Sordarins: In Vitro Activities of New Antifungal Derivatives against Pathogenic Yeasts,Pneumocystis carinii,and Filamentous Fungi

GM 193663, GM 211676, GM 222712, and GM 237354 are new semisynthetic derivatives of the sordarin class. The in vitro antifungal activities of GM 193663, GM 211676, GM 222712, and GM 237354 against 111 clinical yeast isolates of Candida albicans, Candida kefyr, Candida glabrata, Candida parapsilosis, Candida krusei, and Cryptococcus neoformans were compared. The in vitro activities of some of these compounds against Pneumocystis carinii, 20 isolates each of Aspergillus fumigatus and Aspergillus flavus, and 30 isolates of emerging less-common mold pathogens and dermatophytes were also compared. The MICs of GM 193663, GM 211676, GM 222712, and GM 237354 at which 90% of the isolates were inhibited (MIC₉₀s) were 0.03, 0.03, 0.004, and 0.015 μg/ml, respectively, for C. albicans, including strains with decreased susceptibility to fluconazole; 0.5, 0.5, 0.06, and 0.12 μg/ml, respectively, for C. tropicalis; and 0.004, 0.015, 0.008, and 0.03 μg/ml, respectively, for C. kefyr. GM 222712 and GM 237354 were the most active compounds against C. glabrata, C. parapsilosis, and Cryptococcus neoformans. Against C. glabrata and C. parapsilosis, the MIC₉₀s of GM 222712 and GM 237354 were 0.5 and 4 μg/ml and 1 and 16 μg/ml, respectively. The MIC₉₀s of GM 222712 and GM 237354 against Cryptococcus neoformans were 0.5 and 0.25 μg/ml, respectively. GM 193663, GM 211676, GM 222712, and GM 237354 were extremely active against P. carinii. The efficacies of sordarin derivatives against this organism were determined by measuring the inhibition of the uptake and incorporation of radiolabelled methionine into newly synthesized proteins. All compounds tested showed 50% inhibitory concentrations of <0.008 μg/ml. Against A. flavus and A. fumigatus, the MIC₉₀s of GM 222712 and GM 237354 were 1 and 32 μg/ml and 32 and >64 μg/ml, respectively. In addition, GM 237354 was tested against the most important emerging fungal pathogens which affect immunocompromised patients. Cladosporium carrioni, Pseudallescheria boydii, and the yeast-like fungi Blastoschizomyces capitatus and Geotrichum clavatum were the most susceptible of the fungi to GM 237354, with MICs ranging from ≤0.25 to 2 μg/ml. The MICs of GM 237354 against Trichosporon beigelii and the zygomycetes Absidia corymbifera, Cunninghamella bertholletiae, and Rhizopus arrhizus ranged from ≤0.25 to 8 μg/ml. Against dermatophytes, GM 237354 MICs were ≥2 μg/ml. In summary, we concluded that some sordarin derivatives, such as GM 222712 and GM 237354, showed excellent in vitro activities against a wide range of pathogenic fungi, including Candida spp., Cryptococcus neoformans, P. carinii, and some filamentous fungi and emerging invasive fungal pathogens.During the past two decades, the incidence of infections caused by opportunistic fungal pathogens in immunocompromised patients has increased substantially (1, 11, 30, 31, 36). Candida albicans is the major opportunistic pathogen, although the incidence of fungal infections caused by non-C. albicans species is increasing (37). Pneumocystis carinii remains an important pathogen in AIDS patients and other immunocompromised individuals (17), and invasive pulmonary aspergillosis remains a frequently fatal complication of bone marrow transplantation and of cancer chemotherapy in patients with hematologic neoplasms (23, 26, 29). Although there has been an expansion in the number of antifungal drugs available (8–10), in many cases, treatment of fungal diseases remains unsatisfactory. This situation has led to an ongoing search for fungicidal agents with different modes of action and fewer side effects and which can be administered both orally and parenterally.One of the major challenges to finding a potent yet safe antifungal agent is the similarity between fungal and mammalian cells. Like mammalian cells, fungi are eukaryotic, so they have many of the same structures and metabolic pathways as mammalian cells, making it more difficult to find targets of differential toxicity. Although protein synthesis is a universal process in living cells, it has always been considered as one of the more attractive targets for the development of antimicrobial agents (8, 12, 36). It is known that fungal protein has exploitable differences relative to its mammalian counterpart, e.g., the two soluble protein factors elongation factor 3 (EF-3) (19, 34) which is absent from mammalian cells, EF-2, which is functionally distinct from its mammalian counterpart (5, 6). On the basis of these differences, a target-based screening program was established, with the objective of isolating selective protein synthesis inhibitors of the fungal machinery (2). As part of this screening program, a novel antifungal compound, GR 135402, was isolated from fermentation broth of Graphium putredinis and characterized (20). This new compound is the first natural product described to date which possesses antifungal activity through inhibition of fungal but not mammalian protein synthesis (20). GR 135402 belongs to the sordarin class (13), and although it has some structural similarity to zofimarin (27), sordarin (33), and sordarin derivatives (3, 28), no mode of action was described for the antifungal activity of these compounds. A synthetic chemical program was initiated to improve the biological properties of GR 135402, and four compounds, designated GM 193663, GM 211676, GM 222712, and GM 237354, were selected for evaluation.In this study, we analyze the in vitro antifungal activities of these four new sordarin derivatives against several groups of clinical isolates.(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [15, 16].)     

Anti-Pneumocystis Activities of Aromatic Diamidoxime Prodrugs

Aromatic dicationic compounds, such as pentamidine, have potent antimicrobial activities. Clinical use of these compounds has been restricted, however, by their toxicity and limited oral activity. A novel approach, using amidoxime derivatives as prodrugs, has recently been proposed to overcome these limitations. Although results were presented for amidoxime derivatives of only one diamidine, pentamidine, the authors in the original proposal claimed that amidoxime derivatives would work as effective prodrugs for all pharmacologically active diamidines. Nine novel amidoxime derivatives were synthesized and tested in the present study for activity against Pneumocystis carinii in corticosteroid-suppressed rats. Only three of the nine compounds had significant oral anti-Pneumocystis activity. The bisbenzamidoxime derivatives of three direct pentamidine analogs had excellent oral and intravenous activities and reduced acute host toxicity. These compounds are not likely candidates for future drug development, however, because they have chronic toxic effects and the active amidine compounds have multiple sites susceptible to oxidative metabolism, which complicates their pharmacology and toxicology. Novel diamidoximes from three other structural classes, containing different groups linking the cationic moieties, lacked significant oral or intravenous anti-Pneumocystis activity, even though the corresponding diamidines were very active intravenously. Both active and inactive amidoximes were readily metabolized to the corresponding amidines by cell-free liver homogenates. Thus, the amidoxime prodrug approach may provide a strategy to exploit the potent antimicrobial and other pharmacological activities of selected, but certainly not all, aromatic diamidines.

