New Antimicrobial Strategies to Treat Multi-Drug Resistant Infections Caused by Gram-Negatives in Cystic Fibrosis.

People with cystic fibrosis (CF) suffer from recurrent bacterial infections which induce inflammation, lung tissue damage and failure of the respiratory system. Prolonged exposure to combinatorial antibiotic therapies triggers the appearance of multi-drug resistant (MDR) bacteria. The development of alternative antimicrobial strategies may provide a way to mitigate antimicrobial resistance. Here we discuss different alternative approaches to the use of classic antibiotics: anti-virulence and anti-biofilm compounds which exert a low selective pressure; phage therapies that represent an alternative strategy with a high therapeutic potential; new methods helping antibiotics activity such as adjuvants; and antimicrobial peptides and nanoparticle formulations. Their mechanisms and in vitro and in vivo efficacy are described, in order to figure out a complete landscape of new alternative approaches to fight MDR Gram-negative CF pathogens.

Mechanochemical Studies on Coupling of Hydrazines and Hydrazine Amides with Phenolic and Furanyl Aldehydes-Hydrazones with Antileishmanial and Antibacterial Activities.

Hydrazone compounds represent an important area of research that includes, among others, synthetic approaches and biological studies. A series of 17 hydrazones have been synthesized by mechanochemical means. The fragments chosen were phenolic and furanyl aldehydes coupled with 12 heterocyclic hydrazines or hydrazinamides. All compounds can be obtained quantitatively when operating on a planetary ball mill and a maximum reaction time of 180 min (6 cycles of 30 min each). Complete spectroscopic analyses of hydrazones revealed eight compounds (3-5, 8-11, 16) present in one geometric form, six compounds (1, 2, 13-15) present in two isomeric forms, and three compounds (6, 7, 12) where one rotation is restricted giving rise to two different forms. The single crystal X-ray structure of one of the hydrazones bearing the isoniazid fragment (8) indicates a crystal lattice consisting of two symmetry-independent molecules with different geometries. All compounds obtained were tested for anti-infectious and antibacterial activities. Four compounds (1, 3, 5 and 8) showed good activity against Mycobacterium tuberculosis, and one (7) was very potent against Staphylococcus aureus. Most interesting, this series of compounds displayed very promising antileishmanial activity. Among all, compound 9 exhibited an IC50 value of 0.3 µM on the Leishmania donovani intramacrophage amastigote in vitro model and a good selectivity index, better than miltefosine, making it worth evaluating in vivo.

Identification by Reverse Vaccinology of Three Virulence Factors in Burkholderia cenocepacia That May Represent Ideal Vaccine Antigens.

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.

Bactericidal and Anti-Biofilm Activity of the FtsZ Inhibitor C109 against Acinetobacter baumannii.

In the last few years, Acinetobacter baumannii has ranked as a number one priority due to its Multi Drug Resistant phenotype. The different metabolic states, such as the one adopted when growing as biofilm, help the bacterium to resist a wide variety of compounds, placing the discovery of new molecules able to counteract this pathogen as a topic of utmost importance. In this context, bacterial cell division machinery and the conserved protein FtsZ are considered very interesting cellular targets. The benzothiadiazole compound C109 is able to inhibit bacterial growth and to block FtsZ GTPase and polymerization activities in Burkholderia cenocepacia, Pseudomonas aeruginosa, and Staphylococcus aureus. In this work, the activity of C109 was tested against a panel of antibiotic sensitive and resistant A. baumannii strains. Its ability to inhibit biofilm formation was explored, together with its activity against the A. baumannii FtsZ purified protein. Our results indicated that C109 has good MIC values against A. baumannii clinical isolates. Moreover, its antibiofilm activity makes it an interesting alternative treatment, effective against diverse metabolic states. Finally, its activity was confirmed against A. baumannii FtsZ.

CodY Is a Global Transcriptional Regulator Required for Virulence in Group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.

Methodological tools to study species of the genus Burkholderia.

Bacteria belonging to the Burkholderia genus are extremely versatile and diverse. They can be environmental isolates, opportunistic pathogens in cystic fibrosis, immunocompromised or chronic granulomatous disease patients, or cause disease in healthy people (e.g., Burkholderia pseudomallei) or animals (as in the case of Burkholderia mallei). Since the genus was separated from the Pseudomonas one in the 1990s, the methodological tools to study and characterize these bacteria are evolving fast. Here we reviewed the techniques used in the last few years to update the taxonomy of the genus, to study gene functions and regulations, to deepen the knowledge on the drug resistance which characterizes these bacteria, and to elucidate their mechanisms to establish infections. The availability of these tools significantly impacts the quality of research on Burkholderia and the choice of the most appropriated is fundamental for a precise characterization of the species of interest.Key points• Updated techniques to study the genus Burkholderia were reviewed.• Taxonomy, genomics, assays, and animal models were described.• A comprehensive overview on recent advances in Burkholderia studies was made.

Role of RND Efflux Pumps in Drug Resistance of Cystic Fibrosis Pathogens.

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.

Antistaphylococcal Activity of the FtsZ Inhibitor C109.

Staphylococcus aureus infections represent a great concern due to their versatility and involvement in different types of diseases. The shortage of available clinical options, especially to treat multiresistant strains, makes the discovery of new effective compounds essential. Here we describe the activity of the previously described cell division inhibitor C109 against methicillin-sensitive and -resistant S. aureus strains. Antibiofilm activity was assessed using microtiter plates, confocal microscopy, and in an in vitro biofilm wound model. The ability of C109 to block FtsZ GTPase activity and polymerization was tested in vitro. Altogether, the results show that the FtsZ inhibitor C109 has activity against a wide range of S. aureus strains and support its use as an antistaphylococcal compound.

Identification of a Novel Brevibacillus laterosporus Strain With Insecticidal Activity Against Aedes albopictus Larvae.

Bacterial species able to produce proteins that are toxic against insects have been discovered at the beginning of the last century. However, up to date only two of them have been used as pesticides in mosquito control strategies targeting larval breeding sites: Bacillus thuringensis var. israelensis and Lysinibacillus sphaericus. Aiming to expand the arsenal of biopesticides, bacterial cultures from 44 soil samples were assayed for their ability to kill larvae of Aedes albopictus. A method to select, grow and test the larvicidal capability of spore-forming bacteria from each soil sample was developed. This allowed identifying 13 soil samples containing strains capable of killing Ae. albopictus larvae. Among the active isolates, one strain with high toxicity was identified as Brevibacillus laterosporus by 16S rRNA gene sequencing and by morphological characterization using transmission electron microscopy. The new isolate showed a larvicidal activity significantly higher than the B. laterosporus LMG 15441 reference strain. Its genome was phylogenomically characterized and compared to the available Brevibacillus genomes. Thus, the new isolate can be considered as a candidate adjuvant to biopesticides formulations that would help preventing the insurgence of resistance.

Molecular Characterization of the Burkholderia cenocepacia dcw Operon and FtsZ Interactors as New Targets for Novel Antimicrobial Design.

The worldwide spread of antimicrobial resistance highlights the need of new druggable cellular targets. The increasing knowledge of bacterial cell division suggested the potentiality of this pathway as a pool of alternative drug targets, mainly based on the essentiality of these proteins, as well as on the divergence from their eukaryotic counterparts. People suffering from cystic fibrosis are particularly challenged by the lack of antibiotic alternatives. Among the opportunistic pathogens that colonize the lungs of these patients, Burkholderia cenocepacia is a well-known multi-drug resistant bacterium, particularly difficult to treat. Here we describe the organization of its division cell wall (dcw) cluster: we found that 15 genes of the dcw operon can be transcribed as a polycistronic mRNA from mraZ to ftsZ and that its transcription is under the control of a strong promoter regulated by MraZ. B. cenocepacia J2315 FtsZ was also shown to interact with the other components of the divisome machinery, with a few differences respect to other bacteria, such as the direct interaction with FtsQ. Using an in vitro sedimentation assay, we validated the role of SulA as FtsZ inhibitor, and the roles of FtsA and ZipA as tethers of FtsZ polymers. Together our results pave the way for future antimicrobial design based on the divisome as pool of antibiotic cellular targets.

Bacillus subtilis as a host for mosquitocidal toxins production.

Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector-borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well-known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG-inducible, auto-inducible or toxin gene-specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second-instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.

Chemical, Metabolic, and Cellular Characterization of a FtsZ Inhibitor Effective Against Burkholderia cenocepacia.

There is an urgent need for new antimicrobials to treat the opportunistic Gram-negative Burkholderia cenocepacia, which represents a problematic challenge for cystic fibrosis patients. Recently, a benzothiadiazole derivative, C109, was shown to be effective against the infections caused by B. cenocepacia and other Gram-negative and-positive bacteria. C109 has a promising cellular target, the cell division protein FtsZ, and a recently developed PEGylated formulation make it an attractive molecule to counteract Burkholderia infections. However, the ability of efflux pumps to extrude it out of the cell represents a limitation for its use. Here, more than 50 derivatives of C109 were synthesized and tested against Gram-negative species and the Gram-positive Staphylococcus aureus. In addition, their activity was evaluated on the purified FtsZ protein. The chemical, metabolic and cellular stability of C109 has been assayed using different biological systems, including quantitative single-cell imaging. However, no further improvement on C109 was achieved, and the role of efflux in resistance was further confirmed. Also, a novel nitroreductase that can inactivate the compound was characterized, but it does not appear to play a role in natural resistance. All these data allowed a deep characterization of the compound, which will contribute to a further improvement of its properties.

