Polycationic Glycopolymer Demonstrates Activity Against Persisters and Biofilms of Non-tuberculosis Mycobacteria Cystic Fibrosis Clinical Isolates in vitro.

Non-tuberculosis Mycobacterium (NTM) is a group of opportunistic pathogens associated with pulmonary infections that are difficult to diagnose and treat. Standard treatment typically consists of prolonged combination antibiotic therapy. Antibiotic resistance and the role of biofilms in pathogen communities, such as NTM persister cells, is an important unmet challenge that leads to increased toxicity, frequent relapse, poor clinical management, and an extended treatment period. Infection recurrence and relapse are not uncommon among individuals with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD), where thick mucus supports bacterial biofilm production and impairs mucociliary clearance. The study evaluates a membrane-active cationic glycopolymer [poly (acetyl, arginyl) glucosamine (PAAG)] being developed to support the safe and effective treatment of NTM biofilm infections. PAAG shows antibacterial activity against a wide range of pathogenic bacteria at concentrations non-toxic to human epithelial cells. Time-kill curves demonstrated PAAG's rapid bactericidal potential at concentrations as low as 1X MIC against all NTM strains tested and compared to the standard of care. PAAG treatment prevents persister formation and eradicates antibiotic-induced persister cells in planktonic NTM cultures below the limit of detection (10 colony-forming unit (CFU)/ml). Further, PAAG showed the ability to penetrate and disperse NTM biofilms formed by both rapidly and slowly growing strains, significantly reducing the biofilm biomass (p < 0.0001) compared to the untreated NTM biofilms. Microscopical examination confirmed PAAG's ability to disrupt and disperse mycobacterial biofilms. A single PAAG treatment resulted in up to a 25-fold reduction in live-labeled NTM and a 78% reduction in biofilm thickness. Similar to other polycationic molecules, PAAG's bactericidal and antibiofilm activities employ rapid permeabilization of the outer membrane of the NTM strains, and subsequently, reduce the membrane potential even at concentrations as low as 50 µg/ml (p < 0.001). The outcomes of these in vitro analyses suggest the importance of this polycationic glycopolymer, PAAG, as a potential therapeutic agent for opportunistic NTM infections.

A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

In Vitro Activity of a Novel Glycopolymer against Biofilms of Burkholderia cepacia Complex Cystic Fibrosis Clinical Isolates.

Burkholderia cepacia complex (Bcc) lung infections in cystic fibrosis (CF) patients are often associated with a steady decline in lung function and death. The formation of biofilms and inherent multidrug resistance are virulence factors associated with Bcc infection and contribute to increased risk of mortality in CF patients. New therapeutic strategies targeting bacterial biofilms are anticipated to enhance antibiotic penetration and facilitate resolution of infection. Poly (acetyl, arginyl) glucosamine (PAAG) is a cationic glycopolymer therapeutic being developed to directly target biofilm integrity. In this study, 13 isolates from 7 species were examined, including Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia gladioli, Burkholderia dolosa, Burkholderia vietnamiensis, and B. cepacia These isolates were selected for their resistance to standard clinical antibiotics and their ability to form biofilms in vitro Biofilm biomass was quantitated using static tissue culture plate (TCP) biofilm methods and a minimum biofilm eradication concentration (MBEC) assay. Confocal laser scanning microscopy (CLSM) visualized biofilm removal by PAAG during treatment. Both TCP and MBEC methods demonstrated a significant dose-dependent relationship with regard to biofilm removal by 50 to 200 µg/ml PAAG following a 1-h treatment (P < 0.01). A significant reduction in biofilm thickness was observed following a 10-min treatment of Bcc biofilms with PAAG compared to that with vehicle control (P < 0.001) in TCP, MBEC, and CLSM analyses. PAAG also rapidly permeabilizes bacteria within the first 10 min of treatment. Glycopolymers, such as PAAG, are a new class of large-molecule therapeutics that support the treatment of recalcitrant Bcc biofilm.

Novel Glycopolymer Eradicates Antibiotic- and CCCP-Induced Persister Cells in Pseudomonas aeruginosa.

Antibiotic treatments often fail to completely eradicate a bacterial infection, leaving behind an antibiotic-tolerant subpopulation of intact bacterial cells called persisters. Persisters are considered a major cause for treatment failure and are thought to greatly contribute to the recalcitrance of chronic infections. Pseudomonas aeruginosa infections are commonly associated with elevated levels of drug-tolerant persister cells, posing a serious threat to human health. This study represents the first time a novel large molecule polycationic glycopolymer, poly (acetyl, arginyl) glucosamine (PAAG), has been evaluated against antibiotic and carbonyl cyanide m-chlorophenylhydrazone induced P. aeruginosa persisters. PAAG eliminated eliminated persisters at concentrations that show no significant cytotoxicity on human lung epithelial cells. PAAG demonstrated rapid bactericidal activity against both forms of induced P. aeruginosa persister cells resulting in complete eradication of the in vitro persister cells within 24 h of treatment. PAAG demonstrated greater efficacy against persisters in vitro than antibiotics currently being used to treat persistent chronic infections such as tobramycin, colistin, azithromycin, aztreonam, and clarithromycin. PAAG caused rapid permeabilization of the cell membrane and caused significant membrane depolarization in persister cells. PAAG efficacy against these bacterial subpopulations suggests it may have substantial therapeutic potential for eliminating recurrent P. aeruginosa infections.

In Vitro activity of novel glycopolymer against clinical isolates of multidrug-resistant Staphylococcus aureus.

The incidence of multidrug-resistant (MDR) organisms, including methicillin-resistant Staphylococcus aureus (MRSA), is a serious threat to public health. Progress in developing new therapeutics is being outpaced by antibiotic resistance development, and alternative agents that rapidly permeabilize bacteria hold tremendous potential for treating MDR infections. A new class of glycopolymers includes polycationic poly-N (acetyl, arginyl) glucosamine (PAAG) is under development as an alternative to traditional antibiotic strategies to treat MRSA infections. This study demonstrates the antibacterial activity of PAAG against clinical isolates of methicillin and mupirocin-resistant Staphylococcus aureus. Multidrug-resistant S. aureus was rapidly killed by PAAG, which completely eradicated 88% (15/17) of all tested strains (6-log reduction in CFU) in ≤ 12-hours at doses that are non-toxic to mammalian cells. PAAG also sensitized all the clinical MRSA strains (17/17) to oxacillin as demonstrated by the observed reduction in the oxacillin MIC to below the antibiotic resistance breakpoint. The effect of PAAG and standard antibiotics including vancomycin, oxacillin, mupirocin and bacitracin on MRSA permeability was studied by measuring propidium iodide (PI) uptake by bacterial cells. Antimicrobial resistance studies showed that S. aureus developed resistance to PAAG at a rate slower than to mupirocin but similar to bacitracin. PAAG was observed to resensitize drug-resistant S. aureus strains sampled from passage 13 and 20 of the multi-passage resistance study, reducing MICs of mupirocin and bacitracin below their clinical sensitivity breakpoints. This class of bacterial permeabilizing glycopolymers may provide a new tool in the battle against multidrug-resistant bacteria.

Novel glycopolymer sensitizes Burkholderia cepacia complex isolates from cystic fibrosis patients to tobramycin and meropenem.

Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 µg/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (≤4 µg/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

Characterization of an extended-spectrum beta-lactamase Enterobacter hormaechei nosocomial outbreak, and other Enterobacter hormaechei misidentified as Cronobacter (Enterobacter) sakazakii.

Enterobacter hormaechei is a Gram-negative bacterium within the Enterobacter cloacae complex, and has been shown to be of clinical significance by causing nosocomial infections, including sepsis. Ent. hormaechei is spread via horizontal transfer and is often associated with extended-spectrum beta-lactamase production, which increases the challenges associated with treatment by limiting therapeutic options. This report considers 10 strains of Ent. hormaechei (identified by 16S rDNA sequencing) that had originally been identified by phenotyping as Cronobacter (Enterobacter) sakazakii. Seven strains were from different neonates during a nosocomial outbreak in a California hospital. PFGE analysis revealed a clonal relationship among six of the seven isolates and therefore a previously unrecognized Ent. hormaechei outbreak had occurred over a three-month period. Antibiotic-resistance profiles were determined and extended-spectrum beta-lactamase activity was detected. The association of the organism with powdered infant formula, neonatal hosts and Cr. sakazakii suggested that the virulence of these organisms may be similar. Virulence traits were tested and all strains were shown to invade both gut epithelial (Caco-2) and blood-brain barrier endothelial cells (rBCEC4), and to persist in macrophages (U937). Due to misidentification we suggest that Ent. hormaechei may be an under-reported cause of bacterial infection, especially in neonates. Also, its isolation from various sources, including powdered infant milk formula, makes it a cause for concern and merits further investigation.

Enterobacter sakazakii invades brain capillary endothelial cells, persists in human macrophages influencing cytokine secretion and induces severe brain pathology in the neonatal rat.