Aromatic dicationic compounds, including bisbenzamidines and dicationically substituted bisbenzimidazoles and carbazoles, have excellent experimental anti-Pneumocystis activities (14, 40, 46, 48, 50, 51) and are also active against other microbial pathogens, including protozoan parasites (2–5, 10, 38, 41, 43, 44), fungi (45), and some viruses (25–27, 49, 53). Aromatic dications also possess other pharmacological properties, including antiinflammatory and anticoagulant activities (29–37). Two problems hindering development of these compounds as new drugs, however, are limited oral bioavailability and toxicity (24, 38, 48, 51).Recent studies of pentamidine metabolism (7–9, 21–23) have led to a novel approach to overcome the limited oral bioavailability and acute toxicity. Aromatic diamidoximes are hypothesized to be orally bioavailable prodrugs that are readily reduced by drug-metabolizing enzymes to the active aromatic amidines (19, 21, 22), resulting in excellent antimicrobial activity with reduced acute host toxicity.Amidoximes were first shown by Lamb and White to be active against experimental African trypanosomiasis (42) and then later were shown to be active against other microorganisms (1, 17, 18, 28). Although activities were often reported for both amidoximes and corresponding amidines, no mention was made in these early publications that metabolic activation was required for in vivo activity of the amidoximes. Moreover, no systematic studies were performed to determine which analogs were orally active and if the amidoxime derivatives had increased oral activity compared to the amidines. Thus, the concept of amidoximes as prodrugs of amidines was not raised in earlier studies.The hypothesis that amidoximes might be useful prodrugs resulted from research examining the metabolism of pentamidine (6–9, 21, 22). Two primary oxidative metabolites identified were the mono- and diamidoximes, formed by N-hydroxylation of pentamidine. Although the diamidoxime derivative of pentamidine has little or no activity against three protozoan parasites in vitro, both the mono- and diamidoximes were active against African trypanosomes and Leishmania spp. when given to experimental animals subcutaneously (19, 21–23, 39). The diamidoxime given orally to rats was absorbed from the gut and converted to pentamidine, a reaction subsequently shown to be catalyzed by an oxygen-independent hepatic reductase activity (21, 22). These observations led to the proposal that amidoxime derivatives, in general, are effective, orally absorbed prodrugs for all pharmacologically active amidine-containing compounds (19). However, the only amidoximes tested were derivatives of pentamidine. We recently demonstrated that two novel amidoximes of 2,5-bis[4-amidinophenyl]furan were highly active orally and intravenously (13). Moreover, Weller and coworkers demonstrated that amidoximes of potent monoamidine fibrinogen receptor antagonists greatly enhanced their oral bioavailability (54).With this promising background, we began to synthesize potential amidoxime prodrugs of our most active, least toxic diamidines. Results presented here, however, demonstrate that amidoximes are not effective prodrugs for all aromatic dicationic compounds. The nature of the linker between the two amidoxime moieties plays a key role in determining if a particular diamidoxime has oral anti-Pneumocystis activity. Diamidoxime derivatives of the very promising bisbenzimidazole and carbazole classes of dications, and bisbenzamidoximes that contain additional nitrogen atoms in the aliphatic linkers, had little or no anti-Pneumocystis activity, even though the parent diamidines had excellent intravenous activity. Variability in activity does not appear to be caused by differences in enzymatic reductase activity, since both active and inactive diamidoximes were metabolized by cell-free liver homogenates.     

β-Lactamase Inhibitors Are Substrates for the Multidrug Efflux Pumps of Pseudomonas aeruginosa

The MexAB-OprM multidrug efflux system exports a number of antimicrobial compounds, including β-lactams. In an attempt to define more fully the range of antimicrobial compounds exported by this system, and, in particular, to determine whether β-lactamase inhibitors were also accommodated by the MexAB-OprM pump, the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance β-lactam susceptibility in intact cells was assessed. MIC determinations clearly demonstrated that all three compounds tested, clavulanate, cloxacillin, and BRL42715, were accommodated by the pump. Moreover, by using β-lactams which were readily hydrolyzed by the Pseudomonas aeruginosa class C chromosomal β-lactamase, it was demonstrated that elimination of the mexAB-oprM-encoded efflux system greatly enhanced the abilities of cloxacillin and BRL42715 (but not clavulanate) to increase β-lactam susceptibility. With β-lactams which were poorly hydrolyzed, however, the inhibitors failed to enhance β-lactam susceptibility in MexAB-OprM⁺ strains, although BRL42715 did enhance β-lactam susceptibility in MexAB-OprM⁻ strains, suggesting that even with poorly hydrolyzed β-lactams this inhibitor was effective when it was not subjected to efflux. MexEF-OprN-overexpressing strains, but not MexCD-OprJ-overexpressing strains, also facilitated resistance to β-lactamase inhibitors, indicating that these compounds are also substrates for the MexEF-OprN pump. These data indicate that an ability to inactivate MexAB-OprM (and like efflux systems in other bacteria) will markedly enhance the efficacies of β-lactam–β-lactamase inhibitor combinations in treating bacterial infections.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to a variety of antimicrobial agents. Previously attributed to a highly impermeable outer membrane (22), this resistance is now recognized to result from the synergy between broadly specific drug efflux pumps and low outer membrane permeability (16). One such efflux system, encoded by the mexAB-oprM operon (8, 28, 29), effluxes a range of antibiotics, including tetracycline, chloramphenicol, quinolones, β-lactams, novobiocin, macrolides, and trimethoprim (8, 9, 12, 29). Expressed constitutively in wild-type cells, where it contributes to intrinsic drug resistance (5, 12, 29), the operon is hyperexpressed in nalB mutants (30), producing elevated levels of resistance to substrate antibiotics (8, 9, 12, 29). Homologous efflux systems encoded by the mexC-mexD-oprJ (27) and mexE-mexF-oprN (10) operons have also been described. Apparently not expressed during growth under normal laboratory conditions, these systems are expressed in nfxB (27) and nfxC (10) multidrug-resistant mutants, respectively. nfxB strains are resistant to chloramphenicol, tetracycline, quinolones, macrolides, novobiocin, and newer cephalosporins such as cefepime and cefpirome but display hypersusceptibility to most β-lactam antibiotics (18). nfxC strains exhibit resistance to chloramphenicol, trimethoprim, quinolones, and carbapenems, including imipenem, although the resistance to imipenem results from the loss of the porin protein OprD in these mutants and not from the overexpression of MexEF-OprN (6, 10).The tripartite efflux pumps consist of an inner membrane component (MexB, MexD, and MexF) which functions as a resistance-nodulation-division family H⁺ antiport exporter (21, 31), an outer membrane, a presumed channel-forming component (OprM, OprJ, and OprN) (16, 23), and a so-called membrane fusion protein predicted to link the membrane-associated efflux components (MexA, MexC, and MexE) (16, 23). Recent data suggest that the operation of MexAB-OprM (and by analogy the remaining efflux systems) is at least partially dependent upon the TonB energy-coupling protein implicated in the opening of outer membrane gated channels responsible for iron-siderophore uptake across the P. aeruginosa outer membrane (36). Thus, the outer membrane components of these efflux pumps may be gated channels.In an effort to further define the range of antibiotic compounds which are accommodated by the known P. aeruginosa efflux systems, we examined β-lactamase inhibitors as possible pump substrates by assessing the influence of pump status (its presence or absence) on the intrinsic antibacterial activities of these compounds and on their abilities to enhance the efficacies of β-lactam compounds.     

