Enhancing the antibacterial activity of polymyxins using a nonantibiotic drug.

Purpose: The rapid emergence of multidrug-resistant (MDR) bacteria and the lack of new therapies to eliminate them poses a major threat to global health. With the alarming rise in antimicrobial resistance (AMR), focus has now shifted to the use of the polymyxin class of antibiotics as the last line of defense for treatment of Gram-negative infections. Unfortunately, the growing resistance of bacteria against polymyxins is threatening the treatment of MDR infections, necessitating the need for novel strategies. The objective of this study was to determine if combination of polymyxin (polymyxin B or colistin) with a nonantibiotic small molecule AR-12, a celecoxib derivative that is devoid of cyclooxygenase 2 (COX-2) inhibitory activities, can be an effective strategy against polymyxin-resistant MDR bacteria. Methods: Growth inhibition studies, time-kill assays and permeability assays were conducted to investigate the effect of AR-12 on the antibacterial activity of polymyxins. Results: Growth studies were performed on a panel of polymyxin-resistant MDR strains using the combination of AR-12 with either colistin or polymyxin B. The combination treatment had no effect on strains that have inherent polymyxin resistance; however, AR-12 was effective in lowering the minimal inhibitory concentration (MIC) of polymyxins by 4-60-fold in several strains that had acquired polymyxin resistance. Time-kill assays using the combination of AR-12 and colistin with select MDR strains suggest rapid killing and bactericidal activity, while the permeability assays using fluorescently labeled dansylated polymyxin and 1-N-phenylnaphthylamine (NPN) in these MDR strains suggest that AR-12 can potentiate the antibacterial activity of polymyxins by possibly altering the bacterial outer membrane via modification of lipopolysaccharide and thereby improving the uptake of polymyxins. Conclusion: Our studies indicate that the combination of AR-12 and polymyxin is effective in targeting select Gram-negative bacteria that have acquired polymyxin resistance. Further understanding of the mechanism of action of AR-12 will provide new avenues for developing narrow-spectrum antibacterials to target select Gram-negative MDR bacteria. Importantly, our studies show that the use of nonantibiotic small molecules in combination with polymyxins is an attractive strategy to counter the growing resistance of bacteria to polymyxins.

Comparing in vitro and in vivo virulence phenotypes of Burkholderia pseudomallei type G strains.

Burkholderia pseudomallei (Bpm) is a saprophytic rod-shaped gram-negative bacterium and the causative agent of melioidosis. This disease has previously been described as endemic in areas such as northern Australia and Southeast Asia, but, more recently, a better understanding of the epidemiology of melioidosis indicated that the disease is distributed worldwide, including regions of the Americas and Africa. A 16S-23S rDNA internal transcribed spacer (ITS) typing system has been developed for Bpm and has revealed that ITS types C, E, and hybrid CE are mainly associated with Australia and Southeast Asia while type G strains are more associated with cases of melioidosis in the Western Hemisphere. The purpose of the current study was to determine the in vitro and in vivo virulence profiles of the understudied Bpm type G strains Ca2009, Ca2013a, Mx2013, and 724644 and compared such phenotypes to the commonly studied Bpm type C strain K96243. We evaluated virulence by measuring invasion/uptake and survival of these Bpm strains in murine respiratory epithelial LA-4 cells and alveolar macrophage MH-S cells using different multiplicity of infections (MOIs of 1 and 10). We also calculated the lethal dose 50 values (LD50) in BALB/c mice that were inoculated intranasally with either Ca2009, Ca2013a, or Mx2013. Overall, the virulence and lethality phenotypes of Bpm type G strains were similar to the Bpm type C strain K96243. Additional comparative analyses between the Bpm ITS types may lead to a better understanding of the contribution of the ITS type to the epidemiology and ecology of Bpm strains.

Innate immune response to Burkholderia mallei.

PURPOSE OF REVIEW: Burkholderia mallei is a facultative intracellular pathogen that causes the highly contagious and often the fatal disease, glanders. With its high rate of infectivity via aerosol and recalcitrance toward antibiotics, this pathogen is considered a potential biological threat agent. This review focuses on the most recent literature highlighting host innate immune response to B. mallei. RECENT FINDINGS: Recent studies focused on elucidating host innate immune responses to the novel mechanisms and virulence factors employed by B. mallei for survival. Studies suggest that pathogen proteins manipulate various cellular processes, including host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement. Immune-signaling molecules such as Toll-like receptors, nucleotode-binding oligomerization domain, myeloid differentiation primary response protein 88, and proinflammatory cytokines such as interferon-gamma and tumor necrosis factor-α, play key roles in the induction of innate immune responses. Modifications in B. mallei lipopolysaccharide, in particular, the lipid A acyl groups, stimulate immune responses via Toll-like receptor4 activation that may contribute to persistent infection. SUMMARY: Mortality is high because of septicemia and immune pathogenesis with B. mallei exposure. An effective innate immune response is critical to controlling the acute phase of the infection. Both vaccination and therapeutic approaches are necessary for complete protection against B. mallei.

Burkholderia mallei CLH001 Attenuated Vaccine Strain Is Immunogenic and Protects against Acute Respiratory Glanders.

Burkholderia mallei is the causative agent of glanders, an incapacitating disease with high mortality rates in respiratory cases. Its endemicity and ineffective treatment options emphasize its public health threat and highlight the need for a vaccine. Live attenuated vaccines are considered the most viable vaccine strategy for Burkholderia, but single-gene-deletion mutants have not provided complete protection. In this study, we constructed the select-agent-excluded B. mallei ΔtonB Δhcp1 (CLH001) vaccine strain and investigated its ability to protect against acute respiratory glanders. Here we show that CLH001 is attenuated, safe, and effective at protecting against lethal B. mallei challenge. Intranasal administration of CLH001 to BALB/c and NOD SCID gamma (NSG) mice resulted in complete survival without detectable colonization or abnormal organ histopathology. Additionally, BALB/c mice intranasally immunized with CLH001 in a prime/boost regimen were fully protected against lethal challenge with the B. mallei lux (CSM001) wild-type strain.

Characterization of the Burkholderia mallei tonB Mutant and Its Potential as a Backbone Strain for Vaccine Development.

BACKGROUND: In this study, a Burkholderia mallei tonB mutant (TMM001) deficient in iron acquisition was constructed, characterized, and evaluated for its protective properties in acute inhalational infection models of murine glanders and melioidosis. METHODOLOGY/PRINCIPAL FINDINGS: Compared to the wild-type, TMM001 exhibits slower growth kinetics, siderophore hyper-secretion and the inability to utilize heme-containing proteins as iron sources. A series of animal challenge studies showed an inverse correlation between the percentage of survival in BALB/c mice and iron-dependent TMM001 growth. Upon evaluation of TMM001 as a potential protective strain against infection, we found 100% survival following B. mallei CSM001 challenge of mice previously receiving 1.5 x 10(4) CFU of TMM001. At 21 days post-immunization, TMM001-treated animals showed significantly higher levels of B. mallei-specific IgG1, IgG2a and IgM when compared to PBS-treated controls. At 48 h post-challenge, PBS-treated controls exhibited higher levels of serum inflammatory cytokines and more severe pathological damage to target organs compared to animals receiving TMM001. In a cross-protection study of acute inhalational melioidosis with B. pseudomallei, TMM001-treated mice were significantly protected. While wild type was cleared in all B. mallei challenge studies, mice failed to clear TMM001. CONCLUSIONS/SIGNIFICANCE: Although further work is needed to prevent chronic infection by TMM001 while maintaining immunogenicity, our attenuated strain demonstrates great potential as a backbone strain for future vaccine development against both glanders and melioidosis.

Monitoring Therapeutic Treatments against Burkholderia Infections Using Imaging Techniques.

Burkholderia mallei, the etiologic agent of glanders, are Category B select agents with biothreat potential, and yet effective therapeutic treatments are lacking. In this study, we showed that CpG administration increased survival, demonstrating protection in the murine glanders model. Bacterial recovery from infected lungs, liver and spleen was significantly reduced in CpG-treated animals as compared with non-treated mice. Reciprocally, lungs of CpG-treated infected animals were infiltrated with higher levels of neutrophils and inflammatory monocytes, as compared to control animals. Employing the B. mallei bioluminescent strain CSM001 and the Neutrophil-Specific Fluorescent Imaging Agent, bacterial dissemination and neutrophil trafficking were monitored in real-time using multimodal in vivo whole body imaging techniques. CpG-treatment increased recruitment of neutrophils to the lungs and reduced bioluminescent bacteria, correlating with decreased bacterial burden and increased protection against acute murine glanders. Our results indicate that protection of CpG-treated animals was associated with recruitment of neutrophils prior to infection and demonstrated, for the first time, simultaneous real time in vivo imaging of neutrophils and bacteria. This study provides experimental evidence supporting the importance of incorporating optimized in vivo imaging methods to monitor disease progression and to evaluate the efficacy of therapeutic treatment during bacterial infections.

In vivo Bioluminescence Imaging of Burkholderia mallei Respiratory Infection and Treatment in the Mouse Model.

Bioluminescent imaging (BLI) technology is a powerful tool for monitoring infectious disease progression and treatment approaches. BLI is particularly useful for tracking fastidious intracellular pathogens that might be difficult to recover from certain organs. Burkholderia mallei, the causative agent of glanders, is a facultative intracellular pathogen and has been classified by the CDC as a Category B select agent due to its highly infectious nature and potential use as a biological weapon. Very little is known regarding pathogenesis or treatment of glanders. We investigated the use of bioluminescent reporter constructs to monitor the dynamics of infection as well as the efficacy of therapeutics for B. mallei in real-time. A stable luminescent reporter B. mallei strain was created using the pUTmini-Tn5::luxKm2 plasmid and used to monitor glanders in the BALB/c murine model. Mice were infected via the intranasal route with 5 × 10(3) bacteria and monitored by BLI at 24, 48, and 72 h. We verified that our reporter construct maintained similar virulence and growth kinetics compared to wild-type B. mallei and confirmed that it maintains luminescent stability in the presence or absence of antibiotic selection. The luminescent signal was initially seen in the lungs, and progressed to the liver and spleen over the course of infection. We demonstrated that antibiotic treatment 24 h post-infection resulted in reduction of bioluminescence that can be attributed to decreased bacterial burden in target organs. These findings suggest that BLI can be used to monitor disease progression and efficacy of therapeutics during glanders infections. Finally, we report an alternative method to mini-Tn5::luxKm2 transposon using mini-Tn7-lux elements that insert site-specifically at known genomic attachment sites and that can also be used to tag bacteria.

Characterization of Clostridium species utilizing liquid chromatography/mass spectrometry of intact proteins.

A method for biomarker candidate discovery and strain level pathogen characterization using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization is described. This method was applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Seven marker proteins per species (fourteen total) were successfully implemented to speciate unknowns during a blind study and could enhance serological and genetic approaches by serving as new targets for detection. Two sets of C. perfringens isolates that were 100% similar by pulsed-field gel electrophoresis (PFGE) were distinguished using LC/MS, demonstrating the high specificity of this approach. The use of LC/MS is less labor intensive than PFGE, affords greater specificity than real-time PCR, and requires no primers or antibodies.

Liquid chromatography/mass spectrometry characterization of Escherichia coli and Shigella species.

Liquid chromatography/quadrupole time of flight mass spectrometry (LC/QTOF MS) utilizing electrospray ionization was employed to monitor protein expression in Escherichia coli and Shigella organisms. Comparison with MALDI/TOF-MS revealed more proteins, particularly above 15 kDa. A combination of automated charge state deconvolution, spectral mirroring, and spectral subtraction was used to reveal subtle differences in the LC/MS data. Reproducible intact protein biomarker candidates were discovered based on their unique mass, retention time, and relative intensity. These marker candidates were implemented to differentiate closely related strain types, (e.g., two distinct isolates of E. coli O157:H7) and to correctly identify unknown pathogens. This LC/MS approach is less labor-intensive than pulsed-field gel electrophoresis, affords greater specificity than real-time PCR, and requires no primers or antibodies. Additionally, this approach would be beneficial during outbreaks of foodborne disease or bioterrorism investigations by complementing methods typically used in diagnostic microbiology laboratories.

Insulin regulates aging and oxidative stress in Anopheles stephensi.

Observations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7 x 10(-4) and 1.7 x 10(-3) micromol l(-1), doses 0.3-fold and 3.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (SOD) activity and manganese SOD activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable SOD mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a 'signaling center' for mosquito aging.