inhibition activity by nitrate. It means that the NarX-NarL two-component system is a global regulator of biofilm formation [46].

4. ANTIBIOTIC EFFICACY FOR Burkholderia INFECTIONS

Resistance to antibiotics is one of the characteristics of biofilm bacteria, including Burkholderia biofilm [47]. Caraher et al. [2007] reported that antimicrobial agents were active against all the Burkholderia strains when cultured planktonically; however, antimicrobial agents' activity diminished when the Burkholderia strains were grown as biofilms [48]. Following the result, the next study reported multidrug-resistance in Burkholderia following the use of beta-lactams, including meropenem, piperacillin/tazobactam, ceftazidime, and imipenem. B. cenocepacia is also reported to be resistant to antimicrobial peptides, such as polymyxin B (PmB) [49]. Burkholderia pseudomallei Bp1651 has also been found to be resistant to several classes of antibiotics that were usually effective for the treatment of Melioidosis, including tetracyclines, sulfonamides, and β-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem) [50]. Therefore, in many cases, antibiotics were only effective when used in combination.

Combination therapy can be used to increase the efficacy of treatment. The summary of several examples of antibiotics used for Burkholderia infections is listed in Table 2. Burkholderia has been resistant to many antibiotics when used as a single therapy, but they are effective in combination, such as Burkholderia is resistant to tobramycin but susceptible when tobramycin and amiloride are used as a combination therapy (Table 2).

5. RECOMMENDATIONS FOR THE FUTURE IN THERAPEUTIC TARGETING BurkholderiaBIOFILMS

Alternative approaches are needed to tackle Burkholderia resistance issues, especially in therapies to improve cystic fibrosis' patients' life expectancy and eradicate Burkholderia infections. The main problem of the resistance is Burkholderia can form biofilm. Therefore, alternative treatment strategies need to be explored to ensure a robust pipeline of effective therapies, especially targeting Burkholderia biofilm.

Many studies have been conducted to find a new strategy therapy that can increase the eradication of Burkholderia infections. As an example, Sidrim et al. found that Promethazine, anefflux pump inhibitor, could improve the antibiotic efficacy and disrupt biofilms of Burkholderia pseudomallei [51]. The disruption of biofilm formation can be done from its eDNA, quorum sensing, etc. (Table 3).

Table 2. List of used antibiotic for Burkholderia infections [49, 61, 62].
Antibiotic Burkholderia Description
Amikacin B.cepacia Resistance
Azithromycin-trimethoprim-sulfamethoxazole Bcc Inhibit
Azithromycin-ceftazidime Bcc Inhibit
Azithromycin-doxycycline Bcc Inhibit
Azithromycin-trimethoprim-sulfamethoxazole Bcc Inhibit
Biapenem B.cepacia Resistance
Carbenicillin B.cepacia Resistance
Cefotaxime B.cepacia Resistance
Cefuroxime B.cepacia Resistance
Chloramphenicol B.cepacia Resistance
Ciprofloxacin, imipenem B.cepacia Resistance
Gentamicin B.cepacia Resistance
Polymyxin (PmB]. B. cenocepacia Resistance
Sulphamethoxazole B.cepacia Resistance
Piperacillin/ tazobactam B. cepacia Resistance
Tobramycin Bcc Resistance
Tobramycin & amiloride B.cepacia Susceptible
Tobramycin & dichloro-isoproterenol & propranolol B.cepacia synergistic with dichloroisoproterenol and propranolol.
Trimethoprim B.cepacia Resistance
Clarithromycin-tobramycin P. aeruginosa Active/effective
Tobramycin & triclosan Bcc reduction of viable cells within biofilms but
no antimicrobial activity
Table 3. List of alternative targets for the treatment of Burkholderia infection.
Target Description
BiofilmReg operon Need to develop a common drug which is effective for treating all these causative agents [63]
DNABII Antiserum [64]
eDNA Tobramycin & DNase (rhDNase) &dispersin [65]
Efflux pump phenylalanine arginine beta-napthylamide (PAbetaN), a universal efflux inhibitor & CTZ and DOX [8]
Polysaccharides Cellulase development resistance [8]
PNAG Immunotherapy. Antisera [67]
LpxC
LpxC-4
LpxC inhibitor, and LpxC-4 in combination with CAZ [68]
sulphonamide derivates [69]
QS Quorum sensing inhibitors (QSIs) [70]
Note: 1) BceSR: BceS, a sensor kinase, BceR, a response regulator.
2) LpxC is UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase, a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A.
Fig. (1). Scheme of targeted therapies via disruption on eDNA and Quorum sensing using bacterial nuclease and anti-quorum sensing (anti QS).

eDNA is an attractive target because it is a matrix component of biofilm. eDNA enzymatic degradation can sensitize biofilm to antimicrobials. In this case, bacterial nucleases can be applied to degrade eDNA (Fig. 1). This means that eDNA could be targeted therapy by disrupting its interactions with other matrix components. It was discovered that eDNA-binding matrix components have come to light. Therefore, targeting the biofilm matrix via eDNA is emerging and promising [52].

