Reduction In Bacterial Burden Research Articles

An RNA-Based Vaccine Platform for Use against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (M.tb), a bacterial pathogen that causes tuberculosis disease (TB), exerts an extensive burden on global health. The complex nature of M.tb, coupled with different TB disease stages, has made identifying immune correlates of protection challenging and subsequently slowing vaccine candidate progress. In this work, we leveraged two delivery platforms as prophylactic vaccines to assess immunity and subsequent efficacy against low-dose and ultra-low-dose aerosol challenges with M.tb H37Rv in C57BL/6 mice. Our second-generation TB vaccine candidate ID91 was produced as a fusion protein formulated with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion) or as a novel replicating-RNA (repRNA) formulated in a nanostructured lipid carrier. Protein subunit- and RNA-based vaccines preferentially elicit cellular immune responses to different ID91 epitopes. In a single prophylactic immunization screen, both platforms reduced pulmonary bacterial burden compared to the controls. Excitingly, in prime-boost strategies, the groups that received heterologous RNA-prime, protein-boost or combination immunizations demonstrated the greatest reduction in bacterial burden and a unique humoral and cellular immune response profile. These data are the first to report that repRNA platforms are a viable system for TB vaccines and should be pursued with high-priority M.tb antigens containing CD4+ and CD8+ T-cell epitopes.

The Effectiveness of Laser Applications and Photodynamic Therapy on Relevant Periodontal Pathogens (Aggregatibacter actinomycetemcomitans) Associated with Immunomodulating Anti-rheumatic Drugs.

Considering the current context of the increasing resistance of bacterial species to antibiotics and other antimicrobial agents, a major objective is to develop other antimicrobial approaches, which would be able to inactivate pathogens with considerable effectiveness. Two such methods are photodynamic disinfection therapy and laser irradiation. In view of the immunocompromised status of some patients under immunosuppressive therapy and potential drug interactions that can be established between systemic antimicrobial agents, the research of local, minimally invasive methods of inactivating periodontal pathogens in the context of these systemic therapies with modifying drugs of the immune response is justified. This in vitro study evaluated the antimicrobial action of a diode laser, wavelength 940 nm, and photodisinfection therapy at 670 nm (photosensitizer, 3,7 dimethyl phenothiazine chloride) on a type strain of Aggregatibacter actinomycetemcomitans, a known periodontal pathogen, in the presence and absence of active substances used in autoimmune disease therapy (Etanercept, Infliximab, Metothrexate). The association of a conventional antirheumatic drug with anti-TNF-α therapy determined a significantly greater inhibition of the strain of A. actinomycetemcomitans compared to monotherapy, in vitro. Photodisinfection caused a significant reduction in bacterial burden after a 30 s exposure in vitro, regardless of the pharmaceutical associations of biological and conventional disease-modifying antirheumatic drugs (DMARDs). Irradiation with a diode laser for 30 s at a power of 5 W caused a greater reduction compared to irradiation with 1 W. The application of laser and photodisinfection induced a significant reduction in Aggregatibacter actinomycetemcomitans in vitro and could be considered important adjunctive measures for the eradication of this oral pathogen in the context of immunomodulating therapy.

Evaluation of Fosfomycin-Sulbactam Combination Therapy against Carbapenem-Resistant Acinetobacter baumannii Isolates in a Hollow-Fibre Infection Model.

Static concentration in vitro studies have demonstrated that fosfomycin- or sulbactam-based combinations may be efficacious against carbapenem-resistant Acinetobacter baumannii (CRAB). In the present study, we aimed to evaluate the bacterial killing and resistance suppression potential of fosfomycin-sulbactam combination therapies against CRAB isolates in a dynamic infection model. We simulated clinically relevant dosing regimens of fosfomycin (8 g every 8 h, 1 h infusion) and sulbactam (12 g continuous infusion or 4 g every 8 h, 4 h infusion) alone and in combination for 7 days in a hollow-fibre infection model (HFIM) against three clinical isolates of CRAB. The simulated pharmacokinetic profiles in the HFIM were based on fosfomycin and sulbactam data from critically ill patients. Fosfomycin monotherapy resulted in limited bacterial killing. Sulbactam monotherapies resulted in ~ 3 to 4 log10 kill within the first 8 to 32 h followed by regrowth of up to 8 to 10 log10 CFU/mL. A combination of fosfomycin and continuous infusion of sulbactam led to a ~2 to 4 log10 reduction in bacterial burden within the first 24 h, which was sustained throughout the duration of the experiments. A combination of fosfomycin and extended infusion of sulbactam produced a ~4 log10 reduction in colony count within 24 h. This study demonstrated that fosfomycin in combination with sulbactam is a promising option for the treatment of MDR A. baumannii. Further studies are needed to further assess the potential clinical utility of this combination.

Role of FruR transcriptional regulator in virulence of Listeria monocytogenes and identification of its regulon

Listeria monocytogenes is a ubiquitous opportunistic foodborne pathogen capable of survival in various adverse environmental conditions. Pathogenesis of L. monocytogenes is tightly controlled by a complex regulatory network of transcriptional regulators that are necessary for survival and adaptations to harsh environmental conditions both inside and outside host cells. Among these regulatory pathways are members of the DeoR-family transcriptional regulators that are known to play a regulatory role in sugar metabolism. In this study, we deciphered the role of FruR, a DeoR family protein, which is a fructose operon transcriptional repressor protein, in L. monocytogenes pathogenesis and growth. Following intravenous (IV) inoculation in mice, a mutant strain with deletion of fruR exhibited a significant reduction in bacterial burden in liver and spleen tissues compared to the parent strain. Further, the ΔfruR strain had a defect in cell-to-cell spread in L2 fibroblast monolayers. Constitutive activation of PrfA, a pleiotropic activator of L. monocytogenes virulence factors, did not restore virulence to the ΔfruR strain, suggesting that the attenuation was not a result of impaired PrfA activation. Transcriptome analysis revealed that FruR functions as a positive regulator for genes encoding enzymes involved in the pentose phosphate pathway (PPP) and as a repressor for genes encoding enzymes in the glycolysis pathway. These results suggested that FruR may function to facilitate NADPH regeneration, which is necessary for full protection from oxidative stress. Interestingly, deletion of fruR increased sensitivity of L. monocytogenes to H2O2, confirming a role for FruR in survival of L. monocytogenes during oxidative stress. Using anti-mouse neutrophil/monocyte monoclonal antibody RB6-8C5 (RB6) in an in vivo infection model, we found that FruR has a specific function in protecting L. monocytogenes from neutrophil/monocyte-mediated killing. Overall, this work clarifies the role of FruR in controlling L. monocytogenes carbon flow between glycolysis and PPP for NADPH homeostasis, which provides a new mechanism allowing metabolic adaptation of L. monocytogenes to oxidative stress.

Preparation and Evaluation of Low-Dose Calcitriol Dry Powder Inhalation as Host-Directed Adjunct Therapy for Tuberculosis.

BackgroundIt is unclear whether Vitamin D is efficacious as a host-directed therapy (HDT) for patients of tuberculosis (TB). We investigated pulmonary delivery of the active metabolite of Vitamin D3, i.e., 1, 25-dihydroxy vitamin D3 (calcitriol) in a mouse model of infection with Mycobacterium tuberculosis (Mtb).MethodsWe optimized a spray drying process to prepare a dry powder inhalation (DPI) of calcitriol using a Quality by Design (QbD) approach. We then compared outcomes when Mtb-infected mice were treated with inhaled calcitriol at 5 ng/kg as a stand-alone intervention versus DPI as adjunct to standard oral anti-tuberculosis therapy (ATT).ResultsThe DPI with or without concomitant ATT markedly improved the morphology of the lungs and mitigated histopathology in both the lungs and the spleens. The number of nodular lesions on the lung surface decreased from 43.7 ± 3.1 to 22.5 ± 3.9 with the DPI alone and to 9.8 ± 2.5 with DPI + ATT. However, no statistically significant induction of host antimicrobial peptide cathelicidin or reduction in bacterial burden was seen with the DPI alone. DPI + ATT did not significantly reduce the bacterial burden in the lungs compared to ATT alone.ConclusionsWe concluded that HDT using the low dose calcitriol DPI contributed markedly to mitigation of pathology, but higher dose may be required to evoke significant induction of bactericidal host response and bactericidal activity in the lung. Supplementary InformationThe online version contains supplementary material available at 10.1007/s11095-022-03360-5.

Superior Efficacy of a TBI-166, Bedaquiline, and Pyrazinamide Combination Regimen in a Murine Model of Tuberculosis.

TBI-166, derived from riminophenazine analogues, shows more potent anti-TB activity than clofazimine and is being assessed against tuberculosis (TB) in a phase IIa clinical trial in China. Preclinical regimen studies containing TBI-166 will support the phase IIb clinical trials of TBI-166. In the present study, we compared the efficacy in three murine TB models of an all-oral drug-resistant TB drug regimen of TBI-166 with bedaquiline (BDQ) and pyrazinamide (PZA) with the first-line regimen of isoniazid (INH) with rifampin (RFP) and PZA (HRZ regimen), the most effective reported TBI-166-containing regimen of TBI-166 with BDQ and linezolid (LZD), and the Nix-TB clinical trial regimen of BDQ with pretomanid and LZD (BPaL regimen). In the C3HeB/FeJ murine TB model, for the TBI-166+BDQ+PZA regimen, the lungs of mice were culture negative at 4 weeks, and there were no relapses at 8 weeks of treatment. The reduction in bacterial burden and relapse rate were greater than those of the HRZ regimen and the TBI-166+BDQ+LZD regimen. Compared with the BPaL regimen, the TBI-166+BDQ+PZA regimen had similar or stronger early bactericidal activity, bactericidal activity, and sterilizing activity in the BALB/c murine TB model. The bacterial burden in the TBI-166+BDQ+PZA regimen group decreased significantly more than that in the BPaL regimen group and was almost or totally relapse free (<13.33% after 8 weeks). In conclusion, oral short-course three-drug regimens, including TBI-166 with high efficacy, were identified. The TBI-166+BDQ+PZA regimen is recommended for further study in a TBI-166 phase IIb clinical trial.

Photo-Crosslinked Antimicrobial Hydrogel Exhibiting Wound Healing Ability and Curing Infections In Vivo.

With the increasing focus on healthcare research in the current times, therapeutic and biomaterial interventions for healing of wounds and mitigation of wound-associated infections have seen expedited progress. Conventional approaches consist of release-active gels, which demonstrate leaching of antimicrobials, such as antibiotics, metal ions, etc. However, these systems suffer from the disadvantages of burst release, reservoir exhaustion, and associated toxicity. In this report, intrinsically antimicrobial hydrogel (HyDex) is developed by one-pot UV crosslinking of methacrylated dextran, polyethylene glycol diacrylate, and cationic lipophilic methacrylate with varied hydrophobic chain, which displays broad-spectrum antimicrobial activity, hemostatic ability, and rapid wound closure efficacy. The optimized hydrogel exhibits potent antimicrobial efficacy against multidrug-resistant Gram-positive and Gram-negative bacteria as well as against pathogenic fungus Candida albicans. The HyDex hydrogel shows rapid arrest of bleeding in mice liver puncture model. The hydrogel kills carbapenem-resistant Acinetobacter baumannii in a murine model of burn wound infection with >99% reduction in bacterial burden. Furthermore, this hydrogel displays significant reduction in inflammatory responses, with accelerated wound healing in rat deep wound model. Collectively, these results imply the excellent promise held by lead hydrogel to be developed for tackling deep tissue wounds, notorious infections, and resulting inflammatory responses.

Treating ‘Septic’ With Enhanced Antibiotics and ‘Arthritis’ by Mitigation of Excessive Inflammation

Bacterial infection within the synovial joint, commonly known as septic arthritis, remains a clinical challenge as it presents two concurrent therapeutic goals of reducing bacterial burden and preservation of articular cartilage from destructive host inflammation. We hypothesized that mitigation of MRSA-induced inflammatory signaling could diminish destruction of articular cartilage in the setting of septic arthritis when used in conjunction with antibiotics. Herein, we provide evidence which supports a new therapeutic notion that concurrent antimicrobial therapy to address the ‘septic’ component of the disease with inflammation mitigation to manage the destructive ‘arthritis’ component. We established a murine model to mimic septic knee arthritis, as well as a variety of other inflammatory joint conditions. This murine septic arthritis model, in conjunction with in vitro and ex-vivo models, was utilized to characterize the inflammatory profile seen in active septic arthritis, as well as post-antibiotic treatment, via transcriptomic and histologic studies. Finally, we provided the clinical rationale for a novel therapeutic strategy combining enhanced antibiotic treatment with rifampin and adjuvant immunomodulation to inhibit post-infectious, excess chondrolysis and osteolysis. We identified that septic arthritis secondary to MRSA infection in our murine model led to increased articular cartilage damage compared to various types of inflammatory arthritis. The activation of the pERK1/2 signaling pathway, which is implicated with the mounting of an immune response and generation of inflammation, was increased in intracellular MRSA-infected synovial tissue and persisted despite antibiotic treatment. Trametinib, an inhibitor of ERK signaling through suppression of MEK1/2, alleviated the inflammation produced by the addition of intra-articular, heat-killed MRSA. Further, when combined with vancomycin and rifampin, mitigation of inflammation by pERK1/2 targeting improved outcomes for MRSA septic arthritis by conferring chondroprotection to articular cartilage and diminishing inflammatory osteolysis within bone. Our results support a new therapeutic notion that cell/biofilm-penetrating antibiotics alongside adjuvant mitigation of excessive intra-articular inflammation accomplish distinct therapeutic goals: reduction of bacterial burden and preservation of articular cartilage integrity.

Glycolysis is critical for granulocytic myeloid-derived suppressor cell (G-MDSC) activity during Staphylococcus aureus biofilm infection

Abstract Staphylococcus aureus (S. aureus) is a leading cause of prosthetic joint infection (PJI). These infections are often intractable due to biofilm formation, which are complex bacterial communities that adhere to biotic and abiotic surfaces. Within a biofilm, there is considerable heterogeneity in bacterial metabolism and gene expression that serves to create an anti-inflammatory milieu at the site of infection. This is due, in part, to the preferential recruitment of granulocytic myeloid-derived suppressor cells (G-MDSCs), a population of pathologically activated immature leukocytes with immunosuppressive features. Our prior work revealed that G-MDSCs are integral for promoting biofilm persistence via the production of interleukin-10 (IL-10) in response to biofilm-derived lactate. However, the mechanism responsible for G-MDSC anti-inflammatory activity in the context of biofilm infection is unknown. Using a mouse model of S. aureus PJI, time-course single-cell RNA sequencing (scRNA-seq) revealed a significant enrichment in glycolytic and hypoxic transcriptional signatures in infiltrating G-MDSCs. G-MDSCs significantly increased their glycolytic activity when co-cultured with S. aureus biofilm in vitro and inhibiting glycolysis during PJI in vivo with 2-DG nanoparticles significantly attenuated G-MDSC suppressive activity concomitant with a significant reduction in bacterial burden. This study demonstrates that glycolysis is required for G-MDSC-mediated immunosuppression and biofilm persistence during S. aureus PJI. Furthermore, our results demonstrate that G-MDSC metabolism can be targeted to improve infection outcomes. Supported by funding from NIH (2P01 AI083211) &amp; UNMC (UNMC Graduate Fellowship)

T cell transcription factor expression evolves over time in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques.

SUMMARYMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a global health concern, yearly resulting in 10 million new cases of active TB. Immunologic investigation of lung granulomas is essential for understanding host control of bacterial replication. Here, we identify and compare the pathological, cellular, and functional differences in granulomas at 4, 12, and 20 weeks post-infection in Chinese cynomolgus macaques. Original granulomas differ in transcription-factor expression within adaptive lymphocytes, with those at 12 weeks showing higher frequencies of CD8+T-bet+ T cells, while CD4+T-bet+ T cells increase at 20 weeks post-infection. The appearance of T-bet+ adaptive T cells at 12 and 20 weeks is coincident with a reduction in bacterial burden, suggesting their critical role in Mtb control. This study highlights the evolution of T cell responses within lung granulomas, suggesting that vaccines promoting the development and migration of T-bet+ T cells would enhance mycobacterial control.

Bi-directional elucidation of Lactiplantibacillus plantarum (RTA 8) intervention on the pathophysiology of gut-brain axis during Salmonella brain infection

BackgroundThere have been reports of patients suffering from typhoid fever, particularly those involving infants and immunocompromised patients, which at times present with Salmonella induced brain infection. Although rare, it has frequently been associated with adverse neurological complications and increased mortality. In this context, the gut-brain axis, involving two-way communication between the gut and the brain, holds immense significance as various gut ailments have been associated with psychiatric complications. In turn, several neurodegenerative diseases have been associated with an altered gut microbiota profile. Given the paucity of effective antimicrobials and increasing incidence of multi-drug resistance in pathogens, alternate treatment therapies such as probiotics have gained significant attention in the recent past.ResultsIn the current study, prophylactic effect of Lactiplantibacillus plantarum (RTA 8) in preventing neurological complications occurring due to Salmonella brain infection was evaluated in a murine model. Along with a significant reduction in bacterial burden and improved histoarchitecture, L. plantarum (RTA 8) administration resulted in amelioration in the level of neurotransmitters such as serotonin, norepinephrine and dopamine in the gut as well as in the brain tissue. Simultaneously, increased gene expression of physiologically essential molecules such as mucin (MUC1 and MUC3) and brain-derived neurotrophic factor (BDNF) was also observed in this group.ConclusionPresent study highlights the potential benefits of a probiotic supplemented diet in improving various aspects of host health due to their multi-targeted approach, thereby resulting in multi-faceted gains.

Multi‐Functional Small Molecules with Temporal Charge‐Switchability Tackle Infection and Inflammation

AbstractThe alarming situation in public healthcare caused by ever‐increasing catastrophe of antimicrobial resistance, recurrent infections, and associated inflammation has accelerated the hunt for novel therapeutics which can address these diverse problems concomitantly. This article introduces a new class of multi‐functional amino acid conjugated small antibacterial molecules (ASAMs) which tackle complicated infections and associated inflammation. These molecules exhibit broad‐spectrum bactericidal activity against multi‐drug‐resistant bacteria. The phenylalanine‐bearing lead molecule (ASAM‐10) tackles bacterial dormant subpopulations, impenetrable biofilms, and intracellular pathogens simultaneously. Importantly, this molecule addresses the problem of toxicity associated with cationic lipopeptides like colistin through the temporal charge switching (cationic to zwitterionic) owing to the degradation of labile ester linkages. However, this does not affect its desired antibacterial action window. The substantial reduction in the overexpression of pro‐inflammatory cytokines (IL‐6, IL‐8, TNF‐α, and IL‐1β) upon treatment of infected macrophages with ASAM‐10 validates its anti‐inflammatory efficacy. Furthermore, bacteria exhibit diminished susceptibility toward resistance development against ASAM‐10 owing to its membrane‐active nature. ASAM‐10 displays significant reduction in bacterial burden (2 Log CFU/g) when administered intraperitoneally in mice for MRSA thigh infection. Overall, this new class of multi‐functional molecules is safe for anticipated advanced therapeutic applications to combat complex bacterial infections and inflammation.

Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

1095. Pharmacokinetics-Pharmacodynamics Evaluation of Tebipenem Pivoxil Hydrobromide Using the 10-Day Hollow-Fiber In Vitro Infection Model

1095. Pharmacokinetics-Pharmacodynamics Evaluation of Tebipenem Pivoxil Hydrobromide Using the 10-Day Hollow-Fiber <i>In Vitro</i> Infection Model

Prospective assessment of catheter-associated bacteriuria clinical presentation, epidemiology, and colonization dynamics in nursing home residents

BACKGROUNDCatheterization facilitates continuous bacteriuria, for which the clinical significance remains unclear. This study aimed to determine the clinical presentation, epidemiology, and dynamics of bacteriuria in a cohort of long-term catheterized nursing home residents.METHODSProspective urine culture, urinalysis, chart review, and assessment of signs and symptoms of infection were performed weekly for 19 study participants over 7 months. All bacteria ≥ 1 × 103 cfu/mL were cultured, isolated, identified, and tested for susceptibility to select antimicrobials.RESULTSIn total, 226 of the 234 urine samples were polymicrobial (97%), with an average of 4.7 isolates per weekly specimen. A total of 228 urine samples (97%) exhibited ≥ 1 × 106 CFU/mL, 220 (94%) exhibited abnormal urinalysis, 126 (54%) were associated with at least 1 possible sign or symptom of infection, and 82 (35%) would potentially meet a standardized definition of catheter-associated urinary tract infection (CAUTI), but only 3 had a caregiver diagnosis of CAUTI. Bacterial isolates (286; 30%) were resistant to a tested antimicrobial agent, and bacteriuria composition was remarkably stable despite a combined total of 54 catheter changes and 23 weeks of antimicrobial use.CONCLUSIONBacteriuria composition was largely polymicrobial, including persistent colonization by organisms previously considered to be urine culture contaminants. Neither antimicrobial use nor catheter changes sterilized the urine, at most resulting in transient reductions in bacterial burden followed by new acquisition of resistant isolates. Thus, this patient population exhibits a high prevalence of bacteriuria coupled with potential indicators of infection, necessitating further exploration to identify sensitive markers of true infection.FUNDINGThis work was supported by the NIH (R00 DK105205, R01 DK123158, UL1 TR001412).

Biomimetic pH/lipase dual responsive vitamin-based solid lipid nanoparticles for on-demand delivery of vancomycin

In this study, ascorbyl tocopherol succinate (ATS) was designed, synthesized and characterized via FT-IR, HR-MS, H1 NMR and C13 NMR, to simultaneously confer biomimetic and dual responsive properties of an antibiotic nanosystem to enhance their antibacterial efficacy and reduce antimicrobial resistance. Therefore, an in silico-aided design (to mimic the natural substrate of bacterial lipase) was employed to demonstrate the binding potential of ATS to lipase (−32.93 kcal/mol binding free energy (ΔGbind) and bacterial efflux pumps blocking potential (NorA ΔGbind: −37.10 kcal/mol, NorB ΔGbind: −34.46 kcal/mol). ATS bound stronger to lipase than the natural substrate (35 times lower Kd value). The vancomycin loaded solid lipid nanoparticles (VM-ATS-SLN) had a hydrodynamic diameter, zeta potential, polydispersity index and entrapment efficiency of 106.9 ± 1.4 nm, −16.5 ± 0.93 mV, 0.11 ± 0.012 and 61.9 ± 1.31%, respectively. In vitro biocompatibility studies revealed VM-ATS-SLN biosafety and non-haemolytic activity. Significant enhancement in VM release was achieved in response to acidified pH and lipase enzyme, compared to controls. VM-ATS-SLN showed enhanced sustained in vitro antibacterial activity for 5 days, 2-fold greater MRSA biofilm growth inhibition and 3.44-fold reduction in bacterial burden in skin infected mice model compared to bare VM. Therefore, ATS shows potential as a novel multifunctional adjuvant for effective and targeted delivery of antibiotics.

Semi-mechanistic PK/PD modelling of meropenem and sulbactam combination against carbapenem-resistant strains of Acinetobacter baumannii.

Due to limited treatment options for carbapenem-resistant Acinetobacter baumannii (CR-AB) infections, antibiotic combinations are commonly used. In this study, we explored the potential efficacy of meropenem-sulbactam combination (MEM/SUL) against CR-AB. The checkerboard method was used to screen for synergistic activity of MEM/SUL against 50 clinical CR-AB isolates. Subsequently, time-kill studies against two CR-AB isolates were performed. Time-kill data were described using a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model. Subsequently, Monte Carlo simulations were performed to estimate the probability of 2-log kill, 1-log kill or stasis at 24-h following combination therapy. The MEM/SUL demonstrated synergy against 28/50 isolates. No antagonism was observed. The MIC50 and MIC90 of MEM/SUL were decreased fourfold, compared to the monotherapy MIC. In the time-kill studies, the combination displayed synergistic killing against both isolates at the highest clinically achievable concentrations. At concentrations equal to the fractional inhibitory concentration, synergism was observed against one isolate. The PK/PD model adequately delineated the data and the interaction between meropenem and sulbactam. The effect of the combination was driven by sulbactam, with meropenem acting as a potentiator. The simulations of various dosing regimens revealed no activity for the monotherapies. At best, the MEM/SUL regimen of 2 g/4 g every 8 h demonstrated a probability of target attainment of 2-log10 kill at 24 h of 34%. The reduction in the MIC values and the achievement of a moderate PTA of a 2-log10 reduction in bacterial burden demonstrated that MEM/SUL may potentially be effective against some CR-AB infections.

Sustained release of peptides and proteins from polymeric nanocarriers produced by inverse Flash NanoPrecipitation

Peptide and protein therapeutics generally exhibit high potency and specificity and are increasingly important segments of the pharmaceutical market. However, their clinical applications are limited by rapid clearance and poor membrane permeability. Encapsulation of the peptide or protein into a nano-scale carrier can modify its pharmacokinetics and biodistribution. This might be employed to promote uptake in desired cell types or tissues, to limit systemic exposure, or to reduce the need for frequent injections. We have recently described inverse Flash NanoPrecipitation (iFNP), a scalable technique to encapsulate water-soluble therapeutics into polymeric nanocarriers, and have demonstrated improvements in therapeutic loading of an order of magnitude over comparable approaches. Here, we describe the formulation parameters that control release rates of encapsulated model therapeutics polymyxin B, lysozyme, and bovine serum albumin from nanocarriers produced using iFNP. Using a neutropenic lung infection mouse model with a multi-drug resistant Acinetobacter baumannii clinical isolate, we demonstrate enhanced therapeutic effect and safety profile afforded by nanocarrier-encapsulated polymyxin B following pulmonary administration. The encapsulated formulation reduced toxicity observed at elevated doses and resulted in up to 2.7-log10 reduction in bacterial burden below that of unencapsulated polymyxin B. These results establish the promise of iFNP as a platform for nanocarrier delivery of water-soluble therapeutics.

Synergistic Killing and Re-Sensitization of Pseudomonas aeruginosa to Antibiotics by Phage-Antibiotic Combination Treatment.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections pose a serious health threat. Bacteriophage–antibiotic combination therapy is a promising candidate for combating these infections. A 5-phage P. aeruginosa cocktail, PAM2H, was tested in combination with antibiotics (ceftazidime, ciprofloxacin, gentamicin, meropenem) to determine if PAM2H enhances antibiotic activity. Combination treatment in vitro resulted in a significant increase in susceptibility of MDR strains to antibiotics. Treatment with ceftazidime (CAZ), meropenem, gentamicin, or ciprofloxacin in the presence of the phage increased the number of P. aeruginosa strains susceptible to these antibiotics by 63%, 56%, 31%, and 81%, respectively. Additionally, in a mouse dorsal wound model, seven of eight mice treated with a combination of CAZ and PAM2H for three days had no detectable bacteria remaining in their wounds on day 4, while all mice treated with CAZ or PAM2H alone had ~107 colony forming units (CFU) remaining in their wounds. P. aeruginosa recovered from mouse wounds post-treatment showed decreased virulence in a wax worm model, and DNA sequencing indicated that the combination treatment prevented mutations in genes encoding known phage receptors. Treatment with PAM2H in combination with antibiotics resulted in the re-sensitization of P. aeruginosa to antibiotics in vitro and a synergistic reduction in bacterial burden in vivo.

AtxA-Controlled Small RNAs of Bacillus anthracis Virulence Plasmid pXO1 Regulate Gene Expression in trans.

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.