Imcroporin,a New Cationic Antimicrobial Peptide from the Venom of the Scorpion Isometrus maculates

The pace of resistance against antibiotics almost exceeds that of the development of new drugs. As many bacteria have become resistant to conventional antibiotics, new drugs or drug resources are badly needed to combat antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial peptides, rich sources existing in nature, are able to effectively kill multidrug-resistant pathogens. Here, imcroporin, a new antimicrobial peptide, was screened and isolated from the cDNA library of the venomous gland of Isometrus maculates. The MIC of imcroporin against MRSA was 50 μg/ml, 8-fold lower than that of cefotaxime and 40-fold lower than that of penicillin. Imcroporin killed bacteria rapidly in vitro, inhibited bacterial growth, and cured infected mice. These results revealed that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.The war between human beings and bacteria is still going on. On one hand, conventional antibiotics defend against bacterial infection. On the other hand, antibiotic resistance fights back. Among the pathogens of concern, methicillin-resistant Staphylococcus aureus (MRSA) takes priority. From 1999 to 2005, the estimated number of MRSA-related hospitalizations in the United States more than doubled, from 127,036 to 278,203, and MRSA-related deaths averaged about 5,500 per year (range, 3,809 to 7,372) (27). Vancomycin was most commonly used in the past 2 decades to treat MRSA infections. However, vancomycin-intermediate S. aureus has emerged (1, 3, 34). Therefore, new kinds of antimicrobial agents are badly needed.Cationic host defense peptides play an important role in the innate immune response. These peptides have potent antimicrobial activity against gram-positive and gram-negative bacteria, fungi, parasites, and some viruses (19). Such peptides can be constitutively expressed or induced by bacteria in many organisms (20, 23, 29, 38). They are widely distributed in nature, from insects and plants to highly evolved animal species with more complex immune systems (6, 7). More than 2 decades ago, these defense molecules were initially isolated from insect lymph, the skin of frogs, and mammalian neutrophil granules and were demonstrated to have antibacterial properties. Since then, interest in the distribution and application of these peptides has been escalating, leading to the discovery of more than 1,300 cationic peptides from numerous species (16, 31, 37).Cationic antimicrobial peptides are defined as peptides of 12 to 50 amino acids with a net positive charge of 2 to 9 (5, 7, 20). Despite their small size and common physicochemical features, cationic antimicrobial peptides exhibit a range of structures. The peptides can be divided into four groups according to their secondary structures: amphipathic α-helices, amphiphilic peptides with two to four β-strands, loop structures, and extended structures (7, 17, 26). Moreover, many cationic antimicrobial peptides have activity against antibiotic-resistant bacteria. As antibiotic-resistant pathogens have become a threat to human health (2, 4, 25, 36), the cationic antimicrobial peptides are one of the new strategies against infective diseases (21, 22, 31, 33).Several cationic antimicrobial peptides have been found in scorpion hemolymph and venom (9, 15), including hadrurin (35), scorpine (10), opistoporins (30), parabutoporin (30), IsCTs (13, 28), pandinins (11), and mucroporin (12). In the present study, we isolated a cationic antimicrobial peptide, termed imcroporin, from the cDNA library of the venomous gland of the scorpion Isometrus maculates. Imcroporin showed potent growth-inhibitory activity against antibiotic-resistant gram-positive pathogens, but not gram-negative bacteria, and relatively low hemolytic activity against human erythrocytes. What is more, imcroporin killed the bacteria rapidly and cured infected mice. These results indicate that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.     

Intracellular Activity of Antibiotics against Staphylococcus aureus in a Mouse Peritonitis Model

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log₁₀ number of CFU/ml (Δlog₁₀ CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Δlog₁₀ CFU/ml) > cefuroxime (3.56 Δlog₁₀ CFU/ml) > rifampin (1.86 Δlog₁₀ CFU/ml) > gentamicin (0.61 Δlog₁₀ CFU/ml) > azithromycin (0.21 Δlog₁₀ CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.Staphylococcus aureus is a major human pathogen that causes both community- and hospital-acquired infections (35). It causes a diverse array of infections ranging from relatively minor skin and wound infections to more serious and life-threatening diseases such as pneumonia (20, 46), endocarditis (48), osteomyelitis (17, 29), arthritis (1), and meningitis (40). Some of these types of S. aureus infections, e.g., endocarditis, are associated with high rates of mortality (25 to 50%), despite antimicrobial treatment (48, 49, 57). Furthermore, S. aureus infections are often persistent and are associated with treatment difficulties, such as a slow response to antibiotic treatment and recurrences, that lead to an extended duration of antimicrobial therapy (11, 13, 31). The antimicrobial treatment of S. aureus infections has also become more difficult due to the emergence of multidrug-resistant strains (3, 4).Several factors may help explain the capacity of staphylococci to avoid the actions of antibiotics. Biofilm formation might be the main reason for a deficient antibiotic effect when foreign bodies are involved in the staphylococci infections (12, 15, 53). Otherwise, the intracellular presence of the bacteria could offer a good explanation for the slow response to antibiotics, since bacteria located intracellularly might be protected from the effects of antibiotics (55).S. aureus has classically been classified as an extracellular pathogen (21). Conversely, several reports have established that S. aureus internalizes and survives within professional and even nonprofessional mammalian phagocytes (7, 19, 24, 25, 26, 27). The attitude is therefore changing toward classifying S. aureus as a facultative/opportunistic intracellular pathogen (13, 36, 41, 42, 55).Having an intracellular target for antimicrobial therapy is more complex than having an extracellular target, because intracellular antimicrobial activity further depends on the penetration into and accumulation in the cell, cellular metabolism, the subcellular disposition, and the bioavailability of the drug. The bacterial responsiveness to antibiotics can also change intracellularly (54, 55). Antimicrobial activity is therefore often impaired intracellularly (6, 56).To date, this knowledge of the intracellular presence of S. aureus has not influenced the choice of antibiotic to be used for the treatment for S. aureus infections. Penicillinase-stable penicillins, for instance, are considered the mainstay of treatment for methicillin-susceptible S. aureus infections (5, 23, 35), even though penicillins are usually considered not to penetrate cells (8, 30, 50).Recurrent S. aureus infections may also, at least partly, be explained by the intracellular presence of the bacteria. Gresham et al. demonstrated that polymorphonuclear neutrophils with intracellular S. aureus isolated from the peritoneums of infected mice could cause a new infection by intraperitoneal injection of these cells into healthy mice (24). They also demonstrated that intracellular survival was linked to the global regulator sar, which regulates multiple virulence factors in S. aureus. These two observations could together indicate that intracellular survival is a part of the pathogenesis of S. aureus.Appropriate models for the testing of the intracellular activities of antimicrobials against S. aureus are needed. Several in vitro models that use various cells and cell lines are available for the study of intracellular S. aureus (6, 10, 18, 24, 44, 51), but only a few in vivo models have been developed.Here we present an in vivo model that can be used to study the intracellular activities of antimicrobials against S. aureus.(Part of this study was presented at the 16th European Congress of Clinical Microbiology and Infectious Diseases, Nice, France.)     