Enterobacter sakazakii is an opportunistic pathogen associated with contaminated powdered infant formula and a rare cause of Gram-negative sepsis that can develop into meningitis and brain abscess formation in neonates. Bacterial pathogenesis remains to be fully elucidated. In this study, the host inflammatory response was evaluated following intracranial inoculation of Ent. sakazakii into infant rats. Infiltrating macrophages and neutrophils composed multiple inflammatory foci and contained phagocytosed bacteria. Several genotypically distinct Ent. sakazakii strains (16S cluster groups 1-4) were shown to invade rat capillary endothelial brain cells (rBCEC4) in vitro. Further, the persistence of Ent. sakazakii in macrophages varied between strains. The presence of putative sod genes and SOD activity may influence the survival of acidic conditions and macrophage oxidase and contribute to Ent. sakazakii intracellular persistence. The influence of macrophage uptake of Ent. sakazakii on immunoregulatory cytokine expression was assessed by ELISA. This demonstrated that the IL-10/IL-12 ratio is high after 24 h. This is suggestive of a type 2 immune response which is inefficient in fighting intracellular infections. These findings may help explain how the diversity in virulence traits among Ent. sakazakii isolates and an unsuccessful immune response contribute to the opportunistic nature of this infection.

fliP influences Citrobacter koseri macrophage uptake, cytokine expression and brain abscess formation in the neonatal rat.

Citrobacter koseri causes neonatal meningitis frequently complicated with multiple brain abscesses. During C. koseri central nervous system infection in the neonatal rat model, previous studies have documented many bacteria-filled macrophages within the neonatal rat brain and abscesses. Previous studies have also shown that C. koseri is taken up by, survives phagolysosomal fusion and replicates in macrophages in vitro and in vivo. In this study, in order to elucidate genetic and cellular factors contributing to C. koseri persistence, a combinatory technique of differential fluorescence induction and transposon mutagenesis was employed to isolate C. koseri genes induced while inside macrophages. Several banks of mutants were subjected to a series of enrichments to select for gfp : : transposon fusion into genes that are turned off in vitro but expressed when intracellular within macrophages. Further screening identified several mutants attenuated in their recovery from macrophages compared with the wild-type. A mutation within an Escherichia coli fliP homologue caused significant attenuation in uptake and hypervirulence in vivo, resulting in death within 24 h. Furthermore, analysis of the immunoregulatory interleukin (IL)-10/IL-12 cytokine response during infection suggested that C. koseri fliP expression may alter this response. A better understanding of the bacteria-macrophage interaction at the molecular level and its contribution to brain abscess formation will assist in developing preventative and therapeutic strategies.

TraJ-dependent Escherichia coli K1 interactions with professional phagocytes are important for early systemic dissemination of infection in the neonatal rat.

Escherichia coli is a major cause of neonatal bacterial sepsis and meningitis. We recently identified a gene, traJ, which contributes to the ability of E. coli K1 to penetrate the blood-brain barrier in the neonatal rat. Because very little is known regarding the most critical step in disease progression, translocation to the gut and dissemination to the lymphoid tissues after a natural route of infection, we assessed the ability of a traJ mutant to cause systemic disease in the neonatal rat. Our studies determined that the traJ mutant is significantly less virulent than the wild type in the neonatal rat due to a decreased ability to disseminate from the mesenteric lymph nodes to the deeper tissues of the liver and spleen and to the blood during the early stages of systemic disease. Histopathologic studies determined that although significantly less or no mutant bacteria were recovered from the spleen and livers of infected neonatal rats, the inflammatory response was considerably greater than that in wild-type-colonized tissues. In vitro studies revealed that macrophages internalize the traJ mutant less frequently than they do the wild type and by a morphologically distinct process. Furthermore, we determined that tissue macrophages and dendritic cells within the liver and spleen are the major cellular targets of E. coli K1 and that TraJ significantly contributes to the predominantly intracellular nature of E. coli K1 within these professional phagocytes exclusively during the early stages of systemic disease. These data indicate that, contrary to earlier indications, E. coli K1 resides within professional phagocytes, and this is essential for the efficient progression of systemic disease.

Citrobacter koseri brain abscess in the neonatal rat: survival and replication within human and rat macrophages.

A unique feature of Citrobacter koseri is the extremely high propensity to initiate brain abscesses during neonatal meningitis. Previous clinical reports and studies on infant rats have documented many Citrobacter-filled macrophages within the ventricles and brain abscesses. It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by which C. koseri subverts the host response and elicits chronic infection, resulting in brain abscess formation. In this study, we showed that C. koseri causes meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation. Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-term C. koseri infection. To better understand C. koseri pathogenesis, we have characterized the interactions of C. koseri with human macrophages. We found that C. koseri survives and replicates within macrophages in vitro and that uptake of C. koseri increases in the presence of human pooled serum in a dose-dependent manner. EM studies lend support to the hypothesis that C. koseri uses morphologically different methods of uptake to enter macrophages. FcgammaRI blocking experiments show that this receptor primarily facilitates the entry of opsonized C. koseri into macrophages. Further, confocal fluorescence microscopy demonstrates that C. koseri survives phagolysosomal fusion and that more than 90% of intracellular C. koseri organisms are colocalized within phagolysosomes. The ability of C. koseri to survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.