Reduced bacterial contamination rates detected on silicone tourniquets compared to conventional tourniquets in clinical routine.

BACKGROUND: Tourniquets used for peripheral venous vascular access such as blood sampling are regularly contaminated in clinical routine. Although most contaminations are harmless, some pose a possible risk for infection. To improve peripheral venous access infection control standards, tourniquets with no or as few as possible bacterial burden should be used. Conventional tourniquets can be reprocessed by autoclaving or by incubating in disinfectants. However, both methods are time-consuming and not suitable for immediate use between patients. In contrast, silicone tourniquets can be quickly and simply reprocessed with wipe disinfection. In vitro studies from the manufacturer have demonstrated reduced bacterial contamination on silicone tourniquets after usage compared to conventional tourniquets. This study aims to independently investigate the bacterial load on both types of tourniquets in clinical routine. METHODS: In a first trial, new conventional and silicon tourniquets were used for blood sampling in one facility with strict guidelines for reprocessing (after each patient or not at all) for 1 day and tested for bacterial contamination. In a second trial, new tourniquets were used in four facilities while the mode and frequency of tourniquets' reprocessing was defined individually by each facility. The number of treated patients, mode and frequency of reprocessing and other relevant handling measures were documented. RESULTS: Under controlled conditions, with strictly specified reprocessing, slightly fewer bacteria were found on silicone than on conventional tourniquets. In routine clinical practice the reprocessing frequency was not higher for silicone tourniquets in practice. Yet, in all four facilities, there were significantly fewer bacteria found on silicone than on conventional tourniquets. CONCLUSION: Although tourniquets are classified as non-critical medical devices, results show - together with benefits of faster and easier reprocessing - that silicone tourniquets can improve infection control of venous vascular access.

Efficacy of Intraoperative Antiseptic Techniques in the Prevention of Periprosthetic Joint Infection: Superiority of Betadine.

BACKGROUND: Povidone-iodine (PI), chlorhexidine gluconate (CHG), and vancomycin (VANC) powder are common intrawound prophylactic agents to prevent periprosthetic joint infection during primary total joint arthroplasty. The aims of this study are (1) to determine the minimal inhibitory concentration (MIC) and time to death for PI, CHG, and VANC against multiple bacteria and (2) to determine time to death against bacteria dried on titanium discs. METHODS: A standard quantitative suspension assay was performed to determine the MIC for PI, CHG, and VANC against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Haemophilus influenzae, Pseudomonas aeruginosa, Burkholderia cepacia, and Escherichia coli. Time to death assay was performed with time points of 0, 3, 30, and 60 minutes. Concentrations of antiseptic agents for time to death assay were 1% PI, 0.05% CHG, and 5 µg/mL VANC. Dry-phase bacteria on titanium discs were treated in a similar fashion. RESULTS: The MIC of PI was 0.63%, CHG was 0.0031%, and VANC was 1.56 µg/mL. All 7 bacterial isolates were completely killed by PI at all times tested. CHG failed to kill MRSA and B cepacia at 0- and 3-minute exposures. Vancomycin completely killed MRSA and S epidermidis isolates between 18-20 hours of exposure. All bacterial isolates dried on titanium discs were eliminated by PI exposure on contact. E coli and S epidermidis were incompletely eliminated by CHG at 0 minutes, with all isolates eliminated at 3, 10, and 30 minutes. CONCLUSION: Our study suggests that PI kills all bacteria tested immediately on contact and that the exposure time is not the key factor.

Mycobacteria, metals, and the macrophage.

Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here, we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies.

8-Hydroxyquinolines Are Boosting Agents of Copper-Related Toxicity in Mycobacterium tuberculosis.

Copper (Cu) ions are likely the most important immunological metal-related toxin utilized in controlling bacterial infections. Impairment of bacterial Cu resistance reduces viability within the host. Thus, pharmacological enhancement of Cu-mediated antibacterial toxicity may lead to novel strategies in drug discovery and development. Screening for Cu toxicity-enhancing antibacterial molecules identified 8-hydroxyquinoline (8HQ) to be a potent Cu-dependent bactericidal inhibitor of Mycobacterium tuberculosis The MIC of 8HQ in the presence of Cu was 0.16 µM for replicating and nonreplicating M. tuberculosis cells. We found 8HQ's activity to be dependent on the presence of extracellular Cu and to be related to an increase in cell-associated labile Cu ions. Both findings are consistent with 8HQ acting as a Cu ionophore. Accordingly, we identified the 1:1 complex of 8HQ and Cu to be its active form, with Zn, Fe, or Mn neither enhancing nor reducing its Cu-specific action. This is remarkable, considering that the respective metal complexes have nearly identical structures and geometries. Finally, we found 8HQ to kill M. tuberculosis selectively within infected primary macrophages. Given the stark Cu-dependent nature of 8HQ activity, this is the first piece of evidence that Cu ions within macrophages may bestow antibacterial properties to a Cu-dependent inhibitor of M. tuberculosis In conclusion, our findings highlight the metal-binding ability of the 8-hydroxyquinoline scaffold to be a potential focus for future medicinal chemistry and highlight the potential of innate immunity-inspired screening platforms to reveal molecules with novel modes of action against M. tuberculosis.

Stable Phenotypic Changes of the Host T Cells Are Essential to the Long-Term Stability of Latent HIV-1 Infection.

UNLABELLED: The extreme stability of the latent HIV-1 reservoir in the CD4(+) memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or bystander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We demonstrate here that host cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host cell phenotype at a system-wide level and revealed how the stable kinase activity changes networked to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that alter the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host cells are essential to the ability of HIV-1 to establish and maintain latent HIV-1 infection status. IMPORTANCE: The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive antiretroviral therapy. This extreme reservoir stability is somewhat surprising, since the latently HIV-1 infected CD4(+) memory T cells that form the structural basis of the viral reservoir should be exposed to cognate antigen over time. Antigen exposure would trigger a recall response and should deplete the reservoir, likely over a relatively short period. Our data demonstrate that stable and system-wide phenotypic changes to host cells are a prerequisite for the establishment and maintenance of latent HIV-1 infection events. The changes observed are consistent with an unresponsive, anergy-like T cell phenotype of latently HIV-1 infected host cells. An anergy-like, unresponsive state of the host cells of latent HIV-1 infection events would explain the stability of the HIV-1 reservoir in the face of continuous antigen exposure.

Disulfiram and Copper Ions Kill Mycobacterium tuberculosis in a Synergistic Manner.

Tuberculosis is a severe disease affecting millions worldwide. Unfortunately, treatment strategies are hampered both by the prohibitively long treatment regimen and the rise of drug-resistant strains. Significant effort has been expended in the search for new treatments, but few options have successfully emerged, and new treatment modalities are desperately needed. Recently, there has been growing interest in the synergistic antibacterial effects of copper ions (Cu(II/I)) in combination with certain small molecular compounds, and we have previously reported development of a drug screening strategy to harness the intrinsic bactericidal properties of Cu(II/I). Here, we describe the copper-dependent antimycobacterial properties of disulfiram, an FDA-approved and well-tolerated sobriety aid. Disulfiram was inhibitory to mycobacteria only in the presence of Cu(II/I) and exerted its bactericidal activity well below the active concentration of Cu(II/I) or disulfiram alone. No other physiologically relevant bivalent transition metals (e.g., Fe(II), Ni(II), Mn(II), and Co(II)) exhibited this effect. We demonstrate that the movement of the disulfiram-copper complex across the cell envelope is porin independent and can inhibit intracellular protein functions. Additionally, the complex is able to synergistically induce intracellular copper stress responses significantly more than Cu(II/I) alone. Our data suggest that by complexing with disulfiram, Cu(II/I) is likely allowed unfettered access to vulnerable intracellular components, bypassing the normally sufficient copper homeostatic machinery. Overall, the synergistic antibacterial activity of Cu(II/I) and disulfiram reveals the susceptibility of the copper homeostasis system of Mycobacterium tuberculosis to chemical attacks and establishes compounds that act in concert with copper as a new class of bacterial inhibitors.

Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation.

Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation.

Copper complexation screen reveals compounds with potent antibiotic properties against methicillin-resistant Staphylococcus aureus.

Macrophages take advantage of the antibacterial properties of copper ions in the killing of bacterial intruders. However, despite the importance of copper for innate immune functions, coordinated efforts to exploit copper ions for therapeutic interventions against bacterial infections are not yet in place. Here we report a novel high-throughput screening platform specifically developed for the discovery and characterization of compounds with copper-dependent antibacterial properties toward methicillin-resistant Staphylococcus aureus (MRSA). We detail how one of the identified compounds, glyoxal-bis(N4-methylthiosemicarbazone) (GTSM), exerts its potent strictly copper-dependent antibacterial properties on MRSA. Our data indicate that the activity of the GTSM-copper complex goes beyond the general antibacterial effects of accumulated copper ions and suggest that, in contrast to prevailing opinion, copper complexes can indeed exhibit species- and target-specific activities. Based on experimental evidence, we propose that copper ions impose structural changes upon binding to the otherwise inactive GTSM ligand and transfer antibacterial properties to the chelate. In turn, GTSM determines target specificity and utilizes a redox-sensitive release mechanism through which copper ions are deployed at or in close proximity to a putative target. According to our proof-of-concept screen, copper activation is not a rare event and even extends to already established drugs. Thus, copper-activated compounds could define a novel class of anti-MRSA agents that amplify copper-dependent innate immune functions of the host. To this end, we provide a blueprint for a high-throughput drug screening campaign which considers the antibacterial properties of copper ions at the host-pathogen interface.

An AP-1 binding site in the enhancer/core element of the HIV-1 promoter controls the ability of HIV-1 to establish latent infection.

Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon.

Copper resistance is essential for virulence of Mycobacterium tuberculosis.

Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.

Porins increase copper susceptibility of Mycobacterium tuberculosis.

Copper resistance mechanisms are crucial for many pathogenic bacteria, including Mycobacterium tuberculosis, during infection because the innate immune system utilizes copper ions to kill bacterial intruders. Despite several studies detailing responses of mycobacteria to copper, the pathways by which copper ions cross the mycobacterial cell envelope are unknown. Deletion of porin genes in Mycobacterium smegmatis leads to a severe growth defect on trace copper medium but simultaneously increases tolerance for copper at elevated concentrations, indicating that porins mediate copper uptake across the outer membrane. Heterologous expression of the mycobacterial porin gene mspA reduced growth of M. tuberculosis in the presence of 2.5 µM copper by 40% and completely suppressed growth at 15 µM copper, while wild-type M. tuberculosis reached its normal cell density at that copper concentration. Moreover, the polyamine spermine, a known inhibitor of porin activity in Gram-negative bacteria, enhanced tolerance of M. tuberculosis for copper, suggesting that copper ions utilize endogenous outer membrane channel proteins of M. tuberculosis to gain access to interior cellular compartments. In summary, these findings highlight the outer membrane as the first barrier against copper ions and the role of porins in mediating copper uptake in M. smegmatis and M. tuberculosis.

Copper-boosting compounds: a novel concept for antimycobacterial drug discovery.

We and others recently identified copper resistance as important for virulence of Mycobacterium tuberculosis. Here, we introduce a high-throughput screening assay for agents that induce a copper hypersensitivity phenotype in M. tuberculosis and demonstrate that such copper-boosting compounds are effective against replicating and nonreplicating M. tuberculosis strains.

Selected drugs with reported secondary cell-differentiating capacity prime latent HIV-1 infection for reactivation.

Reactivation of latent HIV-1 infection is considered our best therapeutic means to eliminate the latent HIV-1 reservoir. Past therapeutic attempts to systemically trigger HIV-1 reactivation using single drugs were unsuccessful. We thus sought to identify drug combinations consisting of one component that would lower the HIV-1 reactivation threshold and a synergistic activator. With aclacinomycin and dactinomycin, we initially identified two FDA-approved drugs that primed latent HIV-1 infection in T cell lines and in primary T cells for reactivation and facilitated complete reactivation at the population level. This effect was correlated not with the reported primary drug effects but with the cell-differentiating capacity of the drugs. We thus tested other cell-differentiating drugs/compounds such as cytarabine and aphidicolin and found that they also primed latent HIV-1 infection for reactivation. This finding extends the therapeutic promise of N'-N'-hexamethylene-bisacetamide (HMBA), another cell-differentiating agent that has been reported to trigger HIV-1 reactivation, into the group of FDA-approved drugs. To this end, it is also noteworthy that suberoylanilide hydroxamic acid (SAHA), a polar compound that was initially developed as a second-generation cell-differentiating agent using HMBA as a structural template and which is now marketed as the histone deacetylase (HDAC) inhibitor vorinostat, also has been reported to trigger HIV-1 reactivation. Our findings suggest that drugs with primary or secondary cell-differentiating capacity should be revisited as HIV-1-reactivating agents as some could potentially be repositioned as candidate drugs to be included in an induction therapy to trigger HIV-1 reactivation.

Kinase control prevents HIV-1 reactivation in spite of high levels of induced NF-κB activity.

Despite its clinical importance, the molecular biology of HIV-1 latency control is at best partially understood, and the literature remains conflicting. The most recent description that latent HIV-1 is integrated into actively expressed host genes has further confounded the situation. This lack of molecular understanding complicates our efforts to identify therapeutic compounds or strategies that could reactivate latent HIV-1 infection in patients, a prerequisite for the eradication of HIV-1 infection. Currently, many therapeutic development efforts operate under the assumption that a restrictive histone code could govern latent infection and that either dissipation of the histone-based restrictions or NF-κB activation could be sufficient to trigger HIV-1 reactivation. We here present data that suggest an additional, higher level of molecular control. During a high-content drug screening effort, we identified AS601245 as a potent inhibitor of HIV-1 reactivation in latently infected primary T cells and T cell lines. In either system, AS601245 inhibited HIV-1 reactivation despite high levels of induced NF-κB activation. This finding suggests the presence of a gatekeeper kinase activity that controls latent HIV-1 infection even in the presence of high levels of NF-κB activity. Potential therapeutic stimuli that do not target this gatekeeper kinase will likely fail to trigger efficient system-wide HIV-1 reactivation.

Repurposing sunscreen as an antibiotic: zinc-activated avobenzone inhibits methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major healthcare concern with associated healthcare costs reaching over ${\$}$1 billion in a single year in the USA. Antibiotic resistance in S. aureus is now observed against last line of defense antibiotics, such as vancomycin, linezolid, and daptomycin. Unfortunately, high throughput drug discovery approaches to identify new antibiotics effective against MRSA have not resulted in much tangible success over the last decades. Previously, we demonstrated the feasibility of an alternative drug discovery approach, the identification of metallo-antibiotics, compounds that gain antibacterial activity only after binding to a transition metal ion and as such are unlikely to be detected in standard drug screens. We now report that avobenzone, the primary active ingredient of most sunscreens, can be activated by zinc to become a potent antibacterial compound against MRSA. Zinc-activated avobenzone (AVB-Zn) potently inhibited a series of clinical MRSA isolates [minimal inhibitory concentration (MIC): 0.62-2.5 µM], without pre-existing resistance and activity without zinc (MIC: >10 µM). AVB-Zn was also active against clinical MRSA isolates that were resistant against the commonly used zinc-salt antibiotic bacitracin. We found AVB-Zn exerted no cytotoxicity on human cell lines and primary cells. Last, we demonstrate AVB-Zn can be deployed therapeutically as lotion preparations, which showed efficacy in a mouse wound model of MRSA infection. AVB-Zn thus demonstrates Zn-activated metallo-antibiotics are a promising avenue for future drug discovery.

Hit-and-run stimulation: a novel concept to reactivate latent HIV-1 infection without cytokine gene induction.

Current antiretroviral therapy (ART) efficiently controls HIV-1 replication but fails to eradicate the virus. Even after years of successful ART, HIV-1 can conceal itself in a latent state in long-lived CD4(+) memory T cells. From this latent reservoir, HIV-1 rebounds during treatment interruptions. Attempts to therapeutically eradicate this viral reservoir have yielded disappointing results. A major problem with previously utilized activating agents is that at the concentrations required for efficient HIV-1 reactivation, these stimuli trigger high-level cytokine gene expression (hypercytokinemia). Therapeutically relevant HIV-1-reactivating agents will have to trigger HIV-1 reactivation without the induction of cytokine expression. We present here a proof-of-principle study showing that this is a possibility. In a high-throughput screening effort, we identified an HIV-1-reactivating protein factor (HRF) secreted by the nonpathogenic bacterium Massilia timonae. In primary T cells and T-cell lines, HRF triggered a high but nonsustained peak of nuclear factor kappa B (NF-kappaB) activity. While this short NF-kappaB peak potently reactivated latent HIV-1 infection, it failed to induce gene expression of several proinflammatory NF-kappaB-dependent cellular genes, such as those for tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and gamma interferon (IFN-gamma). Dissociation of cellular and viral gene induction was achievable, as minimum amounts of Tat protein, synthesized following application of a short NF-kappaB pulse, triggered HIV-1 transactivation and subsequent self-perpetuated HIV-1 expression. In the absence of such a positive feedback mechanism, cellular gene expression was not sustained, suggesting that strategies modulating the NF-kappaB activity profile could be used to selectively trigger HIV-1 reactivation.

Pyrazolyl Thioureas and Carbothioamides with an NNSN Motif against MSSA and MRSA.

A novel series of copper-activatable drugs intended for use against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) were synthesized, characterized, and tested against the MSSA strain Newman and the MRSA Lac strain (a USA300 strain), respectively. These drugs feature an NNSN structural motif, which enables the binding of copper. In the absence of copper, no activity against MSSA and MRSA at realistic drug concentrations was observed. Although none of the novel drug candidates exhibits a stereocenter, sub-micromolar activities against SA Newman and micromolar activities against SA Lac were observed in the presence, but not in the absence, of bioavailable copper. Copper influx is a component of cellular response to bacterial infections, which is often described as nutritional immunity.

A copper-dependent compound restores ampicillin sensitivity in multidrug-resistant Staphylococcus aureus.

Multi-drug resistant Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), has become a worldwide, major health care problem. While initially restricted to clinical settings, drug resistant S. aureus is now one of the key causative agents of community-acquired infections. We have previously demonstrated that copper dependent inhibitors (CDIs), a class of antibiotics that are only active in the presence of copper ions, are effective bactericidal agents against MRSA. A second-generation CDI, APT-6K, exerted bactericidal activity at nanomolar concentrations. At sub-bactericidal concentrations, it effectively synergized with ampicillin to reverse drug resistance in multiple MRSA strains. APT-6K had a favorable therapeutic index when tested on eukaryotic cells (TI: > 30) and, unlike some previously reported CDIs, did not affect mitochondrial activity. These results further establish inhibitors that are activated by the binding of transition metal ions as a promising class of antibiotics, and for the first time, describe their ability to reverse existing drug resistance against clinically relevant antibiotics.

Assaying for Inorganic Polyphosphate in Bacteria.

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many bacteria. There are a variety of methods for quantifying polyP in biological materials, many of which are either labor-intensive or insensitive, limiting their usefulness. We present here a streamlined method for polyP quantification in bacteria, using a silica membrane column extraction optimized for rapid processing of multiple samples, digestion of polyP with the polyP-specific exopolyphosphatase ScPPX, and detection of the resulting free phosphate with a sensitive ascorbic acid-based colorimetric assay. This procedure is straightforward, inexpensive, and allows reliable polyP quantification in diverse bacterial species. We present representative polyP quantification from the Gram-negative bacterium (Escherichia coli), the Gram-positive lactic acid bacterium (Lactobacillus reuteri), and the mycobacterial species (Mycobacterium smegmatis). We also include a simple protocol for nickel affinity purification of mg quantities of ScPPX, which is not currently commercially available.

Differential Susceptibility of Mycoplasma and Ureaplasma Species to Compound-Enhanced Copper Toxicity.

RATIONALE: Mycoplasmas represent important etiologic agents of many human diseases. Due to increasing antimicrobial resistance and slow rate of novel discovery, unconventional methods of drug discovery are necessary. Copper ions are utilized in host microbial killing, and bacteria must regulate intracellular Cu concentrations to avoid toxicity. We hypothesized that human mollicutes may have susceptibility to Cu-induced toxicity, and compounds that augment copper-dependent killing. METHODS: Mycoplasma pneumoniae (Mpn), Ureaplasma parvum (Up), Ureaplasma urealyticum (Uu), and Mycoplasma hominis (Mh) were exposed to CuSO4 to determine minimal inhibitory concentrations (MICs). Once inhibitory concentrations had been determined, bacteria were treated with an FDA-approved drug disulfiram (DSF), glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), and 2,9-dimethyl-1,10-phenanthroline (neocuproine), with or without Cu2+, to determine compound MICs. RESULTS: Ureaplasma species and Mh were able to tolerate 30-60 µM CuSO4, while Mpn tolerated over 10-fold higher concentrations (>1 mM). GTSM inhibited growth of all four organisms, but was unaffected by Cu2+ addition. Inhibition by GTSM was reduced by addition of the cell-impermeant Cu chelator, bathocuproine disulfonate (BCS). Neocuproine exhibited Cu-dependent growth inhibition of all organisms. DSF exhibited Cu-dependent growth inhibition against Mh at low micromolar concentrations, and at intermediate concentrations for Mpn. CONCLUSION: MICs for CuSO4 differ widely among human mollicutes, with higher MICs for Mpn compared to Mh, Uu, and Up. DSF and Neocuproine exhibit Cu-dependent inhibition of mollicutes with copper concentrations between 25 and 50 µM. GTSM has copper-dependent anti-microbial activity at low levels of copper. Drug enhanced copper toxicity is a promising avenue for novel therapeutic development research with Mycoplasma and Ureaplasma species.