In Vitro Antifungal Susceptibilities and Amplified Fragment Length Polymorphism Genotyping of a Worldwide Collection of 350 Clinical,Veterinary, and Environmental Cryptococcus gattii Isolates

The in vitro susceptibilities of a worldwide collection of 350 Cryptococcus gattii isolates to seven antifungal drugs, including the new triazole isavuconazole, were tested. With amplified fragment length polymorphism (AFLP) fingerprinting, human, veterinary, and environmental C. gattii isolates were subdivided into seven AFLP genotypes, including the interspecies hybrids AFLP8 and AFLP9. The majority of clinical isolates (n = 215) comprised genotypes AFLP4 (n = 76) and AFLP6 (n = 103). The clinical AFLP6 isolates had significantly higher geometric mean MICs for flucytosine and fluconazole than the clinical AFLP4 isolates. Of the seven antifungal compounds examined in this study, isavuconazole had the lowest MIC₉₀ (0.125 μg/ml) for all C. gattii isolates, followed by a 1 log₂ dilution step increase (MIC₉₀, 0.25 μg/ml) for itraconazole, voriconazole, and posaconazole. Amphotericin B had an acceptable MIC₉₀ of 0.5 μg/ml, but fluconazole and flucytosine had relatively high MIC₉₀s of 8 μg/ml.The basidiomycetous yeast Cryptococcus gattii is responsible for life-threatening invasive disease in apparently healthy humans and animals (7, 19). A typical C. gattii infection is acquired through the respiratory tract, from which it can further disseminate to the central nervous system, resulting in fatal meningitis (7, 19, 32). Cryptococcosis caused by the primary pathogenic yeast C. gattii was, until a decade ago, a rarely encountered infection outside tropical and subtropical regions (17, 26, 27). However, this changed due to an unprecedented outbreak that emerged in the temperate climate of Vancouver Island (British Columbia, Canada) that subsequently expanded farther into the Pacific Northwest (1, 8, 10, 16). Its sibling species, Cryptococcus neoformans, differs ecologically and epidemiologically from C. gattii since it occurs on a global scale and is linked with disease occurring in immunocompromised individuals, such as HIV-positive patients and transplant patients who receive immune-suppressive medicines (7, 10, 18, 19, 31).Cryptococcus gattii can be discerned from C. neoformans using a wide range of microbiological and molecular techniques (7, 20). A convenient method is the use of canavanine-glycine-bromothymol blue (CGB) medium, which allows C. gattii but not C. neoformans to grow and which changes the pH indicator in the medium from green-yellowish to blue (18). With the increasing use of molecular techniques, such as PCR fingerprinting, restriction fragment length polymorphism (RFLP) analysis of the PLB1 and URA5 loci, and amplified fragment length polymorphism (AFLP) fingerprint analysis, as well as several multilocus sequence typing (MLST) approaches, it became clear that C. gattii could be divided into five distinct genotypes, named AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV, and AFLP10 (the last one of which is a recently observed novel genotype) (2, 6, 7, 13, 16, 20, 21, 23). Until recently, a serotype agglutination assay was widely used to distinguish C. neoformans (serotypes A and D) from C. gattii (serotypes B and C) (7, 27). In general, serotype B strains are found in each of the five C. gattii AFLP genotypes, but it seems that C. gattii serotype C strains are restricted to genotypes AFLP5/VGIII and AFLP7/VGIV (2, 6, 16, 21, 27).In addition, it was found that C. gattii and C. neoformans can form interspecies hybrids, named genotype AFLP8 (C. neoformans var. neoformans AFLP2/VNIII serotype D × C. gattii AFLP4/VGI serotype B) and AFLP9 (C. neoformans var. grubii AFLP1/VNI serotype A × C. gattii AFLP4/VGI serotype B). These interspecies hybrids have, until now, been isolated only from clinical samples, and they might have a higher virulence potential than regular C. gattii or C. neoformans isolates (4, 5; F. Hagen, K. Tintelnot, and T. Boekhout, unpublished data).Treatment of cryptococcosis depends on, besides the immune status of the patient, the severity and localization of the infection (11). Severe cases of cryptococcosis in immunocompetent and -compromised patients are treated according to the guidelines of the Infectious Diseases Society of America, according to which treatment consists of an induction therapy for 2 weeks with a combination of amphotericin B and flucytosine, followed by a 10-week consolidation therapy with fluconazole (11, 24).Cryptococcus neoformans has been extensively studied for its in vitro susceptibility to a wide variety of antifungal compounds, including the new triazoles posaconazole, voriconazole, ravuconazole, and isavuconazole (12, 14, 28, 29, 33). Despite the ongoing C. gattii outbreak, only a few studies using relatively small sets of C. gattii isolates have been performed to investigate their in vitro susceptibilities to amphotericin B, flucytosine, fluconazole, and the new triazole antifungals (12, 15, 28-30). A few studies divided the C. gattii isolates into groups according to their serotype or genotype (15, 29).Therefore, we studied the in vitro susceptibilities of each of the C. gattii genotypes from a large worldwide collection, subdivided by AFLP genotyping, to amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, posaconazole, and the new experimental broad-spectrum antifungal triazole isavuconazole.     

Highly Conserved Neisseria meningitidis Surface Protein Confers Protection against Experimental Infection

A new surface protein, named NspA, which is distinct from the previously described Neisseria meningitidis outer membrane proteins was identified. An NspA-specific mAb, named Me-1, reacted with 99% of the meningococcal strains tested indicating that the epitope recognized by this particular mAb is widely distributed and highly conserved. Western immunoblotting experiments indicated that mAb Me-1 is directed against a protein band with an approximate molecular mass of 22,000, but also recognized a minor protein band with an approximate molecular mass of 18,000. This mAb exhibited bactericidal activity against four meningococcal strains, two isolates of serogroup B, and one isolate from each serogroup A and C, and passively protected mice against an experimental infection. To further characterize the NspA protein and to evaluate the protective potential of recombinant NspA protein, the nspA gene was identified and cloned into a low copy expression vector. Nucleotide sequencing of the meningococcal insert revealed an ORF of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a predicted molecular weight of 18,404 and a isoelectric point of 9.93. Three injections of either 10 or 20 μg of the affinity-purified recombinant NspA protein efficiently protected 80% of the mice against a meningococcal deadly challenge comparatively to the 20% observed in the control groups. The fact that the NspA protein can elicit the production of bactericidal and protective antibodies emphasize its potential as a vaccine candidate. N eisseria meningitidis causes both endemic and epidemic diseases, principally meningitidis and meningococcemia (1, 2). This pathogenic bacteria primarily affects young children between 6 mo and 2 yr of age, but often infects teenagers (1). The incidence per year of meningococcal diseases during endemic periods is normally ∼1–3 cases per 100,000 in developed countries, but it can be as high as 500 per 100,000 during epidemics (2, 3). N. meningitidis is classified into 12 serogroups based on the immunological characteristics of the capsular polysaccharides found at their surface. Within serogroups, different serotypes, subtypes, and immunotypes can be identified based on the antigenic specificity of the major outer membrane (OM)¹ proteins and LPS (4). Approximately 90% of all meningococcal diseases worldwide are caused by isolates of serogroups A, B, and C (5). Vaccines based on the capsular polysaccharides of serogroups A, C, W-135, and Y were developed and proved efficient to control outbreaks and epidemics of meningococcal diseases (6). However, these vaccines are poorly immunogenic in very young children. Moreover, they do not induce immunological memory and the duration of the protection they provide is relatively short (5, 7–11). Recently, it was demonstrated that conjugation of capsular polysaccharides of serogroups A and C to carrier proteins resulted in a better immunogenicity and a longer persistence of specific antibodies against isolates of these serogroups (12–16). Attempts to develop an efficient vaccine against serogroup B isolates, which are responsible for 50–70% of the meningococcal disease in the developed countries were unsuccessful because the group B capsular polysaccharide is not a good immunogen in human, inducing only a poor IgM response of low specificity which is not protective (17–19). Furthermore, the presence of closely similar, cross-reactive structures in the glycoproteins of neonatal human brain tissue might discourage attempts to improve the immunogenicity of serogroup B polysaccharide (10).To develop a vaccine effective against meningococci of serogroup B several non-capsular surface structures are under investigation (6, 10). Importantly, the presence of bactericidal antibodies against N. meningitidis have been strongly correlated with human immunity and protection (20–22). For that reason, it is believed that non-capsular surface antigens shown to stimulate bactericidal antibodies should be considered as the prime vaccines candidates (6). Early studies using sera of immunized volunteers and convalescent patients indicated that certain meningococcal surface proteins such as the ones responsible for serotype specificity and LPS could induce bactericidal antibodies and be involved in protection (23, 24). mAbs were then used to clearly establish the protective potential of certain meningococcal major surface proteins such as the PorA (class 1), PorB (class 2/3), and Opc (class 5C) (25–28).Different vaccines based on OM proteins were recently evaluated in clinical trials and efficiency between 50 and 80% were recorded (6, 10). These first generation OM proteins vaccines often induced protection against a limited number of strains. Thus, these vaccines could be used during meningococcal epidemics when the antigenic variation of the meningococci causing diseases is relatively low. The specificity of the bactericidal antibodies induced by these vaccines was determined to be directed mainly against PorA and Opc proteins (29, 30). However, the PorA-specific bactericidal antibodies were found to be directed against epitopes located in surface-exposed highly variable regions (31). Moreover, the Opc protein was shown to be produced by only 60% of strains of different serogroups (32), and by ∼20% of serogroup B isolates (33). To improve the protection conferred by the PorA protein, strategies such as multivalent PorA vaccines or the incorporation of additional epitopes on PorA protein are presently under study (34–36). Proteins induced by iron limitation such as FrpB and Tbp-2 are also likely vaccine candidates, but they also show type specificity with respect to the induction of bactericidal antibodies (37–40). Antigenically conserved proteins such as the Lip (or H.8) (41, 42) and the Rmp (or class 4) (43) proteins were identified in the meningococcal OM, but antibodies directed against these proteins were found to be nonbactericidal. Moreover, high concentrations of antibodies to the Rmp protein were also reported to block the bactericidal activity of antibodies directed against PorA protein and could prevent the efficient killing of meningococcal cells (43).In the present report, we described a new highly conserved protein, called NspA for Neisserial surface protein A, which was shown to be present in the OM of all N. meningitidis strains tested. An mAb, named Me-1, which is directed against the NspA protein was found to be bactericidal and to passively protect BALB/c mice against experimental infections. To further characterize this protein and to clearly establish its protective potential, the nspA gene was identified, sequenced and cloned into a low copy expression vector to obtain large quantities of the recombinant protein. In addition, we present data that demonstrate that the injection of purified recombinant NspA protein efficiently protect BALB/c mice against a bacterial challenge with a lethal dose of a meningococcal strain of serogroup B.     

Thirteen-Year Evolution of Azole Resistance in Yeast Isolates and Prevalence of Resistant Strains Carried by Cancer Patients at a Large Medical Center

Drug resistance is emerging in many important microbial pathogens, including Candida albicans. We performed fungal susceptibility tests with archived isolates obtained from 1984 through 1993 and fresh clinical isolates obtained from 1994 through 1997 by testing their susceptibilities to fluconazole, ketoconazole, and miconazole and compared the results to the rate of fluconazole use. All isolates recovered prior to 1993 were susceptible to fluconazole. Within 3 years of widespread azole use, we detected resistance to all agents in this class. In order to assess the current prevalence of resistant isolates in our hematologic malignancy and transplant patients, we obtained rectal swabs from hospitalized, non-AIDS, immunocompromised patients between June 1995 and January 1996. The swabs were inoculated onto sheep’s blood agar plates containing 10 μg of vancomycin and 20 μg of gentamicin/ml of agar. One hundred one yeasts were recovered from 97 patients and were tested for their susceptibilities to amphotericin B, fluconazole, flucytosine, ketoconazole, and miconazole. The susceptibility pattern was then compared to those for all clinical isolates obtained throughout the medical center. The antifungal drug histories for each patient were also assessed. The yeasts from this surveillance study were at least as susceptible as the overall hospital strains. There did not appear to be a direct linkage between prior receipt of antifungal agent therapy and carriage of a new, drug-resistant isolate. Increased resistance to newer antifungal agents has occurred at our medical center, but it is not focal to any high-risk patient population that we studied. Monitoring of susceptibility to antifungal agents appears to be necessary for optimizing clinical therapeutic decision making.Institutions across the United States have reported an increase in their rate of nosocomial fungal infections (3). In the 1980s Candida species were responsible for approximately three-quarters of these fungal infections (3, 24), with Candida albicans being the most commonly isolated (59.7%) species (3). The greatest increase has been noted in bloodstream infections (2, 3), focused primarily in critical care units (3, 35). The rise in fungemia has been striking, ranging from 75% in small (≤200 beds) nonteaching hospitals to 487% in large (>500 beds) teaching hospitals (2). High rates of morbidity and mortality are associated with candidal infections in immunocompromised patients (3, 15, 23, 24, 35, 37, 38), and early diagnosis can be difficult. With Candida bloodstream infections accounting for the highest rates of mortality, Pittet and colleagues found that even single positive cultures could not be ignored (26). Mindful of these factors, the use of antifungal agents for prophylaxis and therapy has grown (1, 28). Recent reports have also suggested an increasing prevalence of yeast isolates resistant to the newer azole class of antifungal agents (27, 29), implying that future antifungal treatment and prophylaxis may be more difficult.We undertook the current study to (i) determine the prevalence of resistance in yeast isolates colonizing non-AIDS, immunocompromised patients at Northwestern Memorial Hospital (NMH) and (ii) to assess any association of increased use of imidazoles, particularly fluconazole, to the increasing rate of resistance of yeasts to these agents.     

Entry of Sanfetrinem into Human Polymorphonuclear Granulocytes and Its Cell-Associated Activity against Intracellular,Penicillin-Resistant Streptococcus pneumoniae

The entry of antibiotics into phagocytes is necessary for activity against intracellular pathogens. The ability of sanfetrinem, the first member of a new class of antibiotics, to penetrate human polymorphonuclear granulocytes and its consequences upon subsequent phagocytosis and killing of ingested penicillin-resistant Streptococcus pneumoniae have been evaluated. Sanfetrinem penetrated into human polymorphonuclear leukocytes (PMNs) at all concentrations tested, with cellular concentration/extracellular concentration ratios of 6.6 to 5.03 and 4.21 when sanfetrinem was used at 0.25 to 0.5 and 1 μg/ml, respectively, within 30 min of incubation. The uptake was complete within 5 min and was not energy dependent, since it was not affected by cell viability, environmental temperature, or the addition of a metabolic inhibitor. At a concentration of one-half the MIC, sanfetrinem significantly enhanced human PMN phagocytosis and increased intracellular bactericidal activity against penicillin-resistant S. pneumoniae. Following preexposure of PMNs to a concentration of one-half the MIC of sanfetrinem, there was a significant increase in both phagocytosis and killing compared with that for the controls, indicating the ability of sanfetrinem to interact with biological membranes and remain active within PMNs. Preexposure of streptococci to sanfetrinem made penicillin-resistant S. pneumoniae more susceptible to the bactericidal mechanisms of human PMNs than untreated organisms.It is only in recent years that the emerging concept of immunomodulation by antimicrobial agents has received worldwide interest (4, 11, 15, 17, 24, 30, 34). Thus, the current trend of therapy requires the use of antibiotics which combine a high level of in vitro antibacterial activity with the capacity to act in concert with the immune system in a way that potentiates the host’s defense mechanisms. Among the several multifacetted aspects of the antibiotic-phagocyte interaction, only antibiotic entry into phagocytes and, subsequently, its bioactivity are considered to be clinically relevant and beneficial for the treatment of infections caused by pathogens that are capable of survival and replication within phagocytic cells, constituting a significant cause of human infections. Several antimicrobial agents have been reported to accumulate to high levels in phagocytes: macrolides, fluoroquinolones, and some antitubercular drugs (1, 5, 8, 10, 14, 25, 35, 37). Unfortunately, among the cell wall-acting antibiotics, most β-lactams do not efficiently penetrate phagocytes and the newer β-lactam drugs, such as cefotaxime, ceftizoxime, cefonicid, ceftriaxone, and ceftazidime, are similar to penicillin in their relative inabilities to penetrate phagocytes (4, 11, 22, 23). Among the cell wall inhibitors, only teicoplanin (3) and carbapenem antibiotics, such as imipenem and meropenem (6, 12, 13, 21), have been shown to have high levels of penetration into different phagocytic cells. Although imipenem binds to phagocytes, the concentration of cell-associated drug declines steadily during an incubation period of 1 h (21); on the other hand, meropenem is able to penetrate human phagocytes and remain intracellularly active (13).In the present study we have investigated the uptake of sanfetrinem, the first member of a new class of antibiotics (trinems), by human polymorphonuclear cells (PMNs), the crucial phagocytes that offer protection against most bacterial pathogens, and further examined the in vitro consequences of its uptake on subsequent PMN activities toward a penicillin-resistant strain of Streptococcus pneumoniae, a pathogen that is known to be a leading cause of invasive infections and that is often associated with considerable morbidity and mortality (18, 28).(This work was presented in part at the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, Canada, 28 September to 1 October 1997 [14a].)     

Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.Influenza virus infections remain a serious seasonal health concern and the potential of severe pandemics due to the emergence of new influenza strains, such as the H5N1 “bird flu” recently identified in Hong Kong (39), provides additional impetus to develop potent and effective antiviral agents (24). At present, only amantadine and, in some countries, rimantadine are approved for the treatment and prophylaxis of influenza A infections. However, the usefulness of these two compounds is limited by their lack of activity against influenza B viruses and their rapid selection of drug-resistant mutants which remain transmissible and pathogenic (10, 25).The influenza neuraminidase, which is expressed on the virus surface, has long been considered a valid target for antiviral therapy. This enzyme, which cleaves terminal sialic acid residue from glycoconjugates, is essential for virus proliferation and infectivity (3, 17, 19, 27, 28). The observation that the structural and catalytic amino acids which line the enzyme active site are highly conserved among different influenza neuraminidase types and subtypes (reviewed in reference 6) suggests that inhibitors of this enzyme would be active against a broad range of influenza viruses.Based on information gained from crystallographic studies of influenza neuraminidases complexed with sialic acid or the transition state analog Neu5Ac2en (2, 41, 43), several potent and selective inhibitors of the influenza neuraminidases have been synthesized (15, 16, 43). Two of these, GS 4071 ([3R,4R,5S]-4-acetamido-5-amino-3-[1-ethylpropoxy]1-cyclohexene-1-carboxylic acid), in the form of its oral prodrug GS 4104, and zanamivir (GG167, 4-guanidino-Neu5Ac2en) are currently under clinical development for the prophylaxis and treatment of influenza virus infections. Both compounds have demonstrated efficacy against influenza A and B viruses in vitro (13, 23, 40, 43, 45), in animal models of influenza virus infection (23, 31, 32, 34), and in experimental influenza virus infection in humans (11, 12, 14) when GS 4104 is taken orally and zanamivir is delivered topically to the respiratory tract as an inhalant.Although the development of resistance to zanamivir in animals or people treated with the drug has not been reported, influenza variants resistant to zanamivir due to mutations within their hemagglutinin or neuraminidase genes have been selected in vitro (1, 7, 8, 20, 38). In general, zanamivir-resistant hemagglutinin mutants have been easier to generate than neuraminidase mutations. These hemagglutinin mutants commonly contain amino acid substitutions in or near the sialic acid binding site and are believed to make the virus replication less dependent on neuraminidase activity (7, 20, 33). However, these mutations likely only affect the in vitro, not the in vivo, susceptibility to zanamivir (29).The most common neuraminidase mutation which arises in vitro under selective pressure of zanamivir has been that of the conserved Glu119 residue in the neuraminidase active site (1, 7, 38). Mutations of Glu119, which interacts with the guanidino side chain of zanamivir but not with the natural substrate (43), cause a 100-fold reduction in the sensitivity of the enzyme to zanamivir (1, 7, 38). Viruses containing mutations at this position remain infectious (8) and capable of inducing a febrile response in ferrets (5). Recently, a Lys substitution for the conserved Arg at position 292 has also been reported for a variant selected in the presence of zanamivir (8). The neuraminidase containing this mutation exhibited only a 10-fold reduction in sensitivity to zanamivir. A reassortant virus containing the mutant neuraminidase was 500-fold less infectious than wild-type virus in mice (8).In this report, we describe the first in vitro isolation and characterization of variants of a human influenza virus, A/Victoria/3/75 (H3N2), selected in the presence of GS 4071. In contrast with the experience with amantadine and rimantadine, with which drug-resistant variants can be selected after one or two passages in culture (26), variants with decreased susceptibility to GS 4071 did not readily occur. In the eighth passage, a variant containing two mutations in the stalk region of the hemagglutinin was isolated. This variant exhibited a minor decrease in susceptibility to neuraminidase inhibitors in general. A second variant, containing a conservative substitution of a Lys for an Arg at amino acid 292 of the neuraminidase enzyme active site, was isolated later in the selection process. This mutation caused a marked decrease in the susceptibility of the virus and the sensitivity of the enzyme to GS 4071. However, this mutation also adversely affected neuraminidase enzyme activity, compromised the ability of the virus to replicate in tissue culture, and reduced the infectivity of the virus 10,000-fold in mice.     

Antibody-mediated Modulation of Cryptococcus neoformans Infection Is Dependent on Distinct Fc Receptor Functions and IgG Subclasses

Coupling of an antibody response to effector cells through the Fc region of antibodies is a fundamental objective of effective vaccination. We have explored the role of the Fc receptor system in a murine model of Cryptococcus neoformans protection by infecting mice deleted for the common γ chain of FcRs. Passive administration of an IgG1 mAb protects FcRγ^+/− mice infected with C. neoformans, but fails to protect FcRγ^−/− mice, indicating that the γ chain acting through FcγRI and/or III is essential for IgG1-mediated protection. In contrast, passive administration of an IgG3 mAb with identical specificity resulted in enhanced pathogenicity in γ chain–deficient and wild-type mice. In vitro studies with isolated macrophages demonstrate that IgG1-, IgG2a-, and IgG2b-opsonized C. neoformans are not phagocytosed or arrested in their growth in the absence of the FcRγ chain. In contrast, opsonization of C. neoformans by IgG3 does not require the presence of the γ chain or of FcRII, and the internalization of IgG3-treated organisms does not arrest fungal growth. C ryptococcus neoformans is an encapsulated fungus that infects immunosuppressed individuals and is responsible for the death of 6–8% of AIDS patients (1). Antibodies to the glucuronoxylomannan (GXM)¹ portion of the capsular polysaccharide modulate the infection (2). We have previously demonstrated that anticryptococcal IgG3 mAbs are not protective in mouse models of cryptococcal infection (3–5). However, when the nonprotective IgG3 mAbs were switched in vitro to other downstream IgG isotypes, these antibodies became protective (4, 5). Since the IgG3 antibodies and their switch variants have identical antigen binding sites (5), this observation underscores the importance of Fc-mediated functions for antibody efficacy against C. neoformans. In principle, the Fc domain of IgG immune complexes can interact with a variety of soluble and cell-bound molecules that may be involved in mediating the protective capacity of this antibody. Thus, complement C1q binding can lead to the activation of C3, which leads in turn to either association with CR1/2 or generation of a membrane attack complex through C5 (6). Direct interaction of IgG1 complexes with cognate FcRs could mediate either fungal killing, or the arrest of fungal growth through NK or macrophage-mediated antibody-dependent cytotoxicity (ADCC) (7), macrophage phagocytosis, or neutrophil activation (8). Finally, IgG1, through its interaction with the FcRn transporter (for review see reference 9), could be involved in clearance of opsonized cells.In this study, we have begun the dissection of the mechanisms by which IgG subclasses mediate their biological responses by comparing the capacity of the IgG3 and the IgG1 isotypes to (a) modulate cryptococcal infection in FcR-deficient mice in vivo and (b) mediate macrophage phagocytosis and the arrest of fungal growth in vitro. Three different classes of murine Fc receptors for IgG (FcγRI, FcγRII and FcγRIII) have been identified on immune effector cells (for review see references 10–12). Activation by cross-linking of FcγRI and III on macrophages, NK cells, mast cells, neutrophils, and other myeloid cells by antigen–antibody complexes leads to activation of diverse biological responses including phagocytosis, ADCC (for review see references 10, 12, 13), and release of inflammatory mediators (for review see reference 14). The γ subunit of Fc receptors is an essential component of type I and III receptors for IgG antibodies and is required for both receptor assembly and signal transduction (15, 16). FcRγ chain deletion leads to impaired macrophage phagocytosis of IgG-coated SRBCs despite persistent binding, and leads to defects in NK cell– mediated ADCC (17). In contrast, cross-linking of FcγRII does not elicit a biological response on effector immune cells. However, when coligated to an activation receptor, such as FcγRIII, B cell receptor, or TCR, FcγRII inhibits the activation signal generated in those cells by inhibiting calcium influx to the cell through the recruitment of the SH2 inositol polyphosphate phosphatase SHIP (18, 19). Deletion of FcγRII results in mice with hyperresponsive B cells, mast cells, and macrophages (20).Here, we report that the FcRγ chain–deficient mice and heterozygous littermate controls are equally susceptible to cryptococcal infection and that γ chain is essential for IgG1-mediated passive protection against this pathogen. In addition, IgG3 mediated the phagocytosis of C. neoformans and SRBCs in the absence of FcγRI, II, or III function, suggesting that it either uses a different FcR for IgG3 or a signal transduction pathway that is different from the other FcγRs. In contrast to the ability of IgG1 and IgG2a mAbs to inhibit fungal growth, IgG3-mediated phagocytosis is not associated with the arrest of fungal replication. The findings in this study provide a rationale for the different protective efficacies of IgG3 and IgG1 mAbs in mice infected with C. neoformans.     

TRAF-interacting Protein (TRIP): A Novel Component of the Tumor Necrosis Factor Receptor (TNFR)- and CD30-TRAF Signaling Complexes That Inhibits TRAF2-mediated NF-κB Activation

Through their interaction with the TNF receptor–associated factor (TRAF) family, members of the tumor necrosis factor receptor (TNFR) superfamily elicit a wide range of biological effects including differentiation, proliferation, activation, or cell death. We have identified and characterized a novel component of the receptor–TRAF signaling complex, designated TRIP (TRAF-interacting protein), which contains a RING finger motif and an extended coiled-coil domain. TRIP associates with the TNFR2 or CD30 signaling complex through its interaction with TRAF proteins. When associated, TRIP inhibits the TRAF2-mediated NF-κB activation that is required for cell activation and also for protection against apoptosis. Thus, TRIP acts as a receptor–proximal regulator that may influence signals responsible for cell activation/proliferation and cell death induced by members of the TNFR superfamily.Members of the TNF receptor (TNFR)¹ superfamily play important roles in the induction of diverse signals leading to cell growth, activation, and apoptosis (1). Whether the signals induced by a given receptor leads to cell activation or death is, however, highly cell-type specific and tightly regulated during differentiation of cells. For example, the TNFRs can exert costimulatory signals for proliferation of naive lymphocytes but also induce death signals required for deletion of activated T lymphocytes (1). The cytoplasmic domains of these receptors lack intrinsic catalytic activity and also exhibit no significant homology to each other or to other known proteins. Exceptions to this include Fas(CD95) and TNFR1 that share a significant homology within an 80–amino acid region of their cytoplasmic tails (called the “death domain”; 2, 3). Therefore, it is suggested that the TNFR family members can initiate different signal transduction pathways by recruiting different types of intracellular signal transducers to the receptor complex (1).Indeed, several types of intracellular signal transducers have been identified that initiate distinct signal transduction pathways when recruited to the members of TNFR superfamily (4–19). Recent biochemical and molecular studies showed that a class of signal-transducing molecules are recruited to Fas(CD95) or TNFR1 via interaction of the death domains (2, 3, 6, 12, 17, 20). For example, Fas(CD95) and TNFR1 recruit FADD(MORT1)/RIP or TRADD/FADD (MORT1)/RIP through the interactions of their respective death domains (2, 3, 6, 12, 17, 20, 21). Clustering of these signal transducers leads to the recruitment of FLICE/ MACH, and subsequently, to cell death (13, 14).The TNFR family members can also recruit a second class of signal transducers called TRAFs (TNFR-associated factor), some of which are responsible for the activation of NF-κB or JNK (9, 20, 22). TRAF proteins were identified by their biochemical ability to interact with TNFR2, CD40, CD30, or LT-βR (4, 5, 10, 11, 15, 23–27). These receptors interact directly with TRAFs via a short stretch of amino acids within their cytoplasmic tails, but do not interact with the death domain containing proteins (4, 5, 15, 24–27). To date, five members of the TRAF family have been identified as signaling components of the TNFR family members. All TRAF members contain a conserved TRAF domain, ∼230 amino acids in length, that is used for either homo- or heterooligomerization among the TRAF family, for interactions with the cytoplasmic regions of the TNFR superfamily, or for interactions with downstream signal transducers (4, 5, 8, 10, 11, 19, 23–25, 28). In addition to the TRAF domain, most of the TRAF family members contain an NH₂-terminal RING finger and several zinc finger structures, which appear to be important for their effector functions (4, 5, 10, 11, 23–25).Several effector functions of TRAFs were revealed by recent experiments based on a transfection system. TRAF2, first identified by its interaction with TNFR2 (4), was subsequently shown to mediate NF-κB activation induced by two TNF receptors, CD40 and CD30 (9, 28–30). TRAF5 was also implicated in NF-κB activation mediated by LTβR (10), whereas TRAF3 (also known as CRAF1, CD40bp, or LAP1; references 5, 11, 24, and 25) was shown to be involved in the regulation of CD40-mediated CD23 upregulation in B cells (5). The role of other TRAF members in the TNFR family–mediated signal transduction is not clear. They may possess some effector functions as yet to be revealed, or work as adapter proteins to recruit different downstream signal transducers to the receptor complex. For example, TRAF1 is required for the recruitment of members of the cellular inhibitor of apoptosis protein (c-IAP) family to the TNFR2-signaling complex (7). In addition to the signal transduction by the TNFR family members, TRAFs may regulate other receptor-mediated signaling pathways. For example, TRAF6 is a component of IL-1 receptor (IL1R)–signaling complex, in which it mediates the activation of NF-κB by IL-1R (31). Since TRAFs form homo- or heterooligomers, it is suggested that the repertoire of TRAF members in a given cell type may differentially affect the intracellular signals triggered by these receptors. This may be accomplished by the selective interaction of TRAFs with a specific set of downstream signal transducers. Although many aspects of TRAF-mediated effector functions leading to cellular activation have been defined, it needs to be determined whether TRAF proteins will also mediate the apoptotic signals induced by the “death-domain-less” members of the TNFR superfamily (1, 27, 32–36).Here we report the isolation and characterization of a novel component of the TNFR superfamily/TRAFs signaling complex, named TRIP (TRAF-interacting protein). TRIP associates with the receptor/TRAF signaling complex, and inhibits the TRAF2-mediated NF-κB activation. Biochemical studies indicate that TRIP associates with the TNFR2 or CD30 receptor complex via its interaction with TRAF proteins, suggesting a model which can explain why the ligation of these receptors can promote different cell fates: proliferation or death.     

Anemia,red blood cell transfusion,and outcomes after severe traumatic brain injury

In the previous issue of Critical Care, Sekhon and colleagues report that mean 7-day hemoglobin concentration <90 g/l was associated with increased mortality among patients with severe traumatic brain injury (TBI). The adverse relationship between reduced hemoglobin concentrations and outcomes among those with TBI has been an inconsistent finding across available studies. However, as anemia is common among adults with severe TBI, and clinical equipoise may exist between specialists as to when to transfuse allogeneic red blood cells, randomized controlled trials of liberal versus restricted transfusion thresholds are indicated.In the previous issue of Critical Care, Sekhon and colleagues conducted a single-center retrospective cohort study to determine whether hemoglobin concentration was associated with outcomes among 273 critically ill adults with severe traumatic brain injury (TBI) [1]. After adjusting for age, Glasgow Coma Scale scores, external ventricular drain insertion, and allogeneic red blood cell (RBC) transfusion, the authors report that the estimated odds of in-hospital mortality among patients with a mean 7-day hemoglobin concentration <90 g/l was 3.1 (95% confidence interval, 1.5 to 6.3) times the estimated odds of in-hospital mortality among those with a mean 7-day hemoglobin concentration ≥90 g/l.Anemia is common among ICU patients [2]. The etiology of ICU anemia is multifactorial and includes the negative effects of the systemic inflammatory response on hematopoiesis, frequent phlebotomy, and hemodilution from intravenous fluid resuscitation [2]. Among ICU patients with TBI, the prevalence of reduced hemoglobin concentration ranges from 22 to 69%, depending on the presence or absence of extracranial hemorrhage and the timing of hemoglobin measurements [3].Although a hemoglobin transfusion threshold >70 g/l was adopted for ICU patients following publication of the Transfusion Requirements in Critical Care trial [4], this target may be poorly tolerated by those with severe TBI [2]. Anemia-induced compensatory mechanisms result in cerebral arteriolar dilatation and increased brain blood flow [2], which could be detrimental for those with cerebral edema or intracranial hypertension. Moreover, as brain tissue oxygen tension is dependent on systemic hemoglobin, reduced hemoglobin concentrations among those with TBI could decrease cerebral oxygen delivery and contribute to brain hypoxia [2].Although the findings of the study by Sekhon and colleagues provide support for the above physiologic concerns regarding reduced hemoglobin concentrations following brain injury [1], the adverse relationship between anemia and clinical outcomes is an inconsistent finding among available clinical studies [1,5-16]. Of the one randomized controlled trial [10] and the now 14 available cohort studies of which we are aware (two of which were based on post-hoc analyses of similar datasets derived from randomized controlled trials) [1,5-9,11-18], eight reported an association between anemia and an increased risk of poor neurological outcomes or mortality [1,5,9,11,13,14,17,18], while the remaining seven observed no such association. Moreover, in a recent systematic review of comparative studies, insufficient evidence was found to support a difference in outcomes between higher and lower hemoglobin levels among mostly TBI patients [19].Possible explanations for the inconsistency in results across studies include differences in TBI severity among study patients and inadequate consideration of the effects of anemia during critical time periods [20]. Although a set hemoglobin threshold may exist under which harm may occur among those with TBI, adverse outcomes may be more likely to occur during times of low cerebral blood flow, brain hypoxia, and/or ineffective autoregulation [16,20]. Some support for this argument was afforded by the findings of a recent retrospective cohort study, which reported that although anemia alone did not appear to be detrimental among patients with severe TBI, the simultaneous combination of anemia and brain hypoxia was linked with an increased risk of unfavorable outcomes [16].Another significant limitation of the existing literature on this topic has been the absence of a defined disease-exposure relationship among patients with TBI. Although it is plausible that development of reduced hemoglobin concentrations may be most important during the first 7 days following severe TBI [1], the use of the mean as a summary measure of exposure has the potential to result in exposure misclassification. Moreover, as the effects of anemia on outcomes following TBI are likely to be small, and a tremendous amount of brain hypoxia due to anemia would probably be needed to increase mortality, a sensitive measure of neurological performance or outcome is probably a more important outcome variable [20].Possibly the most important limitation of the available literature relating anemia to outcomes among those with TBI, however, is the inadequate consideration of the effects of RBC transfusion [19]. Although RBC transfusion often results in a small incremental increase in brain tissue oxygen tension in this patient population, transfused blood has important differences from the patients'' own blood and does not always improve cerebral metabolism [2]. Moreover, at least five retrospective cohort studies have reported that RBC transfusion increases the risk of death or worsened neurological outcome among those with TBI [9,13,14,21,22]. Admittedly, however, these observations could have been related to selection bias and an unbalanced distribution of outcome determinants between treatment groups [20]. Moreover, as anemia and RBC transfusion are probably highly correlated, those studies that used interaction terms for anemia and RBC transfusion in their regression models probably introduced multicollinearity, and therefore their estimated coefficients and odds ratios may be invalid [20].In summary, although preclinical experiments suggest several potential adverse effects of anemia among patients with TBI, the results of the available clinical studies are conflicting, and it remains unclear whether RBC transfusion may further increase risk of adverse outcomes. However, because anemia is common among adults with severe TBI, and a recent survey reported that clinical equipoise may exist among specialists as to when to transfuse allogeneic RBCs [23], randomized controlled trials of liberal versus restricted transfusion thresholds are required among adults with severe TBI. These trials will probably require use of multimodal monitoring to understand whether improved outcomes are only witnessed among those with simultaneous signs of brain hypoxia or cerebral ischemia.     

Correlation between Azole Susceptibilities,Genotypes, and ERG11 Mutations in Candida albicans Isolates Associated with Vulvovaginal Candidiasis in China

The relationship between susceptibilities to fluconazole and itraconazole and microsatellite CAI genotypes were examined from a total of 154 Candida albicans isolates (97 isolates causing vulvovaginitis in Chinese women and 6 vaginal isolates and 51 oral cavity isolates from asymptomatic carriers). The two dominant genotypes, CAI 30-45 (45 isolates) and CAI 32-46 (33 isolates), associated with vulvovaginitis showed significantly different azole susceptibility patterns with strong statistical support. CAI 32-46 isolates were usually less susceptible to both fluconazole and itraconazole than CAI 30-45 isolates and than the oral isolates with other diversified CAI genotypes. Remarkably different mutation patterns in the azole target gene ERG11 were correspondingly observed among C. albicans isolates representing different genotypes and sources. Isolates with the same or similar CAI genotypes usually possessed identical or phylogenetically closely related ERG11 sequences. Loss of heterozygosity in ERG11 was observed in all the CAI 32-46 isolates but not in the CAI 30-45 isolates and most of the oral isolates sequenced. Compared with the ERG11 sequence of strain SC5314 ({"type":"entrez-nucleotide","attrs":{"text":"X13296","term_id":"2503","term_text":"X13296"}}X13296), two homozygous missense mutations (G487T and T916C) leading to two amino acid changes (A114S and Y257H) in Erg11p were found in CAI 32-46 isolates. The correlation between azole susceptibility and C. albicans genotype may be of potential therapeutic significance.Vulvovaginal candidiasis (VVC) is a common vaginal infection, affecting up to 75% of women of child-bearing age at least once in their lifetime (7, 21, 22). The most frequent cause of VVC is Candida albicans, which is responsible for 70 to 90% of vulvovaginitis cases. Non-C. albicans species of Candida, predominantly Candida glabrata, are responsible for the remainder of cases (21). On the basis of the severity of symptoms, frequency, and causative agents, VVC is usually classified as either uncomplicated (mild and sporadic) or complicated (recurrent, severe, or caused by non-C. albicans species) (7, 21). Ten to 20% of women suffer complicated VVC in their lifetime (21). When properly diagnosed, uncomplicated VVC may be treated easily and reliably. However, complicated VVC often causes long-term physical and mental discomfort, significant economic burden from treatments, and considerable negative effect on sexual relations (21-23).At present, prolonged suppressive therapy using fluconazole is recommended as the standard management for chronic, recurrent Candida vulvovaginitis (23). Therefore, there is a great concern about the emergence and spread of azole resistance of C. albicans isolates associated with VVC. Indeed, susceptibility testing of VVC-causing isolates has been performed in different countries and regions of the world (1, 2, 4, 5, 6, 13-15, 17, 18, 20, 24). Although relatively high frequencies of fluconazole- and/or itraconazole-resistant C. albicans isolates causing VVC have been observed in a few reports (13, 20, 24), most studies failed to identify any clear correlation between azole susceptibility and VVC association among C. albicans isolates (1, 2, 4, 5, 6, 14, 15, 17, 18).Recently, we compared the genotype distribution patterns among independent C. albicans isolates associated with VVC in Chinese women and those from various extragenital sites by using the polymorphic microsatellite locus CAI (8, 11). The results showed that the CAI genotypes of C. albicans isolates from extragenital sites were highly diversified. In contrast, isolates associated with VVC from unrelated patients were more homogeneous and belonged to only a few genotypes, with two genotypes, CAI 30-45 and CAI 32-46, being the most common. These two dominant genotypes were rarely found among isolates from extragenital sites (11). In addition, the distribution of the dominant genotypes correlated positively with the severity of VVC (8, 11). These results suggested that C. albicans isolates with genotypes CAI 30-45 and CAI 32-46 might be more virulent and/or more resistant to the commonly used azole drugs than those with other genotypes as causative agents of vaginal infection.Antifungal susceptibility testing using the Etest method revealed that the C. albicans isolates causing VVC in Chinese women were generally susceptible to fluconazole, amphotericin B, ketoconazole, and flucytosine; however, 19.1% of the isolates could be interpreted as being resistant to itraconazole in vitro. Interestingly, most of the itraconazole-resistant isolates belonged to a specific genotype (13). Contrary to the report described above, recent susceptibility testing and microsatellite typing of vulvovaginitis-causing Candida isolates from Europe did not find an association between azole resistance and any particular genotype cluster among C. albicans isolates (1). In the present study, fluconazole and itraconazole susceptibilities of the C. albicans isolates with the dominant genotypes CAI 30-45 and CAI 32-46 from VVC patients were compared with those of isolates possessing other minor genotypes and of isolates from the oral cavity by using the standard broth microdilution method. Furthermore, ERG11 (encoding lanosterol-14-α-demethylase, the target of azoles) gene sequences of C. albicans isolates representing different genotypes and sources were determined. The correlation between azole susceptibilities, genotypes, and ERG11 mutations was examined.     

Recombinant Expression and Characterization of the Major β-Lactamase of Mycobacterium tuberculosis

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis. Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by which M. tuberculosis expresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M. tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of M. tuberculosis H37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA of M. tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase of M. tuberculosis is a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.Tuberculosis causes 3 million deaths annually, more than any other single infectious agent (2, 19, 35). Multidrug resistance is a growing clinical problem, with strains of Mycobacterium tuberculosis exhibiting resistance to 11 or more antimicrobial agents having been described (25). Although it was shown in the 1940s that under certain culture conditions penicillin inhibits the growth of M. tuberculosis (9, 10, 18, 31), the availability of other effective antimicrobial agents limited efforts to determine whether tuberculosis might respond to treatment with β-lactams. However, the recent rise in infections caused by multidrug-resistant strains has made it necessary to identify alternative treatment regimens, including the determination of whether some older classes of antibiotics such as the β-lactams might be effective in the clinical setting.The cell wall structure of M. tuberculosis contains a thick peptidoglycan layer. Cycloserine, a second-line drug in the treatment of tuberculosis, is a d-alanine analog that interferes with peptidoglycan synthesis (37). Recently, it has been shown that M. tuberculosis makes at least four penicillin-binding proteins (PBPs) that bind ampicillin and other β-lactams at clinically relevant antibiotic concentrations (3). The affinities of these agents for their PBP targets are of the magnitude seen for β-lactams that can be effectively used for the treatment of infections caused by other microbes. Also, the outer cellular structures of tubercle bacilli do not represent a major permeability barrier for β-lactams (3, 22). Therefore, the production of β-lactamase by M. tuberculosis appears to be its major mechanism of resistance to β-lactams.Most and possibly all isolates of M. tuberculosis produce β-lactamase (12, 13, 15, 42); however, data regarding its nature are limited. Opinions differ as to whether it is secreted, cytoplasmic, or bound to the cell membrane and as to whether its production is inducible or constitutive (10, 14, 15, 32, 42). Zhang et al. (42) have reported that isoelectric focusing of Triton X-100 extracts of acetone-precipitated cell pellets of strains of M. tuberculosis reveals two bands exhibiting β-lactamase activity with pI values of 4.9 and 5.1.Most of the information on the kinetic properties of M. tuberculosis β-lactamase comes from studies with relatively impure preparations of enzyme or has been inferred indirectly via the results of susceptibility tests involving β-lactams and β-lactam–β-lactamase inhibitor combinations. However, greater penicillinase activity than cephalosporinase activity is consistently reported (15, 20, 22, 42). M. tuberculosis β-lactamase is inhibited competitively by antistaphylococcal penicillins (13–15, 21, 22, 32) and by conventional β-lactamase inhibitors including clavulanic acid, sulbactam, and tazobactam (5, 8, 33, 38, 41, 42). β-Lactamase inhibitors improve the activities of some penicillins against M. tuberculosis in vitro (5, 8, 14, 33) and in vivo (13). In addition, some cephalosporins including ceforanide and cephapirin as well as carbapenems such as imipenem exhibit potent in vitro activities (23, 30, 36).Because a better understanding of the mechanisms by which M. tuberculosis expresses resistance to β-lactams might ultimately lead to strategies in which these agents could be used in the treatment of tuberculosis, we have worked to characterize its β-lactamase(s). In this report, we describe the isolation of three enzymes with distinct pI values directly from M. tuberculosis and the recombinant expression and kinetic characterization of the major enzyme.(Results of this study were presented in part at the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, La., 15 to 18 September 1996, and at the 32nd U.S.-Japan Conference of Tuberculosis/Leprosy, Cleveland, Ohio, 21 to 23 July 1997.)