Bacterial biofilms are surface-attached communities consisting of non-replicating persister cells encased within an extracellular matrix of biomolecules. Unlike bacteria that have acquired resistance to antibiotics, persister cells enable biofilms to demonstrate innate tolerance toward all classes of conventional antibiotic therapies. It is estimated that 50-80% of bacterial infections are biofilm associated, which is considered the underlying cause of chronic and recurring infections. Herein, we report a modular three-step synthetic route to new halogenated phenazine (HP) analogues from diverse aniline and nitroarene building blocks. The HPs were evaluated for antibacterial and biofilm-killing properties against a panel of lab strains and multidrug-resistant clinical isolates. Several HPs demonstrated potent antibacterial (MIC ≤ 0.39 μM) and biofilm-eradicating activities (MBEC < 10 μM) with 6,8-ditrifluoromethyl-HP 15 demonstrated remarkable biofilm-killing potencies (MBEC = 0.15-1.17 μM) against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus clinical isolates. Confocal microscopy showed HP 15 induced significant losses in the polysaccharide matrix in MRSA biofilms. In addition, HP 15 showed increased antibacterial activities against dormant Mycobacterium tuberculosis (Mtb, MIC = 1.35 μM) when compared to replicating Mtb (MIC = 3.69 μM). Overall, this new modular route has enabled rapid access to an interesting series of potent halogenated phenazine analogues to explore their unique antibacterial and biofilm-killing properties.

Klebsiella pneumoniae infections have become a growing threat for human health. The lack of understanding of the relationship between antibiotic resistance, mucoviscosity, and biofilm formation impedes our abilities to effectively predict K. pneumoniae infection outcomes. The Multidrug-Resistant Organism Repository and Surveillance Network offers a unique opportunity into the genetic and phenotypic variabilities in the K. pneumoniae isolates. To this end, we compared the genetic profiles of these isolates with the phenotypic biofilm formation, percent mucoviscosity, and growth rates. There was a significant phenotype-genotype correlation with decreased biofilm formation and an insertion sequence in the transcriptional activator of the type III fimbrial system. Interestingly, the most mucoid strains in the populations were lacking the genetic element regulating the mucoid phenotype and three of these isolates were able to form robust biofilms. The combination of phenotypic, genomic, and image analyses revealed an intricate relation between growth, mucoviscosity and specific virulence-associated genetic determinants.

ABSTRACTThe DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB, encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae ΔyqjA ΔyghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5–2 mM Ca2+ or Mg2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family.IMPORTANCEKlebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of ΔyqjA and ΔyghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence.

Hypervirulent Klebsiella pneumoniae is known for its increased extracellular polysaccharide production. Biofilm matrices of hypervirulent K. pneumoniae have increased polysaccharide abundance and are uniquely susceptible to disruption by peptide bactenecin 7 (bac7 (1-35)). Here, using confocal microscopy, we show that polysaccharides within the biofilm matrix collapse following bac7 (1-35) treatment. This collapse led to the release of cells from the biofilm, which were then killed by the peptide. Characterization of truncated peptide analogs revealed that their interactions with polysaccharide were responsible for the biofilm matrix changes that accompany bac7 (1-35) treatment. Ultraviolet photodissociation mass spectrometry with the parental peptide or a truncated analog bac7 (10-35) reveal the important regions for bac7 (1-35) complexing with polysaccharides. Finally, we tested bac7 (1-35) using a murine skin abscess model and observed a significant decrease in the bacterial burden. These findings unveil the potential of bac7 (1-35) polysaccharide interactions to collapse K. pneumoniae biofilms.

The rise in antimicrobial resistance and the decline in new antibiotics has created a great need for novel approaches to treat drug resistant bacterial infections. Increasing the burden of antimicrobial resistance, bacterial virulence factors allow for survival within the host, where they can evade host killing and antimicrobial therapy within their intracellular niches. Repurposing host directed therapeutics has great potential for adjuvants to allow for more effective bacterial killing by the host and antimicrobials. To this end, phosphoinositide 3-kinase inhibitors are FDA approved for cancer therapy, but also have potential to eliminate intracellular survival of pathogens. This review describes the PI3K pathway and its potential as an adjuvant target to treat bacterial infections more effectively.

