The analysis of Bcc epidemiology, particularly of B. cenocepacia, in CF patients is paramount as highlighted by the several methods used for genotyping. Indeed, transmissibility markers were identified in B. cenocepacia epidemic strains, such as the cblA pilin gene (Sun et al., 1995) and the B. cepacia epidemic strain marker (BCESM) belonging to a pathogenicity island (Baldwin et al., 2004), or the IS1363 insertion sequence (Liu et al., 2003). However, even if some epidemic lineages are associated to these genetic markers, others were not, thus genotyping studies are still required for a full understanding of cross-transmission and for epidemiological surveillance.

Among molecular typing methods, the most used are ribotyping (LiPuma et al., 1988; Dasen et al., 1994), pulsed-field gel electrophoresis (PFGE) (Tenover et al., 1995), random amplified polymorphic DNA (RAPD) (Mahenthiralingam et al., 1996), repetitive elements PCR (rep-PCR) (van Belkum et al., 1996) and multilocus sequence typing (MLST) (Coenye and LiPuma, 2002; Urwin and Maiden, 2003; Spilker et al., 2009). Macro-restriction of chromosomal DNA followed by PFGE was considered the gold standard in bacterial typing for a long time, and was widely applied to the molecular epidemiology of Bcc (Coenye et al., 2002). However, as PFGE is quite a laborious technique and typing results are difficult to compare between different laboratories, MLST replaced it as preferred genotyping method also for CF pathogens (Saiman et al., 2014). MLST differentiates bacterial isolates by comparing the sequence of seven housekeeping gene fragments and then characterizing strains by the resulting allelic profile. For each housekeeping gene, sequence variants are designated as distinct alleles and each allele profile defines a specific sequence type (ST). Compared to other genotyping methods, MLST offers several advantages, primarily yielding unambiguous and reproducible results that can be easily and reliably compared between different laboratories. MLST profiles of more than 2000 Bcc isolates are freely available online⁴ (Jolley and Maiden, 2010), however this technique is quite expensive and time consuming, thus limiting its application for routine use in clinical microbiology laboratories or in genotyping large collection of isolates in national surveillance programs.

For the above reasons, alternative methods were developed to allow routine or large-scale analysis, such as the SNaPBcen assay targeting only single nucleotide polymorphisms in MLST genes instead of analyzing full sequences (Eusebio et al., 2013); the PCR assays based on MLST and specific for particularly globally distributed epidemic strain (Dedeckova et al., 2013); or multilocus variable-number tandem-repeat analysis (MLVA) (Segonds et al., 2015).

Evolution and adaptation of pathogens during chronic infections are of great importance for choosing the appropriate therapeutic strategy, however, the underlying molecular bases for Bcc are still poorly understood. As a consequence, the current genotyping methods currently available are not sufficient to assess the genetic diversity of Bcc strains and predict the clinical outcome (Lee et al., 2017). By contrast, a genomic approach could provide more insights in Bcc evolution during chronic lung infections. A recent study describing the whole genome sequence analysis of 215 B. cenocepacia isolates, collected from 16 CF patients at different times of infection, demonstrated a considerable phenotypic and genotypic variability within single patients and confirmed that distinct lineages could follow distinct evolution patterns during chronic lung infection (Lee et al., 2017). Similar results were previously achieved by comparative genomic analysis of clinical isolates of Pseudomonas aeruginosa and Burkholderia dolosa from CF patients, thus confirming that these bacteria are indeed capable of accumulating different mutations at different stages of chronic infections (Lieberman et al., 2011; Markussen et al., 2014; Lee et al., 2017).

Genome-based taxonomic and phylogenetic analyses that have emerged as present identification standards can provide more accurate genotyping data of clinical isolates and also allow the identification of strains that have rapidly evolved after introduction of novel determinants by horizontal gene transfer (Juhas et al., 2009). For instance, a recent genome-based analysis of Bcc clinical isolates in India identified a previously unknown B. cenocepacia clone characterized by a novel genomic island (i.e., BcenGI15), very similar to that found in Burkholderia pseudomallei strain EY1, and so demonstrated transfer of genomic islands also between different pathogenic species within the Burkholderia genus (Patil et al., 2017).

In Gram negative bacteria the cell envelope is composed of an inner membrane, a periplasmic space and an outer membrane containing lipopolysaccharide (LPS) (Hamad et al., 2012). LPS comprises lipid A, core oligosaccharide (OS) and a polymer composed of glycan monomers called O-antigen (O-Ag). The lipid A-core OS and O-Ag are synthetized independently on the cytoplasmic side of the inner membrane, then they are joined in the periplasmic side and finally the complete LPS molecule is transferred to the outer membrane surface by a group of conserved proteins forming the lipopolysaccharide transport machinery (LPT) (Hamad et al., 2012). LPS undergoes further modifications, such as addition or removal of sugars, phosphates, or acyl groups allowing bacterial survival in stress conditions such as presence of antimicrobial peptides (Raetz et al., 2007). Among LPS alterations, the cationic substitution of phosphate groups by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N), decreases the net negative charge of lipid A. B. cenocepacia is extremely resistant to antimicrobial peptides like polymyxin B. In Burkholderia spp. L-Ara4N is the main constituent of the lipid A and of the OS portion of the LPS (Olaitan et al., 2014). Deletion of the OS region leads to an increased binding of polymyxin B to B. cenocepacia cells and so increased sensitivity to polymyxin B (Ortega et al., 2009).

In addition, another important polymyxin B resistance determinant in B. cenocepacia is the alternative sigma factor RpoE involved in controlling the expression of a group of genes which are part of the extra-cytoplasmic stress response (Loutet and Valvano, 2011).

Finally, there are two other minor determinants of antimicrobial peptide resistance in B. cenocepacia. Two secreted zinc metalloproteases, ZmpA and ZmpB, are involved in the degradation of different antimicrobial peptides in vitro, however, B. cenocepacia knocked-out strains for one or both proteases did not show increased sensitivity. Loutet et al. (2011) demonstrated that antimicrobial peptide resistance could be found in a deep-rough LPS B. cenocepacia mutant in which the LPS molecule is truncated through the disruption of the ADP-L-glycero-D-manno-heptose synthesis.

In Gram negative bacteria efflux mechanisms play a major role in antibiotic resistance. Up to five families of transporters can be involved: the major facilitator superfamily (MFS), the ATP-binding cassette family (ABC), the small multidrug resistance family (SMR), the multidrug and toxic compound extrusion family (MATE), and the resistance nodulation division family (RND) (Li et al., 2015).

In B. cenocepacia 16 open reading frames encoding RND efflux pumps were identified. This type of transporters catalyzes substrate efflux via an H⁺ antiport mechanism. Genes are usually organized as operons and encode multimeric structures composed of an RND protein placed in the inner cellular membrane, an outer membrane protein (OMP) in the outer membrane and a membrane adaptor protein in the periplasmic region that connects the first two proteins (Li et al., 2015).

Different studies highlighted the key role of RND efflux pumps in drug resistance of B. cenocepacia, showing that in particular RND-3, RND-4, and RND-9 protect the bacterium from different compounds (Buroni et al., 2009, 2014; Bazzini et al., 2011). For example, B. cenocepacia cells treated with chlorhexidine overexpress several genes coding for drug resistance determinants such as RND efflux systems (Coenye et al., 2011). Analysis of RND mutants confirmed that some efflux transporters are involved in chlorhexidine efflux during planktonic growth (RND-1 and RND-4), while some others (RND-3 and RND-9) during sessile growth (Coenye et al., 2011).