In fact, pathogens that colonized the host cell will produce virulence factors and then do the QS signaling to form a biofilm. This bacteria conversation is broken by using anti-QS agents, making pathogens more susceptible to host immune responses and antibiotics (Fig. 1). The QS disruption strategies can be managed by several methods, including signal synthesis inhibition, receptor inactivation, signaling blockage by antibodies, and signal degradation. This way can be used as a potential therapeutic target for bacterial diseases [53].

Carbonic anhydrase (BpsCAgamma) as crucial enzymes in B. pseudomallei can be interfered with by Carbonic anhydrase inhibitors. The most effective inhibitors of BpsCAgammaare acetazolamide, benzolamide, and metanilamide. Other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame, and saccharin are also active even in a higher range [54].

The migration of bacterial cells is allowed by swarming. Swarming motility might thus represent a form of social behavior and is associated with widespread antibiotic resistance [55]. One strategy to block social behaviour of bacteria is to interfere the flagella as an apparatus to motile, cell-cell interactions and decreases the presence of surfactant. Therefore swarming motility might be useful as a model and target of eradicating biofilm antibiotic resistance [56-66].

CONCLUSION

Biofilm is a complex adherence structure to the surface that has a single type of cell or different bacterial colonies. Extracellular polymeric substances (matrix) submerge these colonies. The matrix is composed of proteins, eDNA, and polysaccharides, showing high resistance to antibiotics. Biofilm formation is regulated by quorum sensing. Several approaches are used to inhibit the biological activities of Burkholderia. Inhibitors of quorum sensing need to be discovered and screened. Conventional antibiotic therapy is not effective enough to remedy Burkholderia infections. Therefore, combining other non-conventional antibiotic approaches is promising to discover effective treatments of Burkholderia infections.

CONSENT FOR PUBLICATION

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

Declared none.

