Detection of intact vancomycin-arginine as the active antibacterial conjugate in E. coli by whole-cell solid-state NMR.

The introduction of new and improved antibacterial agents based on facile synthetic modifications of existing antibiotics represents a promising strategy to deliver urgently needed antibacterial candidates to treat multi-drug resistant bacterial infections. Using this strategy, vancomycin was transformed into a highly active agent against antibiotic-resistant Gram-negative organisms in vitro and in vivo through the addition of a single arginine to yield vancomycin-arginine (V-R). Here, we report detection of the accumulation of V-R in E. coli by whole-cell solid-state NMR using 15N-labeled V-R. 15N CPMAS NMR revealed that the conjugate remained fully amidated without loss of arginine, demonstrating that intact V-R represents the active antibacterial agent. Furthermore, C{N}REDOR NMR in whole cells with all carbons at natural abundance 13C levels exhibited the sensitivity and selectivity to detect the directly bonded 13C-15N pairs of V-R within E. coli cells. Thus, we also present an effective methodology to directly detect and evaluate active drug agents and their accumulation within bacteria without the need for potentially perturbative cell lysis and analysis protocols.

Vancomycin-arginine (STM-001) abrogates ESBL carrier and carbapenem-resistant Escherichia coli burden in a murine complicated urinary tract infection model.

OBJECTIVES: STM-001, a retargeted glycopeptide, is active against MDR E. coli expressing ESBLs including carbapenemases. Herein, we assessed its capability to combat E. coli complicated urinary tract infections (cUTI) in mice driven by clinically important serine (CTX-M-15) and metallo-ß-lactamases (NDM-1). METHODS: Plasma and urine pharmacokinetics following IV administration of STM-001 (1-50 mg/kg) were determined in mice via LC-MS/MS. The effects on bacterial burden (kidney, bladder and urine) were determined in a 7 day mouse cUTI model whereby STM-001 was administered q12h or q24h at 2-100 mg/kg/day from Day 4. Efficacy was assessed by the change in log10 cfu/g or log10 cfu/mL from vehicle-treated infected mice. RESULTS: MICs of STM-001 for CTX-M-15 and NDM-1 E. coli were 8 and 16 mg/L, respectively. Blood pharmacokinetic profile was linear and dose-dependent with low clearance of 9.49 ±â€Š0.31 mL/min/kg, V = 0.63 ±â€Š0.02 L/kg and t½â€Š= 1.16 ±â€Š0.03 h. High STM-001 concentrations were recovered in urine 0-8 h post-administration, reaching up to 120-fold above its MIC. In cUTI efficacy studies, STM-001 (1-50 mg/kg, q12h) reduced CTX-M-15 burden by log10 4.31 (kidney), 3.95 (bladder) and 4.82 (urine) compared with vehicle-treated animals (P < 0.0001). STM-001 also reduced NDM-1 burden by log10 3.89 (kidney), 3.76 (bladder) and 3.08 (urine) (P < 0.0001), with similar inhibitory effects following q24h dosing. CONCLUSIONS: STM-001 was highly effective in reducing E. coli burden in kidney, bladder and urine in mouse cUTI models. The observed efficacy with either dosing regimen indicates potential low humanized doses of 1-5 mg/kg. These data support further development of STM-001 as an innovative, carbapenem-sparing antibiotic to combat human cUTIs.

In Vivo Targeting of Escherichia coli with Vancomycin-Arginine.

The ability of vancomycin-arginine (V-r) to extend the spectrum of activity of glycopeptides to Gram-negative bacteria was investigated. Its MIC towards Escherichia coli, including ß-lactamase expressing Ambler classes A, B, and D, was 8 to 16 µg/ml. Addition of 8 times the MIC of V-r to E. coli was acutely bactericidal and associated with a low frequency of resistance (<2.32 × 10-10). In vivo, V-r markedly reduced E. coli burden by >7 log10 CFU/g in a thigh muscle model. These data warrant further development of V-r in combatting E. coli, including resistant forms.

In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa.

The development of an anti-bacterial drug in the form of a monoclonal antibody (mAb) targeting an exposed virulence factor, represents an innovative therapeutic strategy. Consequently, a fully human IgG1 mAb (LST-007) targeting Pseudomonas aeruginosa (PA) flagellin type b was recombinantly expressed and characterized in vitro and in an infection model driven by a multidrug resistant (MDR) PA strain. LST-007 demonstrated a highly specific binding towards whole PA bacteria harboring flagellin type b and its recombinant counterpart, with a K(D) of 7.4x10(-10) M. In bioactivity assays, LST-007 or titers of Cmax sera derived from pharmacokinetic studies, markedly attenuated PA motility in an equipotent manner. In vivo, parenteral LST-007 (20 mg/kg) given as a single or double-dosing paradigm post-infection, afforded survival (up to 75% at Day 7) in a lethal model of pneumonia driven by the intratracheal (i.t.) instillation of an LD(80) of the MDR PA isolate. This protective effect was markedly superior to that of imipenem (30% survival at Day 7) and totally devoid with an irrelevant, human isotype mAb. These data lay credence that LST-007 may be a valuable adjunct to the limited list of anti-bacterials that can tackle MDR PA strains, thereby warranting its continued development for eventual clinical evaluation.

Colony to colorimetry in 6 h: ELISA detection of a surface-expressed Pseudomonas aeruginosa virulence factor using immobilized bacteria.

A rapid ELISA employing intact Pseudomonas aeruginosa (PA) is described that allows discrimination between strains harboring flagellin type a or b. All 52 PA strains known to harbor flagellin type b were positive in this ELISA when screened with a fully human monoclonal antibody (LST-007) targeting flagellin type b. Completion of this assay in only 6 h, from picking a single bacterial colony to a colorimetric product, could easily be adapted to a clinical laboratory setting and permit the appropriate choice of therapeutic monoclonal antibody versus its homologous flagellin target in PA-infected patients.

Therapy with anti-flagellin A monoclonal antibody limits Pseudomonas aeruginosa invasiveness in a mouse burn wound sepsis model.

BACKGROUND: The aim of this study was to evaluate the effect of an anti-flagellin sub-type monoclonal antibody (anti-fla-a) on Pseudomonas aeruginosa infection in a mouse burn model and to assay bacterial dissemination and invasiveness. METHODS: After immediate post-burn infection with P. aeruginosa, mortality and morbidity (daily weight changes) were monitored in mice treated with anti-fla-a as compared to untreated mice. Bacterial dissemination and invasiveness were monitored by bacterial counts at the burn site and spleen. Three different timing regimens for anti-fla-a treatment were studied: (a) prophylaxis (pre-infection), (b) therapeutic (post-infection), and (c) combined mode. RESULTS: Combined regimen of anti-fla-a markedly improved survival of mice infected with P. aeruginosa from 6% to 96% (p<0.0001), similar to treatment with Imipenem. Furthermore, a significant improvement in survival was obtained when anti-fla-a was given prior to (75% survival) or post-infection (50% survival). It reduced bacterial load in the spleen (p=0.01), preventing bacterial sepsis. CONCLUSION: Anti-fla-a is effective in reducing mortality and morbidity in murine P. aeruginosa-infected burn model.

Efficacy of antibodies against the N-terminal of Pseudomonas aeruginosa flagellin for treating infections in a murine burn wound model.

BACKGROUND: In an era of increasing drug resistance, immunotherapy is a desirable treatment against Pseudomonas aeruginosa infections. The flagellum, which is an important pseudomonal virulence factor, was targeted for immunotherapy. The aim of the study was to evaluate the efficacy of polyclonal immunotherapy targeted against the N-terminal of flagellin (anti-N'-fla-b) for treating severe P. aeruginosa infection in a murine burn wound model. METHODS: Groups of 12 mice were infected (subeschar) with P. aeruginosa strain PA01, and were treated either with systemic anti-N'-fla-b immunoglobulin G (IgG), nonspecific IgG, or imipenem. The control groups included mice with burn alone, mice with untreated infected burn, and mice without burn infected with P. aeruginosa. Three separate regimens were examined: prophylaxis (preinfection), therapeutic (postinfection), and combined. The efficacy of anti-N'-fla-b was evaluated by monitoring the mortality and morbidity (relative weight loss) during a period of 2 weeks. RESULTS: Anti-N'-fla-b IgG immunotherapy significantly decreased the mortality rate of infected burned mice followed by severe P. aeruginosa infection. The mortality rate in the anti-N'-fla-b-treated groups ranged from 0 to 17 percent compared with 58 to 83 percent in nontreated groups infected with 2 to 5 x 10(6) colony-forming units of P. aeruginosa (p < 0.05). The mortality rate in the anti-N'-fla-b-treated groups was similar to that of groups treated with imipenem. The three tested regimens yielded similar results. Morbidity paralleled survival results. Histopathologic examination revealed an earlier reepithelialization of the infected wound in the anti-N'-fla-b-treated mice compared with untreated mice. CONCLUSION: Immunotherapy with anti-N'-fla-b IgG, given either as prophylaxis or therapeutically, effectively reduced mortality and morbidity and improved wound healing in a severely P. aeruginosa-infected murine burn model.

Antibodies raised against N'-terminal Pseudomonas aeruginosa flagellin prevent mortality in lethal murine models of infection.

The goal of this study was to investigate if antibodies raised against N'-terminal Pseudomonas aeruginosa (Pa) flagellin could afford protection in two lethal mouse models of Pa infection. To that end, rabbit polyclonal antibodies were generated against the N'-terminal domains (amino acids 1-156) of recombinant Pa01 or Salmonella muenchen flagellins, termed anti-N'-fla-b and anti-N'-fla-Sm, respectively. In vitro, anti-N'-fla-b but not anti-N'-fla-Sm IgG specifically recognized recombinant and Pa endogenous flagellin type b proteins, total bacterial lysates of Pa type b, and inhibited Pa01 invasion into A549 cells. In vivo, administration of anti-N'-fla-b afforded a remarkable improvement in survival in lethal peritonitis (90% vs. 12% in control; p<0.001) and burn infection (83% vs. 8-17% in control groups; p<0.005) Pa models. These findings would suggest that the N'-terminal domain of Pa flagellin harbors critically important bioactive domains and that an antibody-targeted, neutralization approach directed at this region could provide a novel therapeutic strategy to combat Pa infection.

Cytokines and chemokines in serum and urine as early predictors to identify septic patients on intensive care unit.

The aim of this prospective cohort study was to address the feasibility of measuring cytokines in serum and urine as early predictor tests for the identification of septic Intensive Care Unit (ICU) patients. The study group consisted of 10 septic and 5 non-septic patients at the onset of sepsis according to modified definitions by the American College of Chest Physicians (ACCP)/Society of Critical Care Medicine (SCCM). Serum and urine samples were taken from septic patients at the onset of sepsis and from non-septic patients, every 12 h for 3 days and thereafter every 24 h until day 10. Levels of TNF-alpha, IL-1beta, IL-6, IL-10, IL-18, IFN-gamma, MCP-1, and PCT (procalcitonin) were measured by ELISA. Apart from serum IL-18 and PCT levels, which were elevated in septic patients (p<0.05), levels of all other cytokines and chemokines in the serum of septic patients did not exceed those of the control group. In urine, in contrast with TNF-alpha, IL-1beta, IL-6, IL-10, IFN-gamma, and MCP-1 in which no differences between the two groups were observed, a distinct trend of elevated IL-18 levels was observed only in the septic group. Whereas elevated serum IL-18 and PCT are clear candidate markers for sepsis criteria, the present data indicating elevated urine IL-18 levels albeit from a limited number of septic patients is an interesting observation. The profile of inflammatory mediators in serum and urine from septic patients herein warrants further investigations in a larger group of patients at the onset of sepsis driven by different infectious foci.

Lipopolysaccharides from different bacterial sources elicit disparate cytokine responses in whole blood assays.

The stimulatory effects of different purified lipopolysaccharide (LPS) preparations from E. coli, S. typhosa, P. aeruginosa, and K. pneumoniae on cytokine and chemokine production were measured in whole blood assays by ELISA. Incubation of 0.5 ml whole blood with 10 ng/ml E. coli and S. typhosa resulted in a time-dependent production of TNF-alpha, IL-1beta, IFN-gamma, IL-10 and MCP-1. K. pneumoniae, however, showed preferential effects on IL-1beta, IL-10 and MCP-1 production with less potent effects on TNF-alpha and IFN-gamma. LPS derived from P. aeruginosa showed a similar potency to other LPS preparations on MCP-1 production, yet completely failed to elicit the production of other cytokines. To further investigate potencies of the different LPS preparations, mediator production was determined following stimulation with agonist concentrations of 0.1 ng and 1000 ng per ml over a 24 h time period. Dose-response curves were obtained with LPS derived from E. coli, S. typhosa and K. pneumoniae on all mediators apart from IL-1beta and MCP-1. Most strikingly though, was the ability of LPS derived from P. aeruginosa to selectively elicit a significant dose-response effect on MCP-1 production, despite its very weak stimulatory effects on all other cytokines. These data imply that the bacterial origin of different LPS preparations can exhibit disparate effects on inflammatory mediator production. Furthermore, the potent, selective dose-response effect of P. aeruginosa LPS on MCP-1 production could help to explain the preponderance of a relentless inflammatory cellular infiltrate in diseases such as cystic fibrosis (CF).