Moreover, in order to better clarify the role of each RND efflux pumps in B. cenocepacia, MIC differences for certain antibiotics were evaluated using knock-out mutants for the 16 RND efflux systems, and analyzed during both planktonic and sessile growth. During planktonic growth RND-3 efflux pump was demonstrated to be crucial for resistance to ciprofloxacin and tobramycin, while RND-4 is important for extrusion of ciprofloxacin, tobramycin, minocycline, and chloramphenicol (Buroni et al., 2014). On the other hand, RND-8 and RND-9 play a key role in protection against tobramycin only during sessile growth (Buroni et al., 2014).

Mira et al. (2011) described the adaptive mechanisms that promote long-term colonization of B. cenocepacia in CF lungs by DNA microarrays transcriptomic analysis of two clonal variants isolated during long-term infection. Among the up-regulated genes in the most resistant strain, mdtABC and bpeA, were identified, respectively encoding RND-6 and RND-4 efflux pumps, BCAM0201, encoding an efflux system of the MFS; and BCAM2188, encoding a component of an ABC-transporter (Mira et al., 2011).

RND-4 and RND-9 were also involved in the resistance to two novel anti-Burkholderia experimental compounds (Scoffone et al., 2014, 2015).

A further study showed that in Bcc clinical isolates, RND-3 is the most up-regulated among the RND efflux systems due to mutations in its transcriptional regulator (Tseng et al., 2014).

Other efflux systems are also involved in Burkholderia antibiotic resistance: for example, the overexpression of fsr (fosmidomycin resistance gene) results in the upregulation of an efflux pump which leads to fosmidomycin resistance (Messiaen et al., 2011).

Drug target modification is not the main resistance mechanism described in B. cenocepacia. A recent work analyzed the roles of the class 1 integron, the quinolone resistance-determining regions (QRDRs) of topoisomerases II and IV, in Bcc clinical isolates (Tseng et al., 2014). Levofloxacin resistance is due to accumulation of mutations in the QRDR genes encoding topoisomerases and efflux pump activation (Pope et al., 2008; Nikaido and Pages, 2012). Among 66 Bcc clinical isolates, 6 levofloxacin resistant strains were identified carrying single-base mutations in the QRDR region of the DNA gyrase subunit A (gyrA gene) at codon 81 (Gly81Asp), 83 (Thr83Ile), and 87 (Asp87His), respectively. No mutations were found in the QRDR region of the DNA topoisomerase IV subunit A (parC gene) (Tseng et al., 2014).

This mechanism is commonly used by bacteria to achieve resistance to β-lactams and aminoglycosides.

β-lactams, such as penicillins, cephalosporins, clavams, carbapenems, and monobactams are inactivated by periplasmic β-lactamases through hydrolysis of the β-lactam ring (Bush and Fisher, 2011). Several β-lactamases were identified in B. cenocepacia: AmpC, hydrolysing expanded-spectrum cephalosporins; AmpD, a cell wall recycling enzyme (Holden et al., 2009); PenB, a Class A penicillinase extremely conserved across the Bcc (Poirel et al., 2009). β-lactams block the cell wall recycling system (Cho et al., 2014), but bacteria can detect them using PenR (AmpR), a transcriptional regulator that normally binds a precursor of peptidoglycan (i.e., UDP-MurNAc-pentapeptide) and represses the expression of ampC. PenR becomes a transcriptional activator after binding peptidoglycan degradation products like 1,6-anhydroMurNAc-peptides. Mutations in ampD have a key role in controlling expression of ampC and penB (Lee et al., 2015; Vadlamani et al., 2015). In a recent study, ampD mutations induced by ceftazidime were demonstrated to cause overexpression of PenB and AmpC β-lactamases encoding genes, hence causing a reduction to ceftazidime, cefotaxime, and meropenem susceptibility (Hwang and Kim, 2015).

A therapeutic strategy could be to block the activity of β-lactamases using β-lactamase inhibitors; a large number of class A enzymes are blocked by clavulanic acid, sulbactam or tazobactam, while class C (AmpC β-lactamases) and some class D enzymes are inhibited by avibactam (Drawz and Bonomo, 2010; Lagacé-Wiens et al., 2014). No inhibitors effective against class B β-lactamases have been described yet (Livermore et al., 2011; Aktaş et al., 2012). In this context, a recent work evaluated the effect of β-lactamase inhibitors on Bcc treated with β-lactam antibiotics (Everaert and Coenye, 2016). In B. cenocepacia LMG 16656 the authors did not observe any difference in the MIC for ceftazidime and cefepime when sulbactam, tazobactam, or avibactam were present. A possible explanation could be that in B. cenocepacia LMG 16656 the majority of the β-lactamases belong to the metallo-β-lactamase family (Winsor et al., 2008), so no effective inhibitors are available yet (Everaert and Coenye, 2016).

When chronic infections are established in CF patients, bacterial cells are able to form biofilm, a matrix of extracellular polymeric molecules composed of DNA, polysaccharides, and proteins. In this context, they can further develop antibiotic resistance due to decreased antibiotic diffusion inside the biofilm matrix, reduction of nutrient availability resulting in metabolic changes that decrease antibiotic susceptibility, and, finally, phenotypic differentiation with appearance of persister cells which play key role in long-term infections (Mulcahy et al., 2010). Persister cells are a sub-population of cells that survive antibiotic treatment but, in contrast to resistant bacteria, this group does not express a specific resistance mechanism and their tolerance derives from physiological processes rather than genetic mutations (Allison et al., 2011). This kind of sub-populations is present in both sessile and planktonic cultures, but they are more difficult to eradicate after biofilm formation.

Persister cells are not mutated, but they are phenotypic variants of the wild type. After antibiotic treatment, these cells neither grow nor die and, and after drug removal, they start again to grow causing symptoms of infection. Van Acker et al. (2013) demonstrated that B. cenocepacia biofilms contain tolerant persister cells after tobramycin treatment.

Molecular mechanisms at the base of persistence are still largely unexplored, however, it was demonstrated that toxin–antitoxin modules (TA) play an important role not only in biofilm formation, gene regulation, programmed cell death and regulation of mobile genetic elements, but also in persistence mechanism (Gerdes and Maisonneuve, 2012). TA modules, abundant in bacteria and archaea, are two-component systems formed by a toxin that inhibits cell growth and by an antitoxin that controls toxin activity. There are five known types of TA loci. Toxin modules are always of protein nature in types I and III TA, while antitoxins are small RNAs that block toxin at translational and post-translational levels. Antitoxins of types II, IV, and V TA are proteins (Wang et al., 2012). Type II TA is well-characterized and usually the two genes are organized in an operon. During normal conditions, toxin and antitoxin form a complex that leads to inactivation of the toxin. Stress conditions induce degradation of the antitoxin and the toxin module regulates different cellular functions (Gerdes and Maisonneuve, 2012). Sixteen type II TA modules are present in B. cenocepacia J2315, 12 of which are conserved among B. cenocepacia strains. They were found to be up-regulated during biofilm growth (Van Acker et al., 2014). After treatment with tobramycin or ciprofloxacin, overexpression of toxins belonging to these TA systems contributes to persistence (Van Acker et al., 2014). TA modules could be an interesting target to treat chronic infections, but their redundancy is a challenging obstacle (Van Acker et al., 2014).

A search in the EU Clinical Trials Register⁵ and ClinicalTrials.gov⁶ using “cystic fibrosis and Burkholderia” keywords retrieved 12 and 8 results, respectively. Among the former, one was completed and the results are reported above in the “Antibiotics in clinical use” paragraph (Aztreonam), one had no results available and all the others were not pertaining to the search being not related to CF or to Burkholderia infections. Among the latter, one was about Burkholderia dolosa infections, two were completed and hence reported above in the “Antibiotics in clinical use” paragraph (Aztreonam and Tobramycin), for one the status had not been verified for more than 2 years, one was about P. aeruginosa infections, one was not related to infections but to microbiota of patients, and the last one had been withdrawn prior to enrollment.

For these reasons, the only trial worth reporting is the “Study of OligoG in cystic fibrosis subjects with Burkholderia spp. infection (SMR-2591)” which is ongoing in Germany. This is a randomized double-blind, placebo-controlled cross-over study of inhaled alginate oligosaccharide (OligoG) for the treatment of chronic Burkholderia infections in adults. Information about this trial is available at www.clinicaltrialsregister.eu/ctr-search/trial/2014-002125-35/DE. OligoG is a low-molecular-weight oligosaccharide enriched from sodium alginate polysaccharides. It is an oligomer composed of α-L-guluronic acid (>85%) and β-D-mannuronic acid (<15%). Alginate is produced by P. aeruginosa during its conversion to a mucoid phenotype in the formation of biofilms (Hentzer et al., 2001). Previous studies demonstrated an improved effect of antibiotics used in combination with OligoG due to perturbation of biofilm formation (Khan et al., 2012; Roberts et al., 2013). Patients (both males and females) with at least two sputum cultures positive for Bcc in the 12 months prior to the beginning of the study were recruited. Moreover, the eligibility criteria included the use of inhaled aztreonam. The main objective of this trial is to explore the efficacy of OligoG in reducing Bcc load in patients’ sputum. The study will also evaluate the effect of inhaled OligoG on lung function, quality of life, rheology, safety and patient compliance with treatment.

Molecules already in use to treat other clinical syndromes and products of natural origins are among the alternative approaches that can enhance current therapies or counteract the problem of resistance.

Immunomodulation has been suggested to contribute to the successful response to antibiotics in cases of cepacia syndrome (Gilchrist et al., 2012). This is probably due to the antagonist effect on the host response involved in the pathogenesis of cepacia syndrome. In one case, a patient was successfully treated with four intravenous antibiotics, oral corticosteroids, and cyclosporin (Gilchrist et al., 2012). In another case, four intravenous antibiotics, nebulised meropenem and tobramycin were used in combination with mycophenolate mofetil and tacrolimus (Weidmann et al., 2008). The role of immunosuppressant therapy in the management of Burkholderia infections is not completely understood, nevertheless it is worth considering this alternative approach for patients with poor prognosis.

Cystic fibrosis macrophages show a suboptimal IFN-γ response during B. cenocepacia infection causing deficient autophagosome formation, therefore it has been suggested that IFN-γ administration may help clearance of these bacteria (Assani et al., 2014). So far, this has been demonstrated only in cell culture models and the efficacy in vivo has yet to be determined.

Thiosulfoacid S-esters are organic compounds with established biological activity, already used as medicines, preservatives of vegetables, growth regulators, insecticides, and radioprotectors (Sotirova et al., 2012). Their chemical structure resembles that of natural compounds like garlic and onion derivatives (Block et al., 1996). In an attempt to find new molecules effective both against Gram-positive and negative microorganisms, Lubenets et al. (2017) synthesized S-esters of 4-acetyl, 4-trifluoroacetyl- and 4-(3-chloropropionylamino)-benzenethiosulfoacids and three of them showed MIC values within the micromolar concentration against B. cepacia. These molecules are examples of compounds worth further investigation to assess structure-activity relationships (SARs) and then prompt their practical application.

Cysteamine has been investigated as a potential enhancer of antimicrobial therapy in CF patients due to its ability to disrupt disulfide bonds, thus achieving a mucolytic activity and improving biofilm penetration (Gahl et al., 1985). This molecule is already approved for other diseases and its safety profile is known since 1994 (Gahl et al., 2007). Moreover, cysteamine showed antimicrobial activity against P. aeruginosa, so recently Fraser-Pitt et al. (2016) showed its effect in combination with antimicrobial agents used for the treatment of Burkholderia. In particular, they found that cysteamine was able to enhance the antimicrobial activity of tobramycin (even reversing resistance/insensitivity in 17 out of 36 strains), ciprofloxacin (reversing resistance/insensitivity in 10 strains), trimethoprim-sulfamethoxazole, but not ceftazidime. Furthermore, inhibition of bacterial biofilm was observed in presence of sub-inhibitory concentrations of cysteamine (Fraser-Pitt et al., 2016).

Interestingly, a very recent report showed that fatty acid cysteamine conjugates are able to promote transport of the misfolded CFTR to the surface of epithelial cells (Vu et al., 2017): in this way, besides having antimicrobial properties, cysteamine could be also useful to restore CFTR function in combination with potentiators and activators.

In an attempt to identify compounds able to increase the susceptibility of B. cenocepacia biofilms to tobramycin, Van den Driessche et al. (2017) screened a repurposing library containing non-toxic compounds with already known metabolic properties. A total of 60 compounds were identified. Among them, four antifungal imidazoles (namely econazole, miconazole, oxiconazole, and ketoconazole) were able to significantly decrease the concentration of tobramycin necessary to completely eradicate Bcc biofilms. However, no potentiating effect could be observed in a 3D long epithelial cells model, nor in Galleria mellonella and mouse models of infection (Van den Driessche et al., 2017).

Essential oils (EOs) are complex extracts derived from aromatic plants comprising mixtures of aldehydes, terpenes, and phenols exhibiting broad spectrum antimicrobial activity (Bakkali et al., 2008). EO extracts containing carvacrol and thymol were shown to inhibit the growth of both clinical and environmental Bcc strains (Maida et al., 2014). Their mechanism of action relies on the partitioning of cytoplasmic membranes resulting in increased permeability, depletion of proton gradients, and disruption of ATP synthesis, ultimately leading to the death of bacterial cells.

First evidencies of antibacterial efficacy of thymol/carvacrol-loaded polymer nanoparticles against B. cenocepacia were reported by Amato et al. (2016) using polymer nanoparticles developed to overcome EO hydrophobicity, volatility and instability.

Polyunsaturated fatty acids (PUFAs), such as essential omega-3 and omega-6 fatty acids, were shown to have antimicrobial activity, probably by disturbing cell membrane structures and associated functions like electron transport, proton gradient and enzymatic activities (Desbois and Smith, 2010). In particular, fish oils are a source of the omega-3 PUFA eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). Different papers described the use of omega-3 PUFAs to control P. aeruginosa infections in vivo (Tiesset et al., 2011; Caron et al., 2015) and in vitro (Tiesset et al., 2009), as well as B. cenocepacia infections (Mil-Homens et al., 2012). In particular, Mil-Homens et al. (2016) reported on their efficacy in treating Burkholderia infections and also as prophylactic therapy, using G. mellonella as infection model. Interestingly, as CF patients are deficient in fatty acids metabolism, administration of omega-3 PUFAs could be beneficial both to aid infection treatment and to improve respiratory, inflammatory, and nutritional parameters (Olveira et al., 2010).

Poly (acetyl, arginyl) glucosamine (PAAG) is a polycationic polysaccharide which represents a novel class of glycopolymers with antibacterial properties as well as synergy with antibiotics in vitro (Siedenbiedel and Tiller, 2012). Its mechanism of action relies on the interaction with the outer membrane of Gram negatives causing a depolarization which results in leakage of the intracellular contents and death. In particular, divalent cations located within the bacterial outer membrane electrostatically bind the LPS by the anionic phosphate groups (Rajyaguru and Muszynski, 1997). Recently, Narayanaswamy et al. (2017) showed that PAAG is effective to treat lung infections caused by Bcc in CF patients in combinations with Meropenem and Tobramycin as it enhances susceptibility of resistant strains.

Phage therapy is the therapeutic application of bacterial viruses (bacteriophages) commercially developed in the 1930s to reduce or eliminate infection (Gravitz, 2012). This is considered a valuable alternative to chemotherapeutic agents due to the specificity of phages toward bacterial cells and their exponential replication highly enhancing the therapeutic potential (Alisky et al., 1998).

Semler et al. (2014) compared the activity of phages delivered as aerosol to that of phages delivered via an intraperitoneal (i.p.) route to treat B. cenocepacia infections in a murine model. Their results showed that infected mice receiving aerosolized phage treatments exhibited a significant decrease in bacterial loads within the lungs, which could not be observed in those receiving treatment via i.p. injection. In this way, they demonstrated that aerosolization provides more widespread and uniform particle deposition, while i.p. delivered phages reach only certain areas in the lungs and may be unable to co-localize with the bacteria in the lung lumen (Semler et al., 2014).

Phage-antibiotic synergy (PAS) is the effect that some antibiotics exert on the ability of phages to form larger plaque under sub-lethal concentrations of the compound itself (Comeau et al., 2007). Ciprofloxacin, meropenem, and tetracycline exhibited PAS in combination with two B. cenocepacia phages enlarging plaque size (Kamal and Dennis, 2015). In particular, presence of ciprofloxacin and meropenem leads to the formation of elongated or filamented cells, and so phages may have increased access to phage receptors; moreover, cell clustering in presence of tetracycline allows phages to travel laterally across adjoined cell surfaces, again enhancing contact with phage receptors. PAS effect was not altered when treating antibiotic resistant cells, thus encouraging the use of this alternative method also with drug resistant strains (Kamal and Dennis, 2015).

Cationic antimicrobial peptides (CAMPs) have been isolated from very different organisms such as microorganisms, invertebrates, plants, and mammals. They are able to establish strong non-specific, hydrophobic and electrostatic interactions with bacterial cytoplasmic membranes (Godballe et al., 2011) and prevent cell adhesion via electrostatic bonds (Overhage et al., 2008).

Antimicrobial peptides able to block B. cenocepacia biofilm formation were described in de la Fuente-Núñez et al. (2014). These peptides are similar to CAMPs but have different SAR. They exert their activity by blocking the stringent response mediated through (p)ppGpp, two small signaling nucleotides (guanosine 5′-diphosphate 3′-diphosphate or ppGpp and guanosine 5′-triphosphate 3′-diphosphate or pppGpp) (Potrykus and Cashel, 2008). These antimicrobial peptides showed activity against both Gram negative and positives bacteria. The peptide described by de la Fuente-Núñez et al. (2014) directly interacts with (p)ppGpp and is able to cross bacterial membranes to reach the cytoplasm. It can prevent biofilm formation and promote biofilm dispersal, and also cell death in biofilms at concentrations sub-lethal for planktonic cells (de la Fuente-Núñez et al., 2014).

In order to attenuate bacterial virulence, several quorum sensing inhibitors (QSI) have been developed in the last 15 years. Among them, analogs of the signal molecule Acyl-homoserine lactone (AHL) are obtained by modification of the acyl side chain, the lactone moiety (Ni et al., 2009) or the central amide moiety (Boukraa et al., 2011). Valuable AHL analogs, able to affect B. cenocepacia (as well as P. aeruginosa) QS and, in turn, inhibit and eradicate biofilm, have been described by Brackman et al. (2012).

More recently, another class of QSIs, namely diketopiperazines, was shown to inhibit the activity of the AHL synthase CepI of B. cenocepacia (Scoffone et al., 2016). These compounds interfered with the production of virulence factors, such as proteases and siderophores, as well as with biofilm formation, and showed good in vivo activity using a Caenorhabditis elegans infection model. Study results suggested that they could be considered for in vivo treatments combined with established or novel antimicrobials (Scoffone et al., 2016).