REFERENCES

[1] Talaro K, Chess B, Wiersema DS, Sen P. Foundations in Microbiology, 2012 2013.
[2] Chen YS, Shieh WJ, Goldsmith CS, et al. Alteration of the phenotypic and pathogenic patterns of Burkholderia pseudomallei that persist in a soil environment. Am J Trop Med Hyg 2014; 90(3): 469-79.
[3] Wanger A, Chavez V, Huang R, Wahed A, Dasgupta A, Actor JK. Microbiology and molecular diagnosis in pathology: a comprehensive review for board preparation, certification and clinical practice 2017.
[4] Tauran PM, Wahyunie S, Saad F, et al. Emergence of melioidosis in Indonesia and today’s challenges. Trop Med infect Dis 2018; 3(1): 32.
[5] Donlan RM, Costerton JW. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002; 15(2): 167-93.
[6] Sawasdidoln C, Taweechaisupapong S, Sermswan RW, Tattawasart U, Tungpradabkul S, Wongratanacheewin S. Growing Burkholderia pseudomallei in biofilm stimulating conditions significantly induces antimicrobial resistance. PLoS One 2010; 5(2)e9196
[7] Limmathurotsakul D, Paeyao A, Wongratanacheewin S, et al. Role of Burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis. Clin Microbiol Infect 2014; 20(11): O854-6.
[8] Sirijant N, Sermswan RW, Wongratanacheewin S. Burkholderia pseudomallei resistance to antibiotics in biofilm-induced conditions is related to efflux pumps. J Med Microbiol 2016; 65(11): 1296-306.
[9] Jenney AW, Lum G, Fisher DA, Currie BJ. Antibiotic susceptibility of Burkholderia pseudomallei from tropical northern Australia and implications for therapy of melioidosis. Int J Antimicrob Agents 2001; 17(2): 109-13.
[10] El-Halfawy OM, Naguib MM, Valvano MA. Novel antibiotic combinations proposed for treatment of Burkholderia cepacia complex infections. Antimicrob Resist Infect Control 2017; 6: 120.
[11] Kunyanee C, Kamjumphol W, Taweechaisupapong S, et al. Burkholderia pseudomallei biofilm promotes adhesion, internalization and stimulates proinflammatory cytokines in human epithelial A549 cells. PLoS One 2016; 11(8)e0160741
[12] Panomket P. Burkholderia pseudomallei and biofilms. Asian Biomed 2017; 9(3): 285-90.
[13] Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol 2016; 14(9): 563-75.
[14] Madala NE, Molinaro A, Dubery IA. Distinct carbohydrate and lipid-based molecular patterns within lipopolysaccharides from Burkholderia cepacia contribute to defense-associated differential gene expression in Arabidopsis thaliana. Innate Immun 2012; 18(1): 140-54.
[15] Ieranò T, Cescutti P, Leone MR, et al. The lipid A of Burkholderia multivorans C1576 smooth-type lipopolysaccharide and its pro-inflammatory activity in a cystic fibrosis airways model. Innate Immun 2010; 16(6): 354-65.
[16] Fathy Mohamed Y, Hamad M, Ortega XP, Valvano MA. The LpxL acyltransferase is required for normal growth and penta-acylation of lipid A in Burkholderia cenocepacia. Mol Microbiol 2017; 104(1): 144-62.
[17] Norris MH, Somprasong N, Schweizer HP, Tuanyok A. Lipid A remodeling is a pathoadaptive mechanism that impacts lipopolysaccharide recognition and intracellular survival of Burkholderia pseudomallei. Infect Immun 2018; 86(10)e00360-18
[18] Anuntagool N, Wuthiekanun V, White NJ, et al. Lipopolysaccharide heterogeneity among Burkholderia pseudomallei from different geographic and clinical origins. Am J Trop Med Hyg 2006; 74(3): 348-52.
[19] Chew SC, Kundukad B, Teh WK, et al. Mechanical signatures of microbial biofilms in micropillar-embedded growth chambers. Soft Matter 2016; 12(23): 5224-32.
[20] Ferreira AS, Silva IN, Oliveira VH, Cunha R, Moreira LM. Insights into the role of extracellular polysaccharides in Burkholderia adaptation to different environments. Front Cell Infect Microbiol 2011; 1: 16.
[21] Pellizzoni E, Ravalico F, Scaini D, Delneri A, Rizzo R, Cescutti P. Biofilms produced by Burkholderia cenocepacia: influence of media and solid supports on composition of matrix exopolysaccharides. Microbiology (Reading) 2016; 162(2): 283-94.
[22] Pinweha P, Pumirat P, Cuccui J, et al. Inactivation of bpsl1039-1040 ATP-binding cassette transporter reduces intracellular survival in macrophages, biofilm formation and virulence in the murine model of Burkholderia pseudomallei infection. PLoS One 2018; 13(5)e0196202
[23] Garcia EC, Anderson MS, Hagar JA, Cotter PA. Burkholderia BcpA mediates biofilm formation independently of interbacterial contact-dependent growth inhibition. Mol Microbiol 2013; 89(6): 1213-25.
[24] Huber B, Riedel K, Köthe M, Givskov M, Molin S, Eberl L. Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111. Mol Microbiol 2002; 46(2): 411-26.
[25] Wilton M, Charron-Mazenod L, Moore R, Lewenza S. Extracellular DNA acidifies biofilms and induces aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2015; 60(1): 544-53.
[26] Pakkulnan R, Anutrakunchai C, Kanthawong S, Taweechaisupapong S, Chareonsudjai P, Chareonsudjai S. Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation. PloS one 2019; 14(3): e0213288-.
[27] Fazli M, Almblad H, Rybtke ML, Givskov M, Eberl L, Tolker-Nielsen T. Regulation of biofilm formation in Pseudomonas and Burkholderia species. Environ Microbiol 2014; 16(7): 1961-81.
[28] Liu C, Sun D, Zhu J, Liu W. Two-component signal transduction systems: a major strategy for connecting input stimuli to biofilm formation. Front Microbiol 2019; 9: 3279.
[29] Savoia D, Zucca M. Clinical and environmental Burkholderia strains: biofilm production and intracellular survival. Curr Microbiol 2007; 54(6): 440-4.
[30] Schmid N, Pessi G, Deng Y, et al. The AHL- and BDSF-dependent quorum sensing systems control specific and overlapping sets of genes in Burkholderia cenocepacia H111. PLoS One 2012; 7(11)e49966
[31] Conway B-AD, Venu V, Speert DP. Biofilm formation and acyl homoserine lactone production in the Burkholderia cepacia complex. J Bacteriol 2002; 184(20): 5678-85.
[32] Deng Y, Schmid N, Wang C, et al. Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover. Proc Natl Acad Sci USA 2012; 109(38): 15479-84.
[33] Boyd CD, O’Toole GA. Second messenger regulation of biofilm formation: breakthroughs in understanding c-di-GMP effector systems. Annu Rev Cell Dev Biol 2012; 28: 439-62.
[34] Ryan RP, Tolker-Nielsen T, Dow JM. When the PilZ don’t work: effectors for cyclic di-GMP action in bacteria. Trends Microbiol 2012; 20(5): 235-42.
[35] Fazli M, McCarthy Y, Givskov M, Ryan RP, Tolker-Nielsen T. The exopolysaccharide gene cluster Bcam1330-Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c-di-GMP and Bcam1349. MicrobiologyOpen 2013; 2(1): 105-22.
[36] Fazli M, O’Connell A, Nilsson M, et al. The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia. Mol Microbiol 2011; 82(2): 327-41.
[37] Lee HS, Gu F, Ching SM, Lam Y, Chua KL. CdpA is a Burkholderia pseudomallei cyclic di-GMP phosphodiesterase involved in autoaggregation, flagellum synthesis, motility, biofilm formation, cell invasion, and cytotoxicity. Infect Immun 2010; 78(5): 1832-40.
[38] Desnoyers G, Bouchard M-P, Massé E. New insights into small RNA-dependent translational regulation in prokaryotes. Trends Genet 2013; 29(2): 92-8.
[39] Michaux C, Verneuil N, Hartke A, Giard J-C. Physiological roles of small RNA molecules. Microbiology (Reading) 2014; 160(Pt 6): 1007-19.
[40] Chambers JR, Sauer K. Small RNAs and their role in biofilm formation. Trends Microbiol 2013; 21(1): 39-49.
[41] Van Puyvelde S, Steenackers HP, Vanderleyden J. Small RNAs regulating biofilm formation and outer membrane homeostasis. RNA Biol 2013; 10(2): 185-91.
[42] Sass A, Kiekens S, Coenye T. Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism. Sci Rep 2017; 7(1): 15665.
[43] Merry CR, Perkins M, Mu L, Peterson BK, Knackstedt RW, Weingart CL. Characterization of a novel two-component system in Burkholderia cenocepacia. Curr Microbiol 2015; 70(4): 556-61.
[44] Cui C, Yang C, Song S, et al. A novel two-component system modulates quorum sensing and pathogenicity in Burkholderia cenocepacia. Mol Microbiol 2018; 108(1): 32-44.
[45] Tabunhan S, Wongratanacheewin S, Wongwajana S, Welbat TU, Faksri K, Namwat W. Characterization of a novel two-component system response regulator involved in biofilm formation and a low-iron response of Burkholderia pseudomallei. Southeast Asian J Trop Med Public Health 2014; 45(5): 1065-79.
[46] Borlee BR, Mangalea MR. The NarX-NarL two-component system is a global regulator of biofilm formation, natural product biosynthesis, and host-associated survival in Burkholderia pseudomallei. bioRxiv 2020.
[47] Kanthawong S, Bolscher JG, Veerman EC, et al. Antimicrobial and antibiofilm activity of LL-37 and its truncated variants against Burkholderia pseudomallei. Int J Antimicrob Agents 2012; 39(1): 39-44.
[48] Caraher E, Reynolds G, Murphy P, McClean S, Callaghan M. Comparison of antibiotic susceptibility of Burkholderia cepacia complex organisms when grown planktonically or as biofilm in vitro. Eur J Clin Microbiol Infect Dis 2007; 26(3): 213-6.
[49] Loutet SA, Valvano MA. Extreme antimicrobial peptide and polymyxin B resistance in the genus Burkholderia. Front Cell Infect Microbiol 2011; 1: 6.
[50] Bugrysheva JV, Sue D, Gee JE, et al. Antibiotic resistance markers in Burkholderia pseudomallei strain Bp1651 identified by genome sequence analysis. Antimicrob Agents Chemother 2017; 61(6)e00010-17
[51] Sidrim JJC, Vasconcelos DC, Riello GB, et al. Promethazine improves antibiotic efficacy and disrupts biofilms of Burkholderia pseudomallei. Biofouling 2017; 33(1): 88-97.
[52] Okshevsky M, Regina VR, Meyer RL. Extracellular DNA as a target for biofilm control. Curr Opin Biotechnol 2015; 33: 73-80.
[53] Jiang Q, Chen J, Yang C, Yin Y, Yao K. Quorum sensing: a prospective therapeutic target for bacterial diseases. BioMed Research International 2019; 15
[54] Del Prete S, Vullo D, Di Fonzo P, et al. Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis. Bioorg Med Chem Lett 2017; 27(3): 490-5.
[55] Lai S, Tremblay J, Déziel E. Swarming motility: a multicellular behaviour conferring antimicrobial resistance. Environ Microbiol 2009; 11(1): 126-36.
[56] Erhardt M. Strategies to block bacterial pathogenesis by interference with motility and chemotaxis 2016; 185-205.
[57] Ortega X, Silipo A, Saldías MS, Bates CC, Molinaro A, Valvano MA. Biosynthesis and structure of the Burkholderia cenocepacia K56-2 lipopolysaccharide core oligosaccharide: truncation of the core oligosaccharide leads to increased binding and sensitivity to polymyxin B. J Biol Chem 2009; 284(32): 21738-51.
[58] Bayliss M, Donaldson MI, Nepogodiev SA, et al. Structural characterisation of the capsular polysaccharide expressed by Burkholderia thailandensis strain E555: wbiI (pKnock-KmR) and assessment of the significance of the 2-O-acetyl group in immune protection. Carbohydr Res 2017; 452: 17-24.
[59] Brett PJ, Burtnick MN, Snyder DS, Shannon JG, Azadi P, Gherardini FC. Burkholderia mallei expresses a unique lipopolysaccharide mixture that is a potent activator of human Toll-like receptor 4 complexes. Mol Microbiol 2007; 63(2): 379-90.
[60] Tavares-Carreón F, Patel KB, Valvano MA. Burkholderia cenocepacia and Salmonella enterica ArnT proteins that transfer 4-amino-4-deoxy-l-arabinose to lipopolysaccharide share membrane topology and functional amino acids. Sci Rep 2015; 5: 10773.
[61] Loutet SA, Mussen LE, Flannagan RS, Valvano MA. A two-tier model of polymyxin B resistance in Burkholderia cenocepacia. Environ Microbiol Rep 2011; 3(2): 278-85.
[62] Maiden MM, Hunt AMA, Zachos MP, et al. Triclosan is an aminoglycoside adjuvant for eradication of Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 2018; 62(6)e00146-18
[63] Voronina OL, Kunda MS, Ryzhova NN, et al. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes. BioMed Res Int 2016; 20166560534
[64] Novotny LA, Amer AO, Brockson ME, Goodman SD, Bakaletz LO. Structural stability of Burkholderia cenocepacia biofilms is reliant on eDNA structure and presence of a bacterial nucleic acid binding protein. PLoS One 2013; 8(6)e67629
[65] Messiaen AS, Nelis H, Coenye T. Investigating the role of matrix components in protection of Burkholderia cepacia complex biofilms against tobramycin. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 2014; 13(1): 56-62.
[66] Rajasekharan SK, Ramesh S. Cellulase inhibits Burkholderia cepacia biofilms on diverse prosthetic materials. Pol J Microbiol 2013; 62(3): 327-30.
[67] Skurnik D, Davis MR Jr, Benedetti D, et al. Targeting pan-resistant bacteria with antibodies to a broadly conserved surface polysaccharide expressed during infection. J Infect Dis 2012; 205(11): 1709-18.
[68] Sengyee S, Saiprom N, Paksanont S, Limmathurotsakul D, Wuthiekanun V, Chantratita N. Susceptibility of clinical isolates of Burkholderia pseudomallei to a lipid a biosynthesis inhibitor. Am J Trop Med Hyg 2017; 97(1): 62-7.
[69] Clements JM, Coignard F, Johnson I, et al. Antibacterial activities and characterization of novel inhibitors of LpxC. Antimicrob Agents Chemother 2002; 46(6): 1793-9.
[70] Santhakumari S, Ravi AV. Targeting quorum sensing mechanism: An alternative anti-virulent strategy for the treatment of bacterial infections. S Afr J Bot 2019; 120: 81-6.