NB-002, a Novel Nanoemulsion with Broad Antifungal Activity against Dermatophytes,Other Filamentous Fungi,and Candida albicans

NB-002 is an oil-in-water emulsion designed for use for the treatment of skin, hair, and nail infections. The activity of NB-002 was compared to the activities of the available antifungal drugs against the major dermatophytes responsible for cutaneous infections, Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum, and Microsporum spp., as well as 12 other genera of filamentous fungi. NB-002 consistently displayed fungicidal activity against all dermatophytes. The comparator compounds were either fungistatic or fungicidal, and for some strain-drug combinations, tolerance was observed. Assessment of the development of spontaneous resistance to NB-002 in different dermatophyte species yielded few stably resistant mutants. For filamentous nondermatophyte fungi, the MIC range varied from 0.06 to 0.5 μg/ml for Alternaria spp. to 2 to 8 μg/ml for Paecilomyes spp. NB-002 had activity against both azole-susceptible and -resistant Candida albicans yeast isolates, with MIC₉₀s of 2 μg/ml, respectively, and minimum fungicidal concentrations at which 90% of isolates are inhibited of 4 and 8 μg/ml, respectively. The kinetics of the fungicidal activity of NB-002 against T. rubrum isolates were compared to those of the other antifungal drugs. NB-002 killed both mycelia and microconidia even when the fungal forms were dormant or not actively growing. Electron micrographs of mycelia and spores treated with NB-002 showed the significant disruption of the fungal structure. The in vitro broad coverage of NB-002 against filamentous fungi, dermatophytes, and C. albicans, as well as its rapid fungicidal activity, warrants further investigations to ascertain if NB-002 would be useful for the treatment of cutaneous mycoses.Superficial fungal infections are found in the top layers of the skin and mucous membranes, the hair, and nails. Examples of fungal infections of the skin and other external surfaces include athlete''s foot, jock itch, ringworm, and other tinea infections. Most of these infections are caused by three genera of dermatophytes: Trichophyton, Epidermophyton, and Microsporum spp. (3, 4, 20, 29, 45, 47).Filamentous fungi that are normal soil saprophytes have also emerged as major opportunistic fungi, especially in immunosuppressed patients (34, 53). Such organisms include Aspergillus spp. (1, 18, 34, 42, 49), Fusarium spp. (18, 22, 28, 34, 36, 49), Scedosporium spp. (18, 34), Paecilomyces spp. (23, 24), Scopulariopsis spp. (18), Scytalidium spp. (11), Chaetomium spp. (13), Alternaria spp. (2, 18, 49), Acremonium spp. (18), and Curvularia spp. (49). Yeasts such as Candida albicans also cause skin infections, generally at sites between the fingers and toes, around the anus, and on the penis or at sites of abrasion where the skin is continuously moist (46).Most cutaneous infections are treated with topical antifungals containing naftidine, tolnaftate, terbinafine, or itraconazole. Oral therapies of griseofulvin, terbinafine, and itraconazole are used to treat tinea capitis. Nail infections can be treated with orally administered agents as well as the topical agent ciclopirox. Terbinafine and azole-like compounds carry the risk of liver and cardiac side effects and drug-drug interactions (14, 31, 38). Topical therapies for inflammatory dermatomycoses often combine an azole and a corticosteroid to rapidly reduce inflammatory symptoms and to increase the bioavailability of the antifungal agent (30).Antimicrobial nanoemulsions are highly stable oil-in-water emulsions composed of nanometer-sized, positively charged droplets that have broad-spectrum activity against enveloped viruses, fungi, and bacteria (5, 17, 25-27, 35, 43, 48). NB-002 contains the cationic quaternary ammonium compound cetylpyridinium chloride (CPC) oriented at its oil-water interface, which stabilizes the nanoemulsion droplets, contributes to the anti-infective activity, and serves as a marker of activity.Studies with NB-002 containing fluorescein have shown that the nanodroplets permeate human cadaver skin by a transfollicular route (6). By the use of a modified Franz cell apparatus, NB-002 was also shown to diffuse laterally from hair follicles and sebaceous glands along tissue planes to reach concentrations in excess of 200 μg/g in human cadaver epidermis as far as 1 cm from the site of application (7). This concentration is significantly above the MIC₉₀ and the minimum fungicidal concentration at which 90% of isolates are inhibited (MFC₉₀) or the ranges of MICs and MFCs determined in this work: ≤4 μg/ml for Trichophyton spp., Microsporum spp., and Epidermophyton floccosum.The studies described here assessed the microbiological activity of NB-002 against fungal pathogens that cause cutaneous infections. Furthermore, we show that the fungicidal activity of NB-002 is rapid and that it kills both the microconidia and mycelia of the dermatophyte Trichophyton rubrum. Consistent with this kill-on-contact mechanism of action, the MICs for the majority of dermatophyte isolates spontaneously resistant to NB-002 were plus or minus twofold of the initial MIC.     

TelA Contributes to the Innate Resistance of Listeria monocytogenes to Nisin and Other Cell Wall-Acting Antibiotics

Nisin is a class I bacteriocin (lantibiotic), which is employed by the food and veterinary industries and exhibits potent activity against numerous pathogens. However, this activity could be further improved through the targeting and inhibition of factors that contribute to innate nisin resistance. Here we describe a novel locus, lmo1967, which is required for optimal nisin resistance in Listeria monocytogenes. The importance of this locus, which is a homologue of the tellurite resistance gene telA, was revealed after the screening of a mariner random mutant bank of L. monocytogenes for nisin-susceptible mutants. The involvement of telA in nisin resistance was confirmed through an analysis of a nonpolar deletion mutant. In addition to being 4-fold-more susceptible to nisin, the ΔtelA strain was also 8-fold-more susceptible to gallidermin and 2-fold-more susceptible to cefuroxime, cefotaxime, bacitracin, and tellurite. This is the first occasion upon which telA has been investigated in a Gram-positive organism and also represents the first example of a link being established between a telA gene and resistance to cell envelope-acting antimicrobials.Nisin is a ribosomally synthesized cationic antimicrobial peptide that has been employed commercially for over 50 years in the preservation of foodstuffs (22) and, more recently, as an antimastitis agent (15). It also exhibits great potency against a number of human clinical pathogens, including many multidrug-resistant strains (33), and as a consequence of the continually diminishing options available to clinicians when targeting such microorganisms, the application of nisin has been the subject of renewed attention. Nisin is the prototypical example of the class I group of bacteriocins, which are also known as lantibiotics by virtue of the presence of unusual posttranslationally introduced structures known as lanthionines (12). In addition to being the most thoroughly characterized lantibiotic, nisin is also an example of a cell envelope-acting antimicrobial, acting through a combination of inhibiting peptidoglycan synthesis and forming pores in the cell membrane of target cells (3, 4, 18, 45).Despite the potency of nisin, there is evidence that suggests that its activity would be even greater were it not for factors that contribute to the innate resistance of some target microorganisms; the deletion of virR and mprF is known to result in 32- and 16-fold reductions in resistance to nisin in Listeria monocytogenes (9). For clarity, we discriminate between the mechanisms underpinning acquired resistance (resistance occurring in a formerly susceptible strain) and innate resistance (resistance intrinsically associated with particular genera or species). One example of a system contributing to innate resistance is DltA, which is required for the d-alanyl decoration of teichoic acid in the cell wall of many Gram-positive microorganisms. Its role in antimicrobial resistance was first noted when a disruption of dltA resulted in the sensitization of Staphylococcus aureus to the lantibiotic gallidermin as a consequence of a reduced capacity to repulse positively charged compounds (30). This susceptibility and, indeed, a susceptibility to a wider range of cationic antimicrobial peptides (CAMPs), including nisin, defensins, vancomycin, polymyxin B, and colistin, are also apparent in dltA mutants of Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecalis, and Listeria monocytogenes (14, 20, 25, 30, 35). An altered cell envelope charge, in this case due to the nonlysinylation of membrane phospholipids, is also the basis for the enhanced susceptibility of mprF mutants of S. aureus, L. monocytogenes, and Bacillus anthracis to nisin and other CAMPs (29, 36, 42). Unsurprisingly, eliminating the VirR regulator component of the two-component signal transduction system (VirRS) that regulates the expression of both dltA and mprF in L. monocytogenes also impacts susceptibility to CAMPs (23, 42). Other loci that play a role in the innate resistance of Gram-positive bacteria to nisin include lisRK, lmo0327, pbp2229, sigB, graS, nsr, and abcAB (1, 11, 16, 24, 34, 37, 40). There have also been a number of loci linked with acquired resistance to nisin through gene expression-based studies. An analysis of nisin-resistant mutants of Lactococcus lactis Il1403 revealed the increased expression of a number of different genes, including cell wall-related loci, operons involved in metabolism, as well as a number of genes involved in transport and stress responses (21). The contribution of some of these genes, i.e., dltD, galKMT, and ahrC, to nisin resistance was subsequently confirmed by using genetic knockouts. The involvement of another set of genes, ysaBC, was confirmed when overexpression was found to lead to increased nisin resistance (21). Expression studies have also established that the histidine kinase-encoding gene (lmo1021), a penicillin binding protein determinant (lmo2229), and a gene encoding a protein of unknown function (lmo2487) are all upregulated in spontaneously nisin-resistant L. monocytogenes strains (16).Here the screening of a mariner transposon bank of L. monocytogenes EGDe has resulted in the identification of a mutant that is susceptible to nisin as a consequence of the disruption of lmo1967, a gene homologous to tellurite resistance loci, designated telA, found in many members of the Firmicutes and Proteobacteria. This is the first occasion in which a telA gene has been associated with resistance to a cell envelope-acting antimicrobial and the first time that it has been studied in a Gram-positive bacterium. The study of this transposon mutant, and of another mutant in which the gene was removed in a nonpolar manner, revealed that telA also contributes to the pathogen''s natural resistance to tellurite and to many cell envelope-acting antimicrobials, including gallidermin, bacitracin, cefuroxime, and cefotaxime.     

Biofilm Formation and Effect of Caspofungin on Biofilm Structure of Candida Species Bloodstream Isolates

Candida biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different Candida species, particularly Candida albicans, C. tropicalis, and C. parapsilosis, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against C. albicans and C. tropicalis cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed Candida species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in C. albicans and C. tropicalis biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in Candida species biofilm populations. The clinical implications of these findings are still unknown.Candida species are opportunistic pathogens that cause superficial and systemic diseases in critically ill patients (8, 22, 44) and are associated with high mortality rates (35%) and costly treatments (8, 19). They rank among the four most common causes of bloodstream infection in U.S. hospitals, surpassing gram-negative rods in incidence (6, 17).Recent studies suggest that the majority of disease produced by this pathogen is associated with a biofilm growth style (7, 16, 28, 48). Biofilms are self-organized communities of microorganisms that grow on an abiotic or biotic surface, are embedded in a self-produced matrix consisting of an extracellular polymeric substance (14, 15, 55), and when associated with implanted medical devices are commonly refractive to antimicrobial therapy.As opportunistic pathogens, Candida species are able to attach to polymeric surfaces and generate a biofilm structure, protecting the organisms from the host defenses and antifungal drugs (11, 16, 45, 48). Candida biofilms are more resistant than their planktonic counterparts to various antifungal agents, including amphotericin B (AMB), fluconazole, itraconazole, and ketoconazole (20, 38, 50). However, the molecular basis for the antifungal resistance of biofilm-related organisms is not completely understood.The complex architecture of Candida biofilms observed both in vitro and in vivo suggests that morphological differentiation to produce hyphae plays an important role in biofilm formation and maturation (7, 32, 33). Baillie and Douglas demonstrated that although mutant cells fixed in either a hyphal or a yeast form can develop into biofilms, the hyphal structure is the essential element for providing the integrity and multilayered architecture of a biofilm (4). It has been reported that Candida parapsilosis, C. glabrata, and C. tropicalis biofilms are not as large as those generated by C. albicans; however, further structural analysis studies are needed to describe biofilm formation by these organisms (30, 31).The mechanisms responsible for the resistance characteristics displayed by Candida biofilms are unclear. Possible mechanisms include a decreased growth rate; nutrient limitation of cells in the biofilm; expression of resistance genes, particularly those encoding efflux pumps; increased cell density; cell aging; or the presence of “persister” cells in the biofilm (1, 3, 5, 29, 34, 36, 38, 43, 46, 48, 50, 51).The echinocandins are a novel class of semisynthetic amphiphilic lipopeptides that display important antifungal activity. The echinocandins that are presently marketed are caspofungin (CAS), micafungin, and anidulafungin. The echinocandins show considerable efficacy in vitro and in vivo in the treatment of candidemia and invasive candidiasis (25, 27, 42). CAS is the first antifungal agent to be licensed that inhibits the synthesis of β-1,3-glucan, the major structural component of Candida cell walls; glucan synthesis might prove to be a particularly effective target for biofilms (29, 31, 38, 48, 50). The paradoxical attenuation of antifungal activity at high echinocandin concentrations is a phenomenon that usually occurs with C. albicans isolates and appears to be specific to CAS among echinocandins. The cells surviving at high concentrations appear to be subject to some drug effect, showing evidence of slowed growth in the presence of CAS (53, 54). Recent studies have described this effect in Candida species biofilms (24, 37, 47); however, we are not aware of studies that have elucidated the effect of CAS on Candida biofilm structure. The present study was designed to (i) characterize the in vitro biofilm growth of Candida species bloodstream isolates and (ii) use scanning electron microscopy (SEM) to obtain visual evidence of the effect of CAS on biofilm morphology changes associated with paradoxical growth (PG).     

Slow Release of Nitric Oxide from Charged Catheters and Its Effect on Biofilm Formation by Escherichia coli

Catheter-associated urinary tract infection is the most prevalent cause of nosocomial infections. Bacteria associated with biofilm formation play a key role in the morbidity and pathogenesis of these infections. Nitric oxide (NO) is a naturally produced free radical with proven bactericidal effect. In this study, Foley urinary catheters were impregnated with gaseous NO. The catheters demonstrated slow release of nitric oxide over a 14-day period. The charged catheters were rendered antiseptic, and as such, were able to prevent bacterial colonization and biofilm formation on their luminal and exterior surfaces. In addition, we observed that NO-impregnated catheters were able to inhibit the growth of Escherichia coli within the surrounding media, demonstrating the ability to eradicate a bacterial concentration of up to 10⁴ CFU/ml.Urinary tract infection (UTI) is the most prevalent cause of nosocomial infections, 80% of which involve catheter-associated urinary tract infection (CAUTI). The risk of acquiring CAUTI depends on the method and duration of catheterization, the quality of catheter care, and host susceptibility (27, 32). Free-floating (planktonic) bacteria can adhere to surfaces on catheters and colonize, creating a tenacious milieu called a biofilm (6). These sessile microcolonies consist of bacteria that are highly differentiated and extremely resilient against standard antibiotics (35). Several innovative approaches have focused on inhibition of biofilm formation in order to prevent CAUTI. These include antiseptic lubricating gels applied at the catheter insertion point, the use of a taped seal applied to the catheter drainage tubing junction, and utilizing an antireflux valve (14, 34). A new approach has recently been emerging whereby catheters are coated with various antiseptic materials (14, 17, 28). For example, catheters coated with silver or silver-containing compounds show clinical promise, but clinical effectiveness varies and appears to be dependent on the silver matrix used. (34). Hydrogel- or silver-hydrogel-coated catheters have been suggested to provide an antiseptic benefit by creating a physical barrier to bacterial infection, thereby preventing adhesion of the bacteria to the catheter (14, 26). The synergistic combination of compounds such as chlorhexidine and protamine sulfate has also been evaluated with some success (8). Antibiotic-coated catheters containing ciprofloxacin, gentamicin, norfloxacin, and nitrofurazone have also been designed (14, 17, 26). However, the risk of developing an antibiotic-resistant strain of bacteria is high (26, 34).Nitric oxide (NO) is a small, naturally produced, hydrophobic, free-radical gas that has a major role in innate immunity. NO exhibits broad reactivity and rapid diffusive properties through biological liquids and lipid membranes, with a short half-life in a physiological milieu (33). Overproduction of NO induced by the enzymatic activity of inducible nitric oxide synthase (iNOS) in various cell types has been shown to play a vital role in several inflammatory and immunoregulatory processes (1). Most notably, NO has been shown to play important roles in vasodilatation, neurotransmission, angiogenesis, modulation of wound healing, and nonspecific responses to infection (19, 29).The antimicrobial activity of NO was demonstrated more than 50 years ago (31), with later in vitro studies showing inhibition of a wide variety of Gram-negative and Gram-positive bacterial species (16). NO was shown to be bacteriostatic (3, 9, 10, 23), with recent in vitro evidence demonstrating bactericidal effects (20). We have shown previously that multiple 30-min treatments of 160 ppm nitric oxide results in over a 5 log₁₀ CFU/ml reduction in the bacterial load of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa (21).A variety of methodologies have been attempted to deliver and study NO therapeutically. They include the use of polymers containing a diazeniumdiolate NO donor (16, 24), exposure chambers for direct topical application of NO (11, 20), and filling a urinary catheter retention balloon with nitrite and ascorbic acid to release NO (4).In this paper, we present a novel approach that creates an antiseptic barrier on urinary catheters by impregnating the catheters with gaseous NO (gNO) using a proprietary technology (2). We show that NO-impregnated Foley urinary catheters slowly release NO into urine over 14 days and are stable under various clinical models and storage. We also provide data showing that these NO-impregnated catheters are rendered antiseptic, prevent bacterial colonization on their exterior and luminal surfaces, and are able to eradicate up to 10⁴ CFU/ml of E. coli in the surrounding media under both stagnant and dynamic conditions.     

Mutational Upregulation of a Resistance-Nodulation-Cell Division-Type Multidrug Efflux Pump,SdeAB, upon Exposure to a Biocide,Cetylpyridinium Chloride,and Antibiotic Resistance in Serratia marcescens

Serratia marcescens is an important opportunistic pathogen in hospitals, where quaternary ammonium compounds are often used for disinfection. The aim of this study is to elucidate the effect of a biocide on the emergence of biocide- and antibiotic-resistant mutants and to characterize the molecular mechanism of biocide resistance in Serratia marcescens. A quaternary ammonium compound-resistant strain, CRes01, was selected by exposing a wild-type strain of S. marcescens to cetylpyridinium chloride. The CRes01 cells exhibited 2- to 16-fold more resistance than the wild-type cells to biocides and antibiotics, including cetylpyridinium chloride, benzalkonium chloride, chlorhexidine gluconate, fluoroquinolones, tetracycline, and chloramphenicol, and showed increased susceptibilities to β-lactam antibiotics and N-dodecylpyridinium iodide. Mutant cells accumulated lower levels of norfloxacin than the parent cells in an energized state but not in a de-energized state, suggesting that the strain produced a multidrug efflux pump(s). To verify this assumption, we knocked out a putative efflux pump gene, sdeAB, in CRes01 and found that the knockout restored susceptibility to most quaternary ammonium compounds and antibiotics, to which the CRes01 strain showed resistance. On the basis of these and other results, we concluded that S. marcescens gains resistance to both biocides and antibiotics by expressing the SdeAB efflux pump upon exposure to cetylpyridinium chloride.Serratia marcescens is a gram-negative bacterium ubiquitous in nature that occurs in hospital environments and is a nosocomial and opportunistic pathogen (10). Infections with this bacterium often cause septicemia, meningitis, endocarditis, and wound infections (2, 10). The high and broad intrinsic resistance of this organism to various antibiotics makes S. marcescens infections difficult to treat (9, 11). Resistance of bacteria to antibiotics is generally attributable to alterations of the drug target, enzymatic modifications of antibiotics, or reduced drug accumulation as a result of efflux pump-mediated drug exclusion or a membrane barrier(s) (17, 19, 20). Expression of the resistance-nodulation-cell division (RND)-type efflux pump is a major mechanism for multidrug resistance in gram-negative bacteria (17, 18).S. marcescens encodes at least three RND-type efflux pumps, SdeAB, SdeCDE, and SdeXY, which play an important role in the resistance of this organism to antibiotics. The SdeAB pump transports ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, and surfactants; SdeCDE transports novobiocin; and SdeXY transports erythromycin, tetracycline, norfloxacin, ampicillin, and biocides (5). The wild-type S. marcescens UOC-67 expresses only SdeAB and SdeCDE (2, 3).Biocides have been widely used in hospitals for disinfection and in the food industry to kill bacteria that cause food poisoning. Despite extensive implementation of new strategies to control multidrug-resistant bacteria, current practices may not be satisfactory (4, 8, 23). Although the mechanisms of antibiotic resistance have been studied extensively, the molecular mechanism of resistance to disinfectants is poorly understood. It is likely that extensive exposure of hospital pathogens to biocides may cause the emergence of bacteria resistant to antibiotics (19, 20, 25), or vice versa.In this study, we isolated an S. marcescens mutant resistant to cetylpyridinium chloride, a quaternary ammonium compound that is widely used for disinfection in hospitals all over the world. The genes and their products responsible for the cetylpyridinium chloride resistance were identified.     

Impact of Cotrimoxazole on Carriage and Antibiotic Resistance of Streptococcus pneumoniae and Haemophilus influenzae in HIV-Infected Children in Zambia

This is a substudy of a larger randomized controlled trial on HIV-infected Zambian children, which revealed that cotrimoxazole prophylaxis reduced morbidity and mortality despite a background of high cotrimoxazole resistance. The impact of cotrimoxazole on the carriage and antibiotic resistance of Streptococcus pneumoniae and Haemophilus influenzae as major causes of childhood mortality in HIV-infected children was investigated since these are unclear. Representative nasopharyngeal swabs were taken prior to randomization for 181 of 534 children (92 on cotrimoxazole and 89 on placebo). Bacterial identification and antibiotic susceptibility were performed by routine methods. Due to reduced mortality, prophylactic cotrimoxazole increased the median time from randomization to the last specimen from 48 to 56 months (P = 0.001). The carriage of H. influenzae was unaltered by cotrimoxazole. Carriage of S. pneumoniae increased slightly in both arms but was not statistically significant in the placebo arm. In S. pneumoniae switching between carriage and no carriage in consecutive pairs of samples was unaffected by cotrimoxazole (P = 0.18) with a suggestion that the probability of remaining carriage free was lower (P = 0.10). In H. influenzae cotrimoxazole decreased switching from carriage to no carriage (P = 0.02). Cotrimoxazole resistance levels were higher in postbaseline samples in the cotrimoxazole arm than in the placebo arm (S. pneumoniae, P < 0.0001; H. influenzae, P = 0.005). Cotrimoxazole decreased switching from cotrimoxazole resistance to cotrimoxazole sensitivity in S. pneumoniae (P = 0.002) and reduced the chance of H. influenzae remaining cotrimoxazole sensitive (P = 0.05). No associations were observed between the percentage of CD4 (CD4%), the change in CD4% from baseline, child age at date of specimen, child gender, or sampling month with carriage of either pathogen.Streptococcus pneumoniae and Haemophilus influenzae are major causes of respiratory tract infections and bacteremia worldwide (5, 29, 34). Children younger than 5 years of age, the elderly, and immunocompromised individuals are most at risk for respiratory infections (3). HIV-infected children younger than 2 years have a 40-fold greater risk of lower respiratory tract infections (34) with a 12-fold greater risk of pneumococcal invasive disease (11, 15, 37), increasing to a 100-fold-higher risk (31) compared to HIV-uninfected children. The prognosis for HIV-infected children developing pneumococcal disease is much worse than that for uninfected children developing pneumococcal disease (7, 21, 35, 54).Invasive infections with S. pneumoniae and H. influenzae are usually preceded by nasopharyngeal colonization (10, 22). Colonizing bacteria are frequently exposed to antibiotics, particularly in regions where there are high burdens of disease. This can lead to the selection of antibiotic-resistant bacteria that can be transmitted and cause invasive disease. Children are the primary reservoir for these bacterial pathogens due to their immature immune systems (24, 33). Bacterial acquisition and carriage are highly variable and depend on age, geographic area, genetic background, and socioeconomic conditions (2, 4, 9, 32, 43). As healthy children age, their immunological responses mature. This results in an increase in the frequency of pneumococcal recolonization and colonization by different serotypes (new acquisitions), with a concomitant decrease in the duration of carriage (23). Furthermore, there is considerable interspecies competition during colonization with constant variation in the nasopharyngeal microbiota (16, 48, 49). The colonizing microbiota may also be altered by antibiotics (17). HIV infection causes a reduction in immunologic responsiveness and a decrease in the secretion of IgA that may affect nasopharyngeal colonization (17, 30). Studies in Africa (45) and in the United States (42) indicate that the increased burden of disease in pediatric HIV infection is not explained by higher rates of nasopharyngeal colonization with the bacterium.Cotrimoxazole (trimethoprim-sulfamethoxazole [CoT]) prophylaxis in children and adults infected with HIV reduces significantly morbidity and mortality, even in regions with high resistance to CoT (7, 25, 38, 50). As a consequence, the World Health Organization (WHO) updated their guidelines for the care of HIV-infected patients in 2006, recommending prophylaxis for HIV-infected children, adolescents, and adults in resource-limited settings (52).Studies undertaken in Zambia and other African countries have shown an increase over recent years in the proportion of resistant isolates to several antimicrobial agents (39). In particular, resistance to CoT has increased in S. pneumoniae and H. influenzae (26, 27, 51). However, these were mainly short-term studies.In light of the recent WHO guidelines and the increasing threat of antibiotic resistance, we monitored over 2 years the impact of CoT prophylaxis on the carriage and resistance on S. pneumoniae and H. influenzae colonization in a double-blind study of a pediatric HIV-infected cohort and compared to children on placebo. This was a planned substudy of the Children with HIV Antibiotic Prophylaxis (CHAP) study (7).     

Population Dynamics of Antibiotic Treatment: a Mathematical Model and Hypotheses for Time-Kill and Continuous-Culture Experiments

The objectives of the study were to develop a quantitative framework for generating hypotheses for and interpreting the results of time-kill and continuous-culture experiments designed to evaluate the efficacy of antibiotics and to relate the results of these experiments to MIC data. A mathematical model combining the pharmacodynamics (PD) of antibiotics with the population dynamics of bacteria exposed to these drugs in batch and continuous cultures was developed, and its properties were analyzed numerically (using computer simulations). These models incorporate details of (i) the functional form of the relationship between the concentrations of the antibiotics and rates of kill, (ii) the density of the target population of bacteria, (iii) the growth rate of the bacteria, (iv) byproduct resources generated from dead bacteria, (v) antibiotic-refractory subpopulations, persistence, and wall growth (biofilms), and (vi) density-independent and -dependent decay in antibiotic concentrations. Each of the factors noted above can profoundly affect the efficacy of antibiotics. Consequently, if the traditional (CLSI) MICs represent the sole pharmacodynamic parameter, PK/PD indices can fail to predict the efficacy of antibiotic treatment protocols. More comprehensive pharmacodynamic data obtained with time-kill and continuous-culture experiments would improve the predictive value of these indices. The mathematical model developed here can facilitate the design and interpretation of these experiments. The validity of the assumptions behind the construction of these models and the predictions (hypotheses) generated from the analysis of their properties can be tested experimentally. These hypotheses are presented, suggestions are made about how they can be tested, and the existing statuses of these tests are briefly discussed.In accordance with the rational design of protocols for antibiotic therapy, dosing regimens are based on the changes in the concentration of the antibiotic during the course of treatment (pharmacokinetics [PK]) and on the in vitro relationship between the concentration of that antibiotic and the growth or death rate of the target bacteria (pharmacodynamics [PD]). Together, these factors comprise the PK/PD indices (1, 19), which are employed as a priori estimates of the potential efficacy of antibiotic treatment regimens. At least three different measures of the PK are used for these indices: (i) peak antibiotic concentration (C_max), (ii) time above the MIC, and (iii) area of the antibiotic concentration curve above the MIC (AUC/MIC) (3, 4, 15, 24, 40, 50). Although antibiotics are classified as time or concentration dependent, the only formal pharmacodynamic parameter used in these indices is the MIC (3, 9, 27), which is also the dominant parameter employed as a measure of the susceptibility (or resistance) of bacteria to antibiotics (14, 21, 39). MICs are estimated in vitro using protocols that are precisely defined for each antibiotic-bacterial species combination (14) and conditions that are optimal for the action of the drug: relatively low (<10⁶) densities of bacteria growing exponentially in liquid media at temperatures and under ionic conditions in which the drug is most effective.It is well established that pharmacodynamic parameters other than MICs measured under the optimal conditions specified by the CLSI protocols are likely to affect the course of antibiotic treatment. Included among these are (i) the functional form of the relationship between the concentration of an antibiotic and the rate of growth or death of bacteria (the pharmacodynamic function) (49), (ii) the density of the bacterial population (18, 52, 57), (iii) ionic conditions (pH and cation concentrations) (2, 5, 9, 16), (iv) the presence of nondividing or slowly dividing subpopulations of bacteria (persistence) (8, 10, 32, 58), (v) the physical structure of the bacterial population, e.g., biofilms (25, 46, 55), and (vi) mortality and delayed replication of antibiotic-exposed bacteria following the elimination of the antibiotic (postantibiotic effects) (16, 34, 35).At least two in vitro procedures have been used to address some of these limitations of MICs as measures of the in vitro efficacy of antibiotics. One is the classical time-kill experiment where, for a defined amount of time, bacteria in liquid culture are exposed to different concentrations of bactericidal antibiotics. The estimated rate of decline in density of viable cells in these experiments is used as a measure of the efficacy of the antibiotic (4, 30, 41, 49, 57). Another approach designed to more comprehensively explore the efficacy of antibiotics in vitro is to add these drugs to bacteria maintained in continuous culture devices (9, 11, 12, 31, 33, 43, 44, 48) and then follow the changes in viable-cell density. With this continuous-culture approach, it is possible to evaluate the efficacy of bacteriostatic as well as bactericidal drugs and to do so in situations in which the concentration of the drug is continually changing. These continuous-culture devices can also be used to explore the role of biofilms, such as those on the surfaces of the vessels (7, 13, 26), and postantibiotic effects on the dynamics of antibiotic action (42).There are vast bodies of MIC data representing different associations of antibiotics with bacteria (2, 14, 22), and it would be particularly useful to be able to compare these single-parameter estimates of the pharmacodynamics of antibiotic-bacterial species interactions with those obtained with time-kill and continuous-culture experiments. In part to achieve this end but also to provide a quantitative framework for generating testable hypotheses and interpreting the results of time-kill and continuous-culture experiments of antibiotic efficacy, we developed and numerically analyzed the properties of mathematical models describing them. These models combine the pharmacodynamics of antibiotics with the population dynamics of bacteria exposed to these drugs in batch and continuous cultures. The models take into account (i) the functional form of the relationship between the concentration of the antibiotics and rates of kill, (ii) the density of the target population of bacteria, (iii) the growth rate of the bacteria, (iv) byproduct resources generated from dead bacteria, (v) antibiotic-refractory subpopulations, wall growing bacteria (biofilm), and persistent states, and (vi) density-independent and density-dependent decay in antibiotic concentrations. On the basis of the results of our analysis of the properties of these models, we present a series of hypotheses regarding the pharmacodynamics and population dynamics of antibiotic treatment that can be tested with time-kill and continuous-culture experiments. We briefly discuss procedures to test these hypotheses and the anticipated results of these experiments.     

Rapamycin Protects Mice from Staphylococcal Enterotoxin B-Induced Toxic Shock and Blocks Cytokine Release In Vitro and In Vivo

Staphylococcal enterotoxins are potent activators for human T cells and cause lethal toxic shock. Rapamycin, an immunosuppressant, was tested for its ability to inhibit staphylococcal enterotoxin B (SEB)-induced activation of human peripheral blood mononuclear cells (PBMC) in vitro and toxin-mediated shock in mice. Stimulation of PMBC by SEB was effectively blocked by rapamycin as evidenced by the inhibition of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, IL-2, gamma interferon (IFN-γ), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, and T-cell proliferation. In vivo, rapamycin protected 100% of mice from lethal shock, even when administered 24 h after intranasal SEB challenge. The serum levels of MCP-1 and IL-6, after intranasal exposure to SEB, were significantly reduced in mice given rapamycin versus controls. Additionally, rapamycin diminished the weight loss and temperature fluctuations elicited by SEB.Staphylococcal exotoxins are among the most common etiological agents that cause toxic shock syndrome (28-30, 38, 44). The disease is characterized by fever, hypotension, desquamation of skin, and dysfunction of multiple organ systems (8, 38, 41). These toxins bind directly to the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells and subsequently stimulate T cells expressing specific Vβ elements on T-cell receptors (9, 15, 24, 29, 35, 42). Staphylococcal enterotoxin B (SEB) and the distantly related toxic shock syndrome toxin 1 are also called superantigens because they induce massive proliferation of T cells (29). In vitro and in vivo studies show that these superantigens induce high levels of various proinflammatory cytokines, and these potent mediators cause lethal shock in animal models (1, 6, 22, 27, 37, 39, 45, 51, 55). SEB also causes food poisoning (4, 21, 52) and is a potential bioterrorism threat agent, as humans are extremely sensitive to this superantigen, especially by inhalation (28). There is currently no effective therapeutic treatment for SEB-induced shock except for the use of intravenous immunoglobulins (11). Various in vitro experiments identified inhibitors to counteract the biological effects of SEB, only some of which were successful in ameliorating SEB-induced shock in experimental models (1, 25-27, 51).Rapamycin is a relatively new FDA-approved drug used to prevent graft rejection in renal transplantation, as it shows less nephrotoxicity than do calcineurin inhibitors (14, 40, 43, 48). Recent studies reveal other uses in animal models of cancer (23, 34), diabetic nephropathy (36), bleomycin-induced pulmonary fibrosis (31), liver fibrosis (5), and tuberous sclerosis (32). Rapamycin binds intracellularly to FK506-binding proteins, specifically FKBP12; the rapamycin-FKBP12 complex then binds to a distinct molecular target called mammalian target of rapamycin (mTOR) (reviewed in reference 48). Rapamycin inhibits mTOR activity, prevents cyclin-dependent kinase activation, and affects G₁-to-S-phase transition (16, 48). Other studies identified mTOR as the conserved serine-threonine kinase for sensing cellular stress, and rapamycin promotes anabolic cellular processes in response to stress signals (20, 47, 50, 54). The mTOR pathway regulates myogenesis (13), cell cycle arrest (20), adipocyte differentiation (3), and insulin signaling (47, 50). The immunological effects of rapamycin include regulation of T-cell activation (48); differentiation, expansion, and preservation of regulatory T cells (2, 10, 19, 46); downregulation of dendritic cells (12, 53); and granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced neutrophil migration (17). Rapamycin impairs dendritic cell maturation and function by inhibiting the expression of adhesion molecule ICAM-1 (12, 53). Thus, rapamycin has a broad spectrum of effects and interferes with the activation of multiple cell types of the immune system.Based on the potent immunosuppressive effects of rapamycin, we investigated the therapeutic impact of rapamycin on SEB-mediated toxic shock. The therapeutic efficacy of rapamycin in SEB-induced toxic shock was investigated by using a lethal murine model with intranasal delivery of SEB (22). This “double-hit” murine model relies on two low doses of SEB without the use of sensitizing agents such as lipopolysaccharide (LPS) or galactosamine to induce lethal shock (6, 27, 33, 37, 45). In this “SEB-only” toxic shock model, SEB was administered intranasally (i.n.) and another dose of SEB was strategically given intraperitoneally (i.p.) 2 h later to induce systemic cytokine release and pulmonary inflammation with lethality as an endpoint. We examined the effect of rapamycin on proinflammatory cytokines and chemokines induced by SEB in vitro using human peripheral blood mononuclear cells (PBMC) as a first step to test its immunological effects on SEB activation.     

Intra- and Extracellular Activities of Dicloxacillin against Staphylococcus aureus In Vivo and In Vitro

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT_MIC]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT_MIC is the index that is the most predictive of the outcome of infection both intra- and extracellularly.Staphylococcus aureus is a major cause of both community- and hospital-acquired infections (28, 30), which range from simple and uncomplicated skin and wound infections (2, 24) to more serious and life-threatening situations such as pneumonia (15, 36), endocarditis (16, 37), osteomyelitis (13, 25), and meningitis (34). S. aureus infections often show poor and slow responses to therapy, with recurrences and ensuing mortality (8, 9, 27, 37, 38, 46). These responses could be caused by the ability of the bacteria to invade and survive inside cells (5, 10, 21, 22, 31, 32). Intracellular antimicrobial activity depends on both drug- and bacterium-related factors (penetration, accumulation, subcellular bioavailability, expression of activity in the local environment, and the state of responsiveness of the organisms [42, 44]). In general, intracellular antimicrobial activity is markedly impaired compared to the activity seen in broth or the extracellular milieu (3, 39, 45), although we know about situations in which the opposite is true (7). Thus, the direct assessment of antibiotic activity in the pertinent models is warranted. Several in vitro models with either human or animal cells have been developed to study the intracellular activities of antibiotics (3, 6, 14, 21, 35, 41), and a corresponding in vivo model (a modified version of a murine peritonitis model) has recently been described (39). We have now combined these models and report here our results obtained by using dicloxacillin (DCX) as a prototype of antistaphylococcal β-lactam antibiotics. Isoxazolyl penicillins have usually been preferred for the treatment of methicillin-susceptible S. aureus (MSSA) infections (2, 20, 26, 30). DCX has been the main choice in Denmark and many other countries due to its stability against penicillinases, low level of toxicity, and availability for both oral and intravenous administration (19). We examined the intra- and extracellular time- and concentration-kill relationships for two MSSA strains in vitro using macrophages and performed corresponding intra- and extracellular dose-kill studies with the murine peritonitis model. In combination with pharmacokinetic (PK) analysis and measurement of the amount of free drug (f) versus protein-bound drug, this allowed us to estimate which PK/pharmacodynamic (PD) index best predicts the efficacy of DCX intra- and extracellularly.(Part of this study was presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 2006, San Francisco, CA.)     

The ABC Transporter AnrAB Contributes to the Innate Resistance of Listeria monocytogenes to Nisin,Bacitracin, and Various β-Lactam Antibiotics

A mariner transposon bank was used to identify loci that contribute to the innate resistance of Listeria monocytogenes to the lantibiotic nisin. In addition to highlighting the importance of a number of loci previously associated with nisin resistance (mprF, virRS, and telA), a nisin-sensitive phenotype was associated with the disruption of anrB (lmo2115), a gene encoding the permease component of an ABC transporter. The contribution of anrB to nisin resistance was confirmed by the creation of nonpolar deletion mutants. The loss of this putative multidrug resistance transporter also greatly enhanced sensitivity to bacitracin, gallidermin, and a selection of β-lactam antibiotics. A comparison of the relative antimicrobial sensitivities of a number of mutants established the ΔanrB strain as being one of the most bacitracin-sensitive L. monocytogenes strains identified to date.Nisin, like all of the antimicrobial peptides termed lantibiotics, is synthesized ribosomally and subjected to posttranslational modification (29, 40, 47). Nisin kills target Gram-positive bacteria by binding to lipid II, preventing cell wall synthesis, and subsequently forming pores in the cell membrane, which facilitates the release of intracellular contents (7-9). Nisin has been used for over 50 years in the food industry to prevent the growth of food spoilage and pathogenic microorganisms (39). Its activity, coupled with its approval by many agencies, e.g., nisin is the only natural food-grade antibacterial preservative approved by the European Union, makes it an attractive option for food producers and consumers (14). In addition, the lantibiotics are also being investigated with a view to their application as chemotherapeutic agents for clinical use, given their desirable properties in this regard, including the ever-increasing problem of resistance to standard antibiotics, the high potency of nisin and other lantibiotics against several multidrug-resistant pathogens (with activity at nanomolar concentrations, on the basis of in vitro investigations [3, 38]), and their stable, noncytotoxic nature (30).The activity of nisin against quite nisin-resistant food pathogens, such as Listeria monocytogenes, may be potentiated through the identification and inhibition of mechanisms that provide these recalcitrant bacteria with a degree of innate resistance to the lantibiotic (i.e., resistance intrinsically associated with particular genera or species rather than that which arises by mutation in a formerly sensitive strain). Previous work on lantibiotic resistance has highlighted a number of these key systems and their associated genetic determinants. The dlt operon is required for d-alanylation of teichoic acids, a decoration which causes the repulsion of cationic antimicrobial peptides (CAMPs), such as nisin, by L. monocytogenes and other pathogens (1, 37). mprF has a similarly important protective role in that its product catalyzes the addition of positively charged residues, via lysinylation, to membrane phospholipids (43). Like the case for other cell-wall-acting antimicrobials, penicillin binding proteins (PBPs), such as the class A PBP Lmo2229 of L. monocytogenes, are also required for optimal resistance to nisin and other lantibiotics (19, 20). Unsurprisingly, regulators that control these and other processes have also been shown to contribute greatly to innate lantibiotic resistance. Examples include both two-component systems, such as LisRK (12), LiaRS (34), and VirRS (32), and sigma factors, such as σ^B (5). Finally, but importantly, one of the approaches most frequently employed by bacteria to survive exposure to antimicrobials is through the removal of such compounds from the cell envelope via ABC transporters (28, 45, 46). Indeed, many lantibiotic producers employ ABC transporters [generically designated LanEF(G)] as immunity proteins (16), and it was recently shown that nonlantibiotic producers can also be protected as a result of the presence of LanEF homologs, in a form of resistance we have designated immune mimicry (15). ABC transporters other than LanEF proteins have also been linked to lantibiotic resistance. Gene expression studies have established that ABC transporter genes are overexpressed in a nisin-hyperresistant Lactococcus lactis strain (ysaBC) (26), a Bacillus subtilis strain subjected to nisin stress (yvcRS) (21), and a Staphylococcus aureus strain exposed to the lantibiotic mersacidin (vraDE) (41). Notably, in Streptococcus pneumoniae, the ABC transporter-encoding genes sp0913 and sp0912 are required for resistance to nisin and other antimicrobials (31).In this study, a mariner transposon bank of L. monocytogenes EGD-e was screened to identify nisin-sensitive mutants. In addition to confirming the importance of a number of genes previously known to be involved in nisin resistance, a number of novel loci of potential significance were identified. Nonpolar deletion of these loci highlighted the importance of lmo2115, encoding the permease component of an ABC transporter which we have designated AnrAB (ABC transporter involved in nisin resistance). Further investigations with the ΔanrB strain revealed an associated increased sensitivity to the lantibiotic gallidermin, to bacitracin, and to a large number of β-lactam antibiotics. In addition to revealing the importance of this novel nisin resistance locus, we have also undertaken the first comparative study of the antimicrobial sensitivities of a number of nisin-sensitive mutants of L. monocytogenes EGD-e.     

Pharmacokinetic Properties of Sulfadoxine-Pyrimethamine in Pregnant Women

To determine the pharmacokinetic disposition of sulfadoxine (SDOX) and pyrimethamine (PYR) when administered as intermittent presumptive treatment during pregnancy (IPTp) for malaria, 30 Papua New Guinean women in the second or third trimester of pregnancy and 30 age-matched nonpregnant women were given a single dose of 1,500 mg of SDOX plus 75 mg of pyrimethamine PYR. Blood was taken at baseline and 1, 2, 4, 6, 12, 18, 24, 30, 48, and 72 h and at 7, 10, 14, 28, and 42 days posttreatment in all women. Plasma samples were assayed for SDOX, N-acetylsulfadoxine (NASDOX), and PYR by high-performance liquid chromatography. Population pharmacokinetic modeling was performed using NONMEM v6.2.0. Separate user-defined mamillary models were fitted to SDOX/NASDOX and PYR. When the covariate pregnancy was applied to clearance, there was a significant improvement in the base model for both treatments. Pregnancy was associated with a significantly lower area under the concentration-time curve from 0 to ∞ for SDOX (22,315 versus 33,284 mg·h/liter), NASDOX (801 versus 1,590 mg·h/liter), and PYR (72,115 versus 106,065 μg·h/liter; P < 0.001 in each case). Because lower plasma concentrations of SDOX and PYR could compromise both curative efficacy and posttreatment prophylaxis in pregnant patients, IPTp regimens incorporating higher mg/kg doses than those recommended for nonpregnant patients should be considered.Treatment strategies for malaria during pregnancy include drug administration for symptomatic episodes and, because asymptomatic infections in areas of malaria endemicity can also be associated with adverse maternal and fetal outcomes, giving antimalarial therapy at prespecified intervals during pregnancy. This latter approach, known as intermittent preventive treatment in pregnancy (IPTp), clears maternal parasitemia and prevents or suppresses subsequent infections (37). Although conventional adult antimalarial treatment doses are recommended, the physiologic changes that take place during pregnancy may significantly alter drug disposition through a variety of mechanisms, including increases in plasma volume, increased clearance, and altered protein binding (2, 16). Despite these considerations, <150 pregnant women have been enrolled in published studies of antimalarial pharmacokinetics (14, 18, 33), and such data have been identified as an urgent priority for optimization of IPTp strategies (33).The treatment with the best evidence for use as IPTp is sulfadoxine (SDOX) plus pyrimethamine (PYR) (SP) (22, 26, 31), but its effectiveness appears paradoxical given the component drugs have a relatively short elimination half-life (t_1/2β), specifically 6 to 11 days for SDOX (12, 19, 21, 24, 25, 32, 34, 35) and 3 to 5 days for PYR (3, 5, 9, 11, 12, 14, 19, 21, 25, 30, 32, 34, 35, 38). SDOX is acetylated by the enzyme N-acetyltransferase 2. Polymorphisms in the N-acetyltransferase 2 gene are associated with rapid or slow acetylation (20), and there is some evidence that the extent of acetylation is linked to treatment failure in malaria (24). In Papua New Guinea (PNG), the national treatment policy is to give SP with chloroquine (CQ) as presumptive treatment at the first antenatal visit. We have investigated the pharmacokinetic properties of SDOX and PYR in pregnant PNG women and in nonpregnant female controls. Since PNG populations comprise predominantly rapid acetylators (8, 23), we measured N-acetylsulfadoxine (NASDOX), the primary metabolite of SDOX, to assess its potential clinical importance.     

Inhibitory and Bactericidal Activities of Daptomycin,Vancomycin, and Teicoplanin against Methicillin-Resistant Staphylococcus aureus Isolates Collected from 1985 to 2007

The inhibitory and bactericidal activities of daptomycin, vancomycin, and teicoplanin against a collection of 479 methicillin-resistant Staphylococcus aureus isolates were assessed. The isolates were collected from U.S. and European hospitals from 1985 to 2007 and were primarily from blood and abscess cultures. The MICs and minimum bactericidal concentrations (MBCs) of the three agents were determined, and the MBC/MIC ratios were calculated to determine the presence or absence of tolerance. Tolerance was defined as an MBC/MIC ratio of ≥32 or an MBC/MIC ratio of ≥16 when the MBC was greater than or equal to the breakpoint for resistance. Tolerance to vancomycin and teicoplanin was observed in 6.1% and 18.8% of the strains, respectively. Tolerance to daptomycin was not observed.Although vancomycin and teicoplanin are the standard therapies for staphylococcal bacteremia, tolerance to vancomycin and teicoplanin has been demonstrated in both coagulase-negative staphylococci and Staphylococcus aureus as well as in various Streptococcus species (2, 3, 7, 10, 13, 15, 20, 21, 23, 25). Daptomycin, a lipopeptide antibiotic, has been demonstrated to have rapid bactericidal activity against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and tolerance to this drug has not been demonstrated (2, 9, 10, 19, 21, 24, 26, 28).The issue of antibiotic tolerance is a complicated one. Some studies have suggested that infections caused by tolerant strains may be more difficult to treat, especially when they cause complicated infections such as endocarditis, meningitis, or osteomyelitis or cause infections in immunocompromised patients (7, 8, 14, 15, 16, 18, 20, 22, 23, 25). Other investigators'' expert analyses do not agree that there is proof of a correlation between tolerant strains and treatment failures or that bactericidal activity is required for the treatment of serious MRSA infections (17, 25, 26, 27, 28). Controversy concerning the appropriate methods for the determination of tolerance in clinical isolates and in the practicality of testing isolates for tolerance in the clinical laboratory also exists.This study looked at MRSA isolates obtained primarily from blood and abscess cultures collected between 1985 and 2007. The main purpose of the study was to determine the in vitro inhibitory and bactericidal activities and the level of tolerance to the three drugs observed by standardized MIC and minimum bactericidal concentration (MBC) tests (4, 5, 19).(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)