The cell division protein FtsZ as a cellular target to hit cystic fibrosis pathogens.

Cystic fibrosis is a rare genetic disease characterized by the production of dehydrated mucus in the lung able to trap bacteria and rendering their proliferation particularly dangerous, thus leading to chronic infections. Among these bacteria, Staphylococcus aureus and Pseudomonas aeruginosa play a major role while, within emerging pathogens, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Burkholderia cepacia complex species, as well as non-tuberculous mycobacteria are listed. Since a common feature of these bacteria is the high level of drug resistance, cell division, and in particular FtsZ, has been explored as a novel therapeutic target for the design of new molecules with antibacterial properties. This review summarizes and provides insight into recent advances in the discovery of compounds targeting FtsZ: the majority of them exhibit anti-staphylococcal activity, while a few were directed against the cystic fibrosis Gram negative pathogens.

PEGylated mucus-penetrating nanocrystals for lung delivery of a new FtsZ inhibitor against Burkholderia cenocepacia infection.

C109 is a potent but poorly soluble FtsZ inhibitor displaying promising activity against Burkholderia cenocepacia, a high-risk pathogen for cystic fibrosis (CF) sufferers. To harness C109 for inhalation, we developed nanocrystal-embedded dry powders for inhalation suspension consisting in C109 nanocrystals stabilized with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) embedded in hydroxypropyl-ß-cyclodextrin (CD). The powders could be safely re-dispersed in water for in vitro aerosolization. Owing to the presence of a PEG shell, the rod shape and the peculiar aspect ratio, C109 nanocrystals were able to diffuse through artificial CF mucus. The promising technological features were completed by encouraging in vitro/in vivo effects. The formulations displayed no toxicity towards human bronchial epithelial cells and were active against planktonic and sessile B. cenocepacia strains. The efficacy of C109 nanosuspensions in combination with piperacillin was confirmed in a Galleria mellonella infection model, strengthening their potential for combined therapy of B. cenocepacia lung infections.

Quorum Sensing as Antivirulence Target in Cystic Fibrosis Pathogens.

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.

Antimicrobial activity of six essential oils against Burkholderia cepacia complex: insights into mechanism(s) of action.

AIM: To investigate the activity and mechanisms of action of six essential oils (EOs) against Burkholderia cepacia complex, opportunistic human pathogens highly resistant to antibiotics. MATERIALS & METHODS: Minimal inhibitory concentration of EOs alone, plus antibiotics or efflux pump inhibitors was determined. RESULTS: Origanum vulgare, Thymus vulgaris and Eugenia caryophyllata EOs resulted to be more active than the other EOs. EOs did not enhance antibiotic activity against the model strain B. cenocepacia J2315. EOs resulted more active in the presence of an efflux pump inhibitor acting on Resistance-Nodulation Cell Division efflux pumps and against B. cenocepacia J2315 Resistance-Nodulation Cell Division knocked-out mutants. CONCLUSION: EOs showed intracellular mechanisms of action and, thus, the efflux pumps inhibitor addition could boost their activity.

Subfunctionalization influences the expansion of bacterial multidrug antibiotic resistance.

BACKGROUND: Antibiotic resistance is a major problem for human health. Multidrug resistance efflux pumps, especially those of the Resistance-Nodulation-Cell Division (RND) family, are major contributors to high-level antibiotic resistance in Gram-negative bacteria. Most bacterial genomes contain several copies of the different classes of multidrug resistance efflux pumps. Gene duplication and gain of function by the duplicate copies of multidrug resistance efflux pump genes plays a key role in the expansion and diversification of drug-resistance mechanisms. RESULTS: We used two members of the Burkholderia RND superfamily as models to understand how duplication events affect the antibiotic resistance of these strains. First, we analyzed the conservation and distribution of these two RND systems and their regulators across the Burkholderia genus. Through genetic manipulations, we identified both the exact substrate range of these transporters and their eventual interchangeability. We also performed a directed evolution experiment, combined with next generation sequencing, to evaluate the role of antibiotics in the activation of the expression of these systems. Together, our results indicate that the first step to diversify the functions of these pumps arises from changes in their regulation (subfunctionalization) instead of functional mutations. Further, these pumps could rewire their regulation to respond to antibiotics, thus maintaining high genomic plasticity. CONCLUSIONS: Studying the regulatory network that controls the expression of the RND pumps will help understand and eventually control the development and expansion of drug resistance.

Biochemical Characterization of Glutamate Racemase-A New Candidate Drug Target against Burkholderia cenocepacia Infections.

The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 µM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia.