Burkholderia cenocepacia is an opportunistic pathogen particularly dangerous for cystic fibrosis (CF) patients. It can cause a severe decline in CF lung function possibly developing into a life-threatening systemic infection known as cepacia syndrome. Antibiotic resistance and presence of numerous virulence determinants in the genome make B. cenocepacia extremely difficult to treat. Better understanding of its resistance profiles and mechanisms is crucial to improve management of these infections. Here we present the clinical distribution of B. cenocepacia described in the last six years and methods for identification and classification of epidemic strains. We also detail new antibiotics, clinical trials and alternative approaches reported in the literature in the last five years to tackle B. cenocepacia resistance issue. All together these findings point out the urgent need of new and alternative therapies to improve CF patients’ life expectancy.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder which leads to the secretion of a viscous mucus layer on the respiratory epithelium that facilitates colonization by various bacterial pathogens. The problem of drug resistance has been reported for all the species able to colonize the lung of CF patients, so alternative treatments are urgently needed. In this context, a valid approach is to investigate new natural and synthetic molecules for their ability to counteract alternative pathways, such as virulence regulating quorum sensing (QS). In this review we describe the pathogens most commonly associated with CF lung infections: Staphylococcus aureus, Pseudomonas aeruginosa, species of the Burkholderia cepacia complex and the emerging pathogens Stenotrophomonas maltophilia, Haemophilus influenzae and non-tuberculous Mycobacteria. For each bacterium, the QS system(s) and the molecules targeting the different components of this pathway are described. The amount of investigations published in the last five years clearly indicate the interest and the expectations on antivirulence therapy as an alternative to classical antibiotics.

Acinetobacter baumannii is considered one of the prioritized ESKAPE microorganisms for the research and development of novel treatments by the World Health Organization, especially because of its remarkable persistence and drug resistance. In this review, we describe how this can be acquired by the enzymatic degradation of antibiotics, target site modification, altered membrane permeability, multidrug efflux pumps, and their ability to form biofilms. Also, the evolution of drug resistance in A. baumannii, which is mainly driven by mobile genetic elements, is reported, with particular reference to plasmid-associated resistance, resistance islands, and insertion sequences. Finally, an overview of existing, new, and alternative therapies is provided.

Drug resistance represents a great concern among people with cystic fibrosis (CF), due to the recurrent and prolonged antibiotic therapy they should often undergo. Among Multi Drug Resistance (MDR) determinants, Resistance-Nodulation-cell Division (RND) efflux pumps have been reported as the main contributors, due to their ability to extrude a wide variety of molecules out of the bacterial cell. In this review, we summarize the principal RND efflux pump families described in CF pathogens, focusing on the main Gram-negative bacterial species (Pseudomonas aeruginosa, Burkholderia cenocepacia, Achromobacter xylosoxidans, Stenotrophomonas maltophilia) for which a predominant role of RND pumps has been associated to MDR phenotypes.

The Burkholderia cepacia complex comprises environmental and clinical Gram-negative bacteria that infect particularly debilitated people, such as those with cystic fibrosis. Their high level of antibiotic resistance makes empirical treatments often ineffective, increasing the risk of worst outcomes and the diffusion of multi-drug resistance. However, the discovery of new antibiotics is not trivial, so an alternative can be the use of vaccination. Here, the reverse vaccinology approach has been used to identify antigen candidates, obtaining a short-list of 24 proteins. The localization and different aspects of virulence were investigated for three of them—BCAL1524, BCAM0949, and BCAS0335. The three antigens were localized in the outer membrane vesicles confirming that they are surface exposed. We showed that BCAL1524, a collagen-like protein, promotes bacteria auto-aggregation and plays an important role in virulence, in the Galleria mellonella model. BCAM0949, an extracellular lipase, mediates piperacillin resistance, biofilm formation in Luria Bertani and artificial sputum medium, rhamnolipid production, and swimming motility; its predicted lipolytic activity was also experimentally confirmed. BCAS0335, a trimeric adhesin, promotes minocycline resistance, biofilm organization in LB, and virulence in G. mellonella. Their important role in virulence necessitates further investigations to shed light on the usefulness of these proteins as antigen candidates.

Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors.