Showing papers in "Antimicrobial Agents and Chemotherapy in 1997"

Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.

Abstract: In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

Assessment of the pharmacodynamic properties of antimalarial drugs in vivo.

Abstract: Malaria is unusual among the systemic infections of humans in that the number of organisms causing the disease may be quantitated with reasonable precision. This applies particularly to those causing benign human malarias, i.e., Plasmodium vivax, P. malariae, and P. ovale, as these parasites are not sequestered in the microcirculation (13). The assessment of parasite burden for the sequestering, potentially lethal parasite P. falciparum is more difficult, but there are some clues which allow a rough estimation of the proportion of parasites circulating in the bloodstream (65). If the number of organisms causing an infection is known, then the pharmacodynamic properties required of an anti-infective drug to produce a cure can be defined. The assessment of the treatment response in malaria rests on the clinical outcome (mortality, speed of recovery from coma, fever clearance, etc.) and the parasitological outcome—the subject of this discussion. Although a great deal remains to be learned about the pharmacodynamic properties of antimalarial drugs in vivo, sufficient information is already available to construct simple models which predict for how long antimalarial treatment should be given and the chances of treatment failure (i.e., recrudescence of the infection). In the management of individual patients, the ratio of the parasitemia at the time of treatment to the count 48 h later (the parasite reduction ratio [PRR]), representing the fractional reduction per asexual life cycle, may be a simple but useful predictive index. General principles. Parasitological recovery from malaria is assessed conventionally by the clearance of parasites from peripheral blood smears (68). In highly drug-resistant infections, parasites do not disappear from the peripheral blood or may increase following the administration of antimalarial drugs. Parasites with lower grades of resistance disappear from the peripheral blood (in fact, the concentration falls below the level of microscopic detection) but recur at a later time, usually in association with a return of symptoms. The efficacy of antimalarial drug treatment is assessed in terms of the speed at which symptoms and signs resolve and parasitemia declines (usually recorded as the parasite clearance time [PCT]) and the proportion of patients in whom infections recur within a defined period (71). Relationship between parasitemia and disease. Assessment of the therapeutic response in falciparum malaria is complicated by the loose relationship between parasitemia (number of parasites per unit volume of blood) and disease severity (10). A patient may be admitted in a deep coma with evidence of liver and renal dysfunction and severe metabolic acidosis, yet parasites are visible only on the thick blood film (,0.02%), whereas in areas where the disease is endemic a child may be able to walk and continues to eat when nearly half of the erythrocytes are parasitized. Several factors explain this discrepancy. As immunity to malaria develops with repeated infections in areas where the disease is endemic, the parasitemia threshold at which symptoms develop rises. This is often termed antitoxic immunity or premunition (2). It reflects a change in the relationship between parasite burden and both the release of and the response to illness-inducing cytokines (25). Interestingly, this relationship differs among the parasites causing human malarias; for example, P. vivax has a lower pyrogenic threshold (ca. 200/ml) than P. falciparum (ca. 10,000/ ml) (23). As a consequence, adults living in areas where malaria is endemic who have been exposed repeatedly to malaria during their lives tolerate detectable parasitemias (total parasite burden, .10) without symptoms, whereas nonimmune persons with such burdens are ill. The second important factor explaining the discrepancy between parasitemia and disease severity relates to the stage and synchronicity of infection (53, 65). In P. falciparum malaria, only the first half of the 48-h life cycle is visible to the microscopist. At approximately 16 to 24 h of asexual development, intraerythrocytic parasites start to induce the expression of adhesins on the infected erythrocyte surface (20). Parasitized erythrocytes aggregate with uninfected erythrocytes (rosetting) and also begin to adhere to vascular endothelium, particularly in the venules. This process is termed cytoadherence, and it leads to sequestration of the mature parasites in the deep vasculature (67). Thus, in P. falciparum malaria, depending on stage and synchronicity, the bulk of the infecting asexual stage parasites may be either circulating (when the mean age of development is in the first half of the cycle) and measurable or sequestered (when the mean age of development is in the second half of the cycle) and not measurable (53). In the expanding phase of the infection, there are usually more circulating than sequestered parasites and there can be considerable differences in synchronous infections (65). As pathophysiological processes in falciparum malaria are thought to relate to the sequestered forms of the parasite and subsequent merogony (67), and not to the younger circulating asexual stages, patients tend to be more ill when the majority of their parasites are sequestered (i.e., not visible to the microscopist) or just after schizont rupture (merogony). In benign human malarias, which synchronize more readily than falciparum malaria, fever and rigors are associated with synchronous merogony (22, 23, 28). These symptoms result from the simultaneous release of malaria-related pyrogens * Mailing address: Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. Phone: 66 2 246 0832. Fax: 66 2 246 7795.

Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus.

Abstract: Resistance to antifungal drugs, specifically azoles such as fluconazole, in the opportunistic yeast Candida albicans has become an increasing problem in human immunodeficiency virus (HIV)-infected individuals. The molecular mechanisms responsible for this resistance have only recently become apparent and can include alterations in the target enzyme of the azole drugs (lanosterol 14alpha demethylase [14DM]), or in various efflux pumps from both the ABC transporter and major facilitator gene families. To determine which of these possible mechanisms was associated with the development of drug resistance in a particular case, mRNA levels have been studied in a series of 17 clinical isolates taken from a single HIV-infected patient over 2 years, during which time the levels of fluconazole resistance of the strain increased over 200-fold. Using Northern blot analysis of steady-state levels of total RNA from these isolates, we observed increased mRNA levels of ERG16 (the 14DM-encoding gene), CDR1 (an ABC transporter), and MDR1 (a major facilitator) in this series. The timing of the increase in mRNA levels of each of these genes correlated with increases in fluconazole resistance of the isolates. Increased mRNA levels were not observed for three other ABC transporters, two other genes in the ergosterol biosynthetic pathway, or the NADPH-cytochrome P-450 oxidoreductase gene that transfers electrons from NADPH to 14DM. Increases in mRNA levels of ERG16 and CDR1 correlated with increased cross-resistance to ketoconazole and itraconazole but not to amphotericin B. A compilation of the genetic alterations identified in this series suggests that resistance develops gradually and is the sum of several different changes, all of which contribute to the final resistant phenotype.

Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.

Abstract: We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.

Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication.

Abstract: We report the development and isolation of a cell line, termed HepAD38, that replicates human hepatitis B virus (HBV) under conditions that can be regulated with tetracycline. In the presence of the antibiotic, this cell line is free of virus due to the repression of pregenomic (pg) RNA synthesis. Upon removal of tetracycline from the culture medium, the cells express viral pg RNA, accumulate subviral particles in the cytoplasm that contain DNA intermediates characteristic of viral replication, and secrete virus-like particles into the supernatant. Since the HepAD38 cell line can produce high levels of HBV DNA, it should be useful for analyses of the viral replication cycle that depend upon viral DNA synthesis in a synchronized fashion. In addition, this cell line has been formatted into a high-throughput, cell-based assay that permits the large-scale screening of diverse compound libraries for new classes of inhibitors of HBV replication.

Itraconazole resistance in Aspergillus fumigatus.

Abstract: Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Abstract: Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.

Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon.

Abstract: Multidrug resistance in bacteria is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (48, 62). However, descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus (Fig. 1) in Escherichia coli (45, 46). Mar mutants of E. coli arose at a frequency of 10 to 10 and were selected by growth on subinhibitory levels of tetracycline or chloramphenicol (45, 46). These mutants exhibited resistance to tetracyclines, chloramphenicol, penicillins, cephalosporins, puromycin, nalidixic acid, and rifampin (45). Later, the resistance phenotype was extended to include fluoroquinolones (25, 105), oxidative stress agents (7, 51), and, more recently, organic solvents (8, 49, 144). The expression of the Mar phenotype is greater at 30°C than 37°C (45, 127). Continued growth in the same or higher antibiotic concentrations led to increased levels of resistance, thus demonstrating an amplifiable multiple antibiotic resistance phenotype (45). Both highand low-level resistances were decreased or completely reversed by a Tn5 insertion into a single locus at 34 min (1,636.7 kb) on the E. coli chromosome, called the mar locus (46). The genetic basis for high-level resistance is only partially attributed to the mar locus, since transduction of the locus from highor low-level mar mutants produced only a low level of multidrug resistance (94). The mar locus consists of two divergently positioned transcriptional units that flank the operator marO (Fig. 1) in E. coli (22, 24, 112) and Salmonella typhimurium (133). One operon encodes MarC, a putative integral inner membrane protein (Fig. 1) without any yet apparent function, but which appears to contribute to the Mar phenotype in some strains (see below) (49, 143, 144). The other operon comprises marRAB, encoding the Mar repressor (MarR), which binds marO and negatively regulates expression of marRAB (22, 90, 127), an activator (MarA), which controls expression of other genes on the chromosome, e.g., the mar regulon (22, 41, 126), and a putative small protein (MarB) of unknown function (Fig. 1). The marRAB operon responds to a variety of compounds (7, 24, 52, 54, 99, 119, 127) including tetracycline and chloramphenicol (54). Deletion or inactivation of the marRAB operon results in increased susceptibility to multiple antibiotics, a variety of oxidative stress agents, and organic solvents (22, 24, 46, 51, 54, 144). Since salicylate and acetylsalicylate induced a reversible “phenotypic antibiotic resistance” to multiple antibiotics (118), a connection was made between phenotypic antibiotic resistance and mar (24); salicylate induced expression of marRAB (24). These studies also extended the spectrum of inducers to include acetaminophen, sodium benzoate, 2,4-dinitrophenol (an uncoupling agent), and cinnamate (a salicylate precursor in plants) (24). The uncoupling agent carbonyl cyanide m-chlorophenylhydrazone and redox-cycling compounds, menadione and plumbagin, are also inducers of marRAB transcription (127). Initially it was thought that 7.8 kb of chromosomal DNA was needed to generate constitutive mar mutants (mar) in a strain bearing a large (39 kb) chromosomal deletion (54). However, this finding was shown to be strain specific, and ;1.1 kb of mar sequence, containing marO, marR, and marA sequences, was sufficient to select a mar mutant (91, 134). mar mediates tetracycline resistance through an energy-dependent efflux system (45). Mar mutants are resistant to fluoroquinolones through a combined decrease in cell influx, e.g., a decrease in the porin OmpF, and an intrinsic efflux system (25). Chloramphenicol resistance in Mar mutants is also attributed to active efflux, which is enhanced over an intrinsic efflux system (96). The Mar phenotype is linked to overexpression of the acrAB locus; deletion of acrAB confers increased susceptibility to multiple drugs (85, 106) and organic solvents (144) in wild-type or Mar strains (85, 106, 144). These findings suggest that the acrAB efflux system is a major mechanism of Mar-mediated resistance (106). However, since the tetracycline (45) and fluoroquinolone (26) efflux systems were only saturated by the respective drug and not by others, it is probable that other drug-specific efflux systems are involved, particularly in high-level multidrug-resistant mutants.

Pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus protease by coadministration with ritonavir.

Abstract: Coadministration with the human immunodeficiency virus (HIV) protease inhibitor ritonavir was investigated as a method for enhancing the levels of other peptidomimetic HIV protease inhibitors in plasma. In rat and human liver microsomes, ritonavir potently inhibited the cytochrome P450 (CYP)-mediated metabolism of saquinavir, indinavir, nelfinavir, and VX-478. The structural features of ritonavir responsible for CYP binding and inhibition were examined. Coadministration of other protease inhibitors with ritonavir in rats and dogs produced elevated and sustained plasma drug levels 8 to 12 h after a single dose. Drug exposure in rats was elevated by 8- to 46-fold. A > 50-fold enhancement of the concentrations of saquinavir in plasma was observed in humans following a single codose of ritonavir (600 mg) and saquinavir (200 mg). These results indicate that ritonavir can favorably alter the pharmacokinetic profiles of other protease inhibitors. Combination regimens of ritonavir and other protease inhibitors may thus play a role in the treatment of HIV infection. Because of potentially substantial drug level increases, however, such combinations require further investigation to establish safe regimens for clinical use.

Why are antibiotic resistance genes so resistant to elimination

Abstract: The euphoria produced by the discovery of antibiotics led to confident predictions that bacterial diseases would soon be conquered and could thus be safely forgotten, leaving scientists free to attack other pressing health problems such as viral diseases and cancer. Instead of witnessing the disappearance of bacterial diseases, however, we are now experiencing a resurgence of them, both in hospital and in community settings. To make matters worse, bacterial pathogens have become increasingly resistant to a variety of antibiotics. A major driving force behind the increase in resistant strains is the ease with which bacteria can acquire resistance genes, even from distantly related genera. Much of what has been written about gene transfer elements has focused on elements acting in isolation. Yet, gene transfer elements can—and often do—hunt as a pack, by interacting with each other in a variety of ways that enhance their collective ability to transfer resistance genes. This interactive capacity needs to be taken into account when considering the potential for horizontal transfer of resistance genes. One aim of this minireview is to consider the consequences of cooperative behavior between different gene transfer elements. The characteristics of the different elements involved in the spread of resistance genes are summarized in Table 1. Although the acquisition of new resistance genes is an important factor in the increasing incidence of resistant strains, it is only part of the resistance story. A critical, but often underappreciated, feature of resistance gene transfer elements is their stability. Their ability to adapt rapidly to new hosts so that they are not readily lost even in the absence of antibiotic selection may explain why increases in resistance can be so hard to reverse. The hope that the cessation of the use of a particular antibiotic will cause resistance to that antibiotic to disappear is proving to be illusory. It is true that decreased use of an antibiotic is usually accompanied by some decrease in the incidence of resistant strains. For example, in a country-wide Cuban program to control the use of certain classes of antibiotics, impressive decreases in resistance to most of the restricted-use antibiotics occurred (7). Similarly, a decrease in penicillin-resistant strains of Streptococcus pneumoniae was seen in Hungary after doctors drastically reduced their use of penicillin for the treatment of pneumococcal infections (17). As encouraging as such reports may seem at first glance, closer inspection reveals a different and more ominous message. Although the incidence of resistant strains may drop, it seldom falls to zero (7, 17). This leaves a residuum of persistently resistant strains that can rebound rapidly to become the predominant strains if antibiotic use is resumed. An example of this phenomenon has been provided by Gerding et al. (11), who described the rapid return of resistance to gentamicin and tobramycin when gentamicin was reintroduced after amikacin use was discontinued in a hospital. Unfortunately, relatively few studies of this type have been done, so it is not clear whether rapid rebound of resistance will occur with all types of antibiotics or only with some antibiotics. Another troubling message comes from studies of nonclinical isolates. Calva et al. (8) found high levels of resistant strains of enteric bacteria in the feces of children from urban areas of Mexico. In this case, episodic use of antibiotics, which in Mexico are available without prescription, may be sufficient to maintain high levels of resistant strains. Another study that compared antibiotic resistance patterns in bacteria from feces from rural and urban Mexican children (2) showed that in general the bacteria from rural children were less resistant than those from urban children, except in the case of antibiotics used agriculturally. There is still relatively little information about the impact of agricultural use of antibiotics on resistance levels. Finally, a study of groundwater isolates from rural Tennessee revealed unexpectedly high levels of resistant enteric bacteria (16). It is difficult to rule out some sort of antibiotic contamination of the groundwater. Nonetheless, it is noteworthy that although chloramphenicol has rarely been used in the United States over the past decade, nearly 17% of the coliforms in the groundwater have been found to be resistant to chloramphenicol. Any successful attempt to curb the spread of resistance will have to take into account the fact that resistance genes and the transmissible elements that carry them are hard to lose as well as easy to get. What we do not know and desperately need to learn is whether the persistence of resistant bacteria in the environment is due to low-level antibiotic contamination, some nonantibiotic selection, or simply the stability of the resistance genes and transfer elements. This minireview surveys some recent work on the “easy-toget” and “hard-to-lose” sides of the resistance gene transfer equation. The purpose of this minireview is not to provide an exhaustive coverage of these topics, a task which would require much more space than is available in a minireview. Rather, we want to provoke a reassessment of some widely held views about antibiotic resistance. Accordingly, only selected examples and selected references are provided to illustrate the points being made. Where possible review articles rather than original papers are cited. Another aim of this minireview is to highlight the fact that virtually all of the studies of resistance patterns done to date have focