Targeting host-specific metabolic pathways-opportunities and challenges for anti-infective therapy.

Microorganisms can takeover critical metabolic pathways in host cells to fuel their replication. This interaction provides an opportunity to target host metabolic pathways, in addition to the pathogen-specific ones, in the development of antimicrobials. Host-directed therapy (HDT) is an emerging strategy of anti-infective therapy, which targets host cell metabolism utilized by facultative and obligate intracellular pathogens for entry, replication, egress or persistence of infected host cells. This review provides an overview of the host lipid metabolism and links it to the challenges in the development of HDTs for viral and bacterial infections, where pathogens are using important for the host lipid enzymes, or producing their own analogous of lecithin-cholesterol acyltransferase (LCAT) and lipoprotein lipase (LPL) thus interfering with the human host's lipid metabolism.

The Role of the Insulin/Glucose Ratio in the Regulation of Pathogen Biofilm Formation.

During the management of patients in acute trauma the resulting transient hyperglycemia is treated by administration of insulin. Since the effect of insulin, a quorum sensing compound, together with glucose affects biofilm formation in a concentration-specific manner, we hypothesize that the insulin/glucose ratio over the physiologic range modulates biofilm formation potentially influencing the establishment of infection through biofilm formation. METHODS: A variety of Gram-positive and Gram-negative bacteria were grown in peptone (1%) yeast nitrogen base broth overnight in 96-well plates with various concentrations of glucose and insulin. Biofilm formation was determined by the crystal violet staining procedure. Expression of insulin binding was determined by fluorescent microscopy (FITC-insulin). Controls were buffer alone, insulin alone, and glucose alone. RESULTS: Overall, maximal biofilm levels were measured at 220 mg/dL of glucose, regardless of insulin concentration (10, 100, 200 µU/mL) of the organism tested. In general, insulin with glucose over the range of 160-180 mg/dL exhibited a pattern of biofilm suppression. However, either above or below this range, the presence of insulin in combination with glucose significantly modulated (increase or decrease) biofilm formation in a microbe-specific pattern. This modulation appears for some organisms to be reflective of the glucose-regulated intrinsic expression of bacterial insulin receptor expression. CONCLUSION: Insulin at physiologic levels (normal and hyperinsulinemic) in combination with glucose can affect biofilm formation in a concentration-specific and microbe-specific manner. These findings may provide insight into the importance of co-regulation of the insulin/glucose ratio in patient management.

Utilization of Existing Human Kinase Inhibitors as Scaffolds in the Development of New Antimicrobials.

The prevalence and continuing expansion of drug resistance, both in clinical and community settings represents a major challenge for current antimicrobial therapy. The different approaches for addressing this challenge include (1) identification of novel antibacterials by repurposing of existing drugs originally that historically target host proteins; and (2) effect target switching through modification of existing antimicrobials. The focus of this manuscript is on these drug discovery strategies, with utility for development of new antimicrobials with different modes of action.

Fragment-Based Lead Discovery Strategies in Antimicrobial Drug Discovery.

Fragment-based lead discovery (FBLD) is a powerful application for developing ligands as modulators of disease targets. This approach strategy involves identification of interactions between low-molecular weight compounds (100-300 Da) and their putative targets, often with low affinity (KD ~0.1-1 mM) interactions. The focus of this screening methodology is to optimize and streamline identification of fragments with higher ligand efficiency (LE) than typical high-throughput screening. The focus of this review is on the last half decade of fragment-based drug discovery strategies that have been used for antimicrobial drug discovery.

Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions.

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 µU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

Evaluation of the Mitragynine Content, Levels of Toxic Metals and the Presence of Microbes in Kratom Products Purchased in the Western Suburbs of Chicago.

Kratom (Mitragyna speciosa, Korth) is a tree-like plant that is indigenous to Southeast Asia. Kratom leaf products have been used in traditional folk medicine for their unique combination of stimulant and opioid-like effects. Kratom is being increasingly used in the West for its reputed benefits in the treatment of pain, depression and opioid use disorder. Recently, the United States Food and Drug Administration and Centers for Disease Control have raised concerns regarding the contamination of some kratom products with toxic metals (Pb and Ni) and microbes such as Salmonella. To further explore this issue, eight different kratom products were legally purchased from various "head"/"smoke" shops in the Western Suburbs of Chicago and then tested for microbial burden, a panel of metals (Ni, Pb, Cr, As, Hg, Cd), and levels of the main psychoactive alkaloid mitragynine. All of the samples contained significant, but variable, levels of mitragynine (3.9-62.1 mg/g), indicating that the products were, in fact, derived from kratom. All but two of the samples tested positive for the presence of various microbes including bacteria and fungi. However, none of the samples tested positive for Salmonella. Seven products showed significant levels of Ni (0.73-7.4 µg/g), Pb (0.16-1.6 µg/g) and Cr (0.21-5.7 µg/g) while the other product was negative for metals. These data indicate that many kratom products contain variable levels of mitragynine and can contain significant levels of toxic metals and microbes. These findings highlight the need for more stringent standards for the production and sale of kratom products.

Comparison of In Vitro Chlamydia muridarum Infection Under Aerobic and Anaerobic Conditions.

Although Chlamydia infects host body regions that are hypoxic to anoxic, standard Chlamydiae culture conditions are in CO2 enriched (5%) atmospheric oxygen (21%). Because of its success in causing disease in principally anaerobic body sites, e.g., vaginal tract, we hypothesize that Chlamydia has an anaerobic life cycle that plays a role in its maintenance in the host. Using a model system developed for the anaerobic culture of mammalian cells, we assessed the anoxic infectious cycle of C. muridarum in anaerobically cultured HeLa 229 cells. In the absence of oxygen, C. muridarum is capable of going through their life cycle, although its cycle is slowed (2 days post-infection anaerobic vs. 1 day aerobic). Interestingly, in addition to a slower rate of replication, there is a reduction in Chlamydia inclusion number and size as compared to aerobic controls. Anaerobic infected host cell physiology also changed with IL-6 and IL-8 production significantly lower (p ≤ 0.05) compared to aerobic infected host cells (day 4 post-infection). These findings demonstrate that Chlamydia are capable of replicating in the absence of oxygen.

C4-Phenylthio ß-lactams: Effect of the chirality of the ß-lactam ring on antimicrobial activity.

C4-phenylthio ß-lactams are a new family of antibacterial agents that have activity against two phylogenetically distant bacteria - Mycobacterium tuberculosis (Mtb) and Moraxella catarrhalis (M. cat). These compounds are effective against ß-lactamase producing Mtb and M. cat unlike the clinically relevant ß-lactam antibiotics. The structure-activity relationship for the C4 phenylthio ß-lactams has not yet been completely defined. Earlier efforts in our laboratories established that the C4-phenylthio substituent is essential for antimicrobial activity, while the N1 carbamyl substituent plays a more subtle role. In this present study, we investigated the role that the stereochemistry at C4 plays in these compounds' antibacterial activity. This was achieved by synthesizing and testing the antimicrobial activity of diastereomers with a chiral carbamyl group at N1. Our findings indicate that a strict stereochemistry for the C4-phenylthio ß-lactams is not required to obtain optimal anti-Mtb and anti-M. cat activity. Furthermore, the structure-bioactivity profiles more closely relate to the electronic requirement of the phenylthiogroup. In addition, the MICs of Mtb are sensitive to growth medium composition. Select compounds showed activity against non-replicating and multi-drug resistant Mtb.

Anaerobic Growth and Maintenance of Mammalian Cell Lines.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2-3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage. Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Differential expression of cytokines and receptor expression during anoxic growth.

OBJECTIVE: Cell density in tumor cell three dimensional (3D) cultures affects secretome expression of components. A microenvironment characteristic shared by high-density 3D cell culture and in vivo tumor masses is poor oxygenation, with anoxia being a natural cell state in tumor centers. Until recently, the ability to study anoxia-adapted cell physiology was not possible. Using a newly-developed methodology, anoxic HeLa cell secretome expression was measured. RESULTS: Anoxic HeLa cell cytokine levels after 3 days' (hypoxia inducible factor, HIF1 positive) and 10 days' growth (HIF1 negative; anaerobic respiration) were significantly (p < 0.01) higher than normoxic controls for: IL-8 (1.8- and 3.4-fold higher, respectively), GRO (1.3- and 1.1-fold higher, respectively), and IL-11 (1.4- and 1.1-fold higher, respectively). In contrast, G-CSF, IFNα2, and CXCL-10 levels decreased over time (day 3 vs. day 10). Thus, metabolically active HeLa cells respond to the lack of oxygen, in part, by regulating the levels of cytokines produced. Cytokines expressed at increased levels, in the absence of oxygen, correspond to a secretomic profile reported for paracrine signaling pathways associated with metastasis. Further studies defining physiologic changes that occur upon anoxic growth may lead to the discovery of novel chemotherapeutic drug targets.

Prevalence of a Cefazolin Inoculum Effect Associated with blaZ Gene Types among Methicillin-Susceptible Staphylococcus aureus Isolates from Four Major Medical Centers in Chicago.

The efficacy of cefazolin with high-inoculum methicillin-susceptible Staphylococcus aureus (MSSA) infections remains in question due to therapeutic failure inferred as being due to an inoculum effect (InE). This study investigated the local prevalence of a cefazolin InE (CInE) and its association with staphylococcal blaZ gene types among MSSA isolates in the Chicago area. Four medical centers in Chicago, IL, contributed MSSA isolates. Cefazolin MICs (C-MIC) were determined at 24 h by the broth microdilution method using a standard inoculum (SI; 5 × 105 CFU/ml) and a high inoculum (HI; 5 × 107 CFU/ml). The CInE was defined as (i) a ≥4-fold increase in C-MIC between SI and HI and/or (ii) a pronounced CInE, i.e., a nonsusceptible C-MIC of ≥16 µg/ml at HI. PCR was used to amplify the blaZ gene, followed by agarose gel electrophoresis and sequencing to determine the gene type. Approximately 269 MSSA isolates were included. All but one isolate were susceptible to cefazolin at SI, and 97% remained susceptible at HI. A total of 196 isolates (73%) were blaZ positive, with the blaZ types led by gene type C (40%). CInE was seen in 45 blaZ-positive isolates (23%), with 44 (22%) presenting a ≥4-fold increase in C-MIC (SI to HI) and 5 (3%) a pronounced CInE. Four of the five met both definitions of CInE, two of which expressed the type A gene. The prevalence of a pronounced CInE associated with the type A blaZ gene from MSSA isolates in Chicago is low. Our predilection for cefazolin use, even early in the management of hospitalized MSSA infections, is tenable.

A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production.

The center of tumors, stem cell niches and mucosal surfaces all represent areas of the body that are reported to be anoxic. However, long-term study of anoxic cell physiology is hindered by the lack of a sustainable method permitting cell cultivation in the complete absence of oxygen. A novel methodology was developed that enabled anoxic cell cultivation (17d maximum time tested) and cell passage. In the absence of oxygen, cell morphology is significantly altered. All cells tested exhibited morphologic changes, i.e., a combination of tethered (monolayer-like) and runagate (suspension-like) morphologies. Both morphologies replicated (Vero and HeLa cells tested) and could be passaged anaerobically. In the absence of exogenous oxygen, anoxic cells produced reactive oxygen species (ROS). Anaerobic runagate HeLa and Vero cells increased ROS production from day 3 to day 10 by 2- and 3-fold, respectively. In contrast, anoxic tethered HeLa and Vero cells either showed no significant change in ROS production between days 3 and 10 or exhibited a 3-fold decrease in ROS, respectively. Detection of ROS was inversely related to detection of hypoxia-inducible factor-1α (HIF1) mRNA and HIF-1 protein expression which cycled over a 10-day period. This methodology has broad applications for the study of tumor and stem cell physiology as well as gastrointestinal cell-microbiome interactions. In addition, sustainable anaerobic cell culture may lead to the identification of novel pathways and targets for chemotherapeutic drug development.

Attenuation of antimicrobial activity by the human steroid hormones.

Upon entering the human host, Staphylococcus aureus is exposed to endogenous steroid hormones. The interaction between S. aureus and dehydroepiandosterone (DHEA) results in an increased resistance to the host cationic defense peptide, ß-1 defensin, as well as vancomycin and other antibiotics that have a positive charge. The increased resistance to vancomycin is phenotypic and appears to correlate with a DHEA-mediated alteration in cell surface architecture. DHEA-mediated cell surface changes include alterations in: cell surface charge, surface hydrophobicity, capsule production, and carotenoid production. In addition, exposure to DHEA results in decreased resistance to lysis by Triton X-100 and lysozyme, indicating activation of murien hydrolase activity. We propose that DHEA is an interspecies quorum-like signal that triggers innate phenotypic host survival strategies in S. aureus that include increased carotenoid production and increased vancomycin resistance. Furthermore, this DHEA-mediated survival system may share the cholesterol-squalene pathway shown to be statin sensitive thus, providing a potential pathway for drug targeting.

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV's effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

Host environmental signals and effects on biofilm formation.

Biofilm matrix formation is a phenotype linked to the ability to survive a hostile host environment that includes the presence of antimicrobial peptides and serum factors. Multiple hormones and other host derived factors have been shown to function as exogenous quorum signaling compound homologs that inform microbes of their in situ presence, thus triggering a shift from a planktonic to the sessile biofilm phenotype. The focus of this review is to describe the impact various host-derived factors have on the initial steps required for biofilm formation, i.e., adherence to host surfaces and multiplication in the host.

Herpes Simplex Virus (HSV) Modulation of Staphylococcus aureus and Candida albicans Initiation of HeLa 299 Cell-Associated Biofilm.

Although herpes simplex virus type-1 (HSV-1), and type-2 (HSV-2), Staphylococcus aureus and Candida albicans co-habit the oral and genital mucosa, their interaction is poorly understood. We determined the effect HSV has on bacterial and/or fungal adherence, the initial step in biofilm formation. HeLa229 cells were infected with HSV-1 (KOS) gL86 or HSV-2 (KOS) 333gJ (-) at a multiplicity of infection (MOI) of 50 and 10. S. aureus (ATCC 25923) and/or C. albicans (yeast forms or germ tube forms) were co-incubated for 30 min (37 °C; 5 % CO2; 5:1 organism: HeLa cell ratio; n = 16) with virus-infected HeLa cells or uninfected HeLa cell controls. Post-incubation, the monolayers were washed (3x; PBS), lysed (RIPA), and the lysate plated onto Fungisel and/or mannitol salts agar for standard colony count. The level of HeLa-associated S. aureus was significantly decreased (P < 0.05) for both HSV-1- and HSV-2-infected cells, as compared to virus-free HeLa cell controls (38 and 59 % of control, respectively). In contrast, HSV-1 and HSV-2 significantly (P < 0.05) enhanced HeLa cell association of C. albicans yeast forms and germ tube approximately two-fold, respectively. The effect of S. aureus on germ tube and yeast form adherence to HSV-1- and HSV-2-infected cells was specific for the Candida phenotype tested. Our study suggests that HSV, while antagonist towards S. aureus adherence enhances Candida adherence. Furthermore, the combination of the three pathogens results in S. aureus adherence that is either unaffected, or partially restored depending on both the herpes viral species and the fungal phenotype present.

Non-transpeptidase binding arylthioether ß-lactams active against Mycobacterium tuberculosis and Moraxella catarrhalis.

The prevalence of drug resistance in both clinical and community settings as a consequence of alterations of biosynthetic pathways, enzymes or cell wall architecture is a persistent threat to human health. We have designed, synthesized, and tested a novel class of non-transpeptidase, ß-lactamase resistant monocyclic ß-lactams that carry an arylthio group at C4. These thioethers exhibit inhibitory and cidal activity against serine ß-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=8) ß-lactamase producing Moraxella catarrhalis clinical isolates.

Antimicrobial and biofilm effects of herbs used in traditional Chinese medicine.

Of the twenty-two components of tea decoctions commonly used to treat infections, only Scutellaria, Taraxacum, Tussilago and Glycyrrhiza exhibited antimicrobial activity. The activity, when present, was organism specific, i.e., anti-Staphylococcus aureus, including anti-MRSA activity under aerobic and/or anaerobic conditions. However, with the exception of Scutellaria, sub-inhibitory concentrations of the herbs exhibited a pattern of inducing enhanced production of biofilm.

C4-alkylthiols with activity against Moraxella catarrhalis and Mycobacterium tuberculosis.

Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infectious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New approaches to drug development are especially needed to target organisms that exhibit broad antibiotic resistance due to expression of ß-lactamases which is the most common mechanism by which bacteria become resistant to ß-lactam antibiotics. We designed and synthesized 20 novel monocyclic ß-lactams with alkyl- and aryl-thio moieties at C4, and subsequently tested these for antibacterial activity. These compounds demonstrated intrinsic activity against serine ß-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=6) ß-lactamase producing Moraxella catarrhalis clinical isolates.

Possible role of sarA in dehydroepiandrosterone-mediated increase in Staphylococcus aureus resistance to vancomycin.

BACKGROUND: Dehydroepiandrosterone (DHEA), a steroid present throughout life, can induce an increase in resistance to vancomycin in methicillin-sensitive and methicillin-resistant clinical isolates of Staphylococcus aureus. METHODS: The in vitro effect of DHEA on vancomycin killing of S. aureus with mutations in sarA and/or agr was determined by standard microtiter protocols and time to kill determinations. RESULTS: Of the isolates tested, the strain with a deletion in sarAderived from a DHEA- responsive parent was not protected from vancomycin killing by DHEA. However, DHEA significantly (p < 0.01) slowed the rate of vancomycin killing of sarA-. CONCLUSION: These data indicate that sarA may play a role in DHEA-mediated protection from vancomycin killing of S. aureus.