Klebsiella pneumoniae biofilm formation is associated with chronic and relapsing infections. Scanning electron microscopy (SEM) is a powerful tool for characterizing biofilm structure and studying their formation. Reliable visualization of biofilm structure requires careful sample preservation, otherwise there may be loss of non-covalent interactions that are susceptible to damage during the dehydration and washing preparation steps. However, no standard procedure has been adopted in the literature to fix K. pneumoniae biofilm for scanning electron microscopy studies. This lack of standardization makes it challenging to compare results between studies and determine the degree to which native structures have been preserved. To advance this critical area of study, we investigated different scanning electron microscopy fixation methods for K. pneumoniae biofilm preservation. Our study reveals the impact preparation steps can have on retaining in biofilm architecture observed using scanning electron microscopy. Using fixation methods developed through our studies, we show that although species that overproduce capsular extracellular polysaccharides produced more robust biofilms, K. pneumoniae can form a developed biofilm in the absence of capsular polysaccharides.

Klebsiella pneumoniae produces a thick capsule layer composed of extracellular polysaccharides protecting the bacterial cells from clearance by innate host immunity during infection. Here we characterize the interactions of a structurally diverse set of host defense peptides with K. pneumoniae extracellular polysaccharides. Remarkably, we found that all host defense peptides were active against a diverse set of K. pneumoniae strains, including hypermucoviscous strains with extensive capsule production, and aggregated with extracted capsule. Interestingly, the polyproline peptide bac7 (1-35), was the most potent antimicrobial and induced the most capsule aggregation. In addition to capsule aggregation, we found that bac7 (1-35) could also disrupt pre-formed hypermucoviscous K. pneumoniae biofilm. Further analysis using scanning electron microscopy revealed the biofilm matrix of a hypermucoviscous strain is removed by bac7 (1-35) exposing associated bacterial cells. This is the first description of a host defense peptide interacting with capsular and biofilm extracellular polysaccharides to expose cells from a K. pneumoniae biofilm matrix and suggests that features of polyproline peptides may be uniquely suited for extracellular polysaccharide interactions. IMPORTANCE Klebsiella pneumoniae bacterial infections are a major threat to human health as mortality rates are steadily on the rise. A defining characteristic of K. pneumoniae is the robust polysaccharide capsule that aids in resistance to the human immune system. We have previously discovered that a synthetic peptide could aggregate with capsule polysaccharides and disrupt the capsule of K. pneumoniae. Here we describe that host defense peptides also aggregate with capsule produced from hypermucoviscous K. pneumoniae, revealing this mechanism is shared by natural peptides. We found the polyproline peptide bac7 (1-35) had the greatest antimicrobial activity and caused the most capsule aggregation. Interestingly, bac7 (1-35) also removed the biofilm matrix of hypermucoviscous K. pneumoniae exposing the associated bacterial cells. This is the first description of a polyproline peptide interacting with capsular and biofilm polysaccharides to expose cells from a K. pneumoniae biofilm matrix.

The rapid development and spread of antibiotic resistance necessitate the development of novel strategies for antibiotic discovery. Symbah-1, a synthetic peptide antibiotic, was identified in a high-throughput antibacterial screen of random peptide sequences. Symbah-1 functions through membrane disruption and contains broad spectrum bactericidal activity against several drug-resistant pathogens. Circular dichroism and high-resolution mass spectrometry indicate symbah-1 has a β-hairpin structure induced by lipopolysaccharide and is cyclized via an intramolecular disulfide bond. Together these data classify symbah-1 as an uncommon synthetic member of the β-hairpin antimicrobial peptide class. Symbah-1 displays low hemolysis but loses activity in human serum. Characterization of a symbah-1 peptide library identified two variants with increased serum activity and protease resistance. The method of discovery and subsequent characterization of symbah-1 suggests large synthetic peptide libraries bias toward macrocyclic β-hairpin structure could be designed and screened to rapidly expand and better understand this rare peptide antibiotic class.

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae. Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

We have previously reported the use of combinatorial chemistry to identify broad-spectrum antibacterial agents. Herein, we extend our analysis of this technology toward the discovery of anti-resistance molecules, focusing on efflux pump inhibitors. Using high-throughput screening against multi-drug resistant Pseudomonas aeruginosa, we identified a polyamine scaffold that demonstrated strong efflux pump inhibition without possessing antibacterial effects. We determined that these molecules were most effective with an amine functionality at R1 and benzene functionalities at R2 and R3. From a library of 188 compounds, we studied the properties of 5 lead agents in detail, observing a fivefold to eightfold decrease in the 90% effective concentration of tetracycline, chloramphenicol, and aztreonam toward P. aeruginosa isolates. Additionally, we determined that our molecules were not only active toward P. aeruginosa, but toward Acinetobacter baumannii and Staphylococcus aureus as well. The specificity of our molecules to efflux pump inhibition was confirmed using ethidium bromide accumulation assays, and in studies with strains that displayed varying abilities in their efflux potential. When assessing off target effects we observed no disruption of bacterial membrane polarity, no general toxicity toward mammalian cells, and no inhibition of calcium channel activity in human kidney cells. Finally, combination treatment with our lead agents engendered a marked increase in the bactericidal capacity of tetracycline, and significantly decreased viability within P. aeruginosa biofilms. As such, we report a unique polyamine scaffold that has strong potential for the future development of novel and broadly active efflux pump inhibitors targeting multi-drug resistant bacterial infections.

There is an acute need for new and effective agents to treat infectious diseases. We conducted a screening program to assess the potential of mangrove-derived endophytic fungi as a source of new antibiotics. Fungi cultured in the presence and absence of small molecule epigenetic modulators were screened against Mycobacterium tuberculosis and the ESKAPE panel of bacterial pathogens, as well as two eukaryotic infective agents, Leishmania donovani and Naegleria fowleri. By comparison of bioactivity data among treatments and targets, trends became evident, such as the result that more than 60% of active extracts were revealed to be selective to a single target. Validating the technique of using small molecules to dysregulate secondary metabolite production pathways, nearly half (44%) of those fungi producing active extracts only did so following histone deacetylase inhibitory (HDACi) or DNA methyltransferase inhibitory (DNMTi) treatment.

We previously reported a series of N2,N4-disubstituted quinazoline-2,4-diamines as dihydrofolate reductase inhibitors with potent in vitro and in vivo antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. In this work, we extended our previous study to the Gram-negative pathogen Acinetobacter baumannii We determined that optimized N2,N4-disubstituted quinazoline-2,4-diamines are strongly antibacterial against multidrug-resistant A. baumannii strains when the 6-position is replaced with a halide or an alkyl substituent. Such agents display potent antibacterial activity, with MICs as low as 0.5 μM, while proving to be strongly bactericidal. Interestingly, these compounds also possess the potential for antibiofilm activity, eradicating 90% of cells within a biofilm at or near MICs. Using serial passage assays, we observed a limited capacity for the development of resistance toward these molecules (4-fold increase in MIC) compared to existing folic acid synthesis inhibitors, such as trimethoprim (64-fold increase) and sulfamethoxazole (128-fold increase). We also identified limited toxicity toward human cells, with 50% lethal doses (LD50s) of ≤23 μM for lead agents 4 and 5. Finally, we demonstrated that our lead agents have excellent in vivo efficacy, with lead agent 5 proving more efficacious than tigecycline in a murine model of A. baumannii infection (90% survival versus 66%), despite being used at a lower dose (2 versus 30 mg kg-1). Together, our results demonstrate that N2,N4-disubstituted quinazoline-2,4-diamines have strong antimicrobial and antibiofilm activities against both Gram-positive organisms and Gram-negative pathogens, suggesting strong potential for their development as antibacterial agents.

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.

Pathogen resistance, appearance of new diseases, and lack of cure for neglected diseases are several motivations driving the search for new treatments [1, 2]. The chemical survival mechanisms used by fungi offer a potential axis for research to find new drugs. The production of metabolites being directly related to the expression of genetic information encoded in DNA, regulating this resource will enable producing of new molecules for the purpose of drug discovery progress. Epigenetic regulation is a key mechanism to orchestrate the expression or suppression of gene activity [3]. Manipulating these mechanisms offers new opportunities to express down-regulated secondary metabolite genes and has the potential to generate new potent and novel metabolites [3].

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are responsible for the majority of nosocomial infections and are resistant to most antibacterial agents [1]. The continuous need for new antibiotics has invigorated drug discovery research, leading us to investigate niche biological sources for new chemodiversity. We focus on mangrove-associated microorganisms which have been found to be a great source of chemically diverse and biologically active metabolites [2]. In particular, we have been interested in the isolation and characterisation of secondary metabolites from Floridian mangrove-associated fungi as well as their biological activity against the ESKAPE pathogens. Moreover, we are interested in enhancing the bioactivity of these secondary metabolites by epigenetic regulation of the fungi using histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors [3].

In an effort to develop novel antimicrobial agents against drug-resistant bacterial infections, 5,6-dihydroimidazo[2,1-b]thiazole compounds were synthesized and tested for their antimicrobial activity. Eight compounds comprised by two sub-scaffolds were identified as hits against methicillin-resistant Staphylococcus aureus (MRSA). These hits were modified at 6-position by replacing (S)-6 to (R)-6 configuration and the (R)-isomers increased their antimicrobial activities by two-fold. The most active compound showed a MIC90 value of 3.7μg/mL against MRSA in a standard microdilution bacterial growth inhibitory assay. This compound protected wax moth worms against MRSA at a dose of 5× MIC using a worm infectious model. This compound also exhibited inhibition of DNA gyrase activity in a DNA gyrase supercoil assay, suggesting the 5,6-dihydroimidazo[2,1-b]thiazoles may target DNA gyrase for the antimicrobial action.

In this issue of Cell Chemical Biology, Choby et al. (2016) use a small molecule inhibitor active against fermenting S. aureus to unravel a unique connection between virulence factor production and central metabolism. In so doing, the authors uncover Fe-S cluster assembly proteins as a novel antibacterial target, and deliver a first-in-class scaffold for optimization against anaerobically growing cells.

Treating bacterial infections can be difficult due to innate or acquired resistance mechanisms, and the formation of biofilms. Cyclic lipopeptides derived from fusaricidin/LI-F natural products represent particularly attractive candidates for the development of new antibacterial and antibiofilm agents, with the potential to meet the challenge of bacterial resistance to antibiotics. A positional-scanning combinatorial approach was used to identify the amino acid residues responsible for driving antibacterial activity, and increase the potency of these cyclic lipopeptides. Screening against the antibiotic resistant ESKAPE pathogens revealed the importance of hydrophobic as well as positively charged amino acid residues for activity of this class of peptides. The improvement in potency was especially evident against bacterial biofilms, since the lead cyclic lipopeptide showed promising in vitro and in vivo anti-biofilm activity at the concentration far below its respective MICs. Importantly, structural changes resulting in a more hydrophobic and positively charged analog did not lead to an increase in toxicity toward human cells.

A new rearranged spongian diterpene, darwinolide, has been isolated from the Antarctic Dendroceratid sponge Dendrilla membranosa. Characterized on the basis of spectroscopic and crystallographic analysis, the central seven-membered ring is hypothesized to originate from a ring-expansion of a spongian precursor. Darwinolide displays 4-fold selectivity against the biofilm phase of methicillin-resistant Staphylococcus aureus compared to the planktonic phase and may provide a scaffold for the development of therapeutics for this difficult to treat infection.

Microbial natural products have historically played an important role in both antibacterial and antiparasitic drug discovery. However, with emerging drug resistance as a global threat to nearly all current antimicrobial treatments on the market, new discovery efforts are needed with increasing urgency. Mangrove endophytic fungi, known as a source of great biological and chemical diversity, were investigated in a large epigenetics based screening program. Cultured under control and epigenetically modified conditions, isolates were extracted and submitted for biological assay against the ESKAPE pathogens, the tropical disease causing parasite Leishmania donovani, and the meningoencephalitis causing amoeba Naeglaria fowlerii. The chemical investigation of one active organism identified in this way yielded a suite of new antimicrobial compounds. Herein, the structure elucidation and biological profiles of these new compounds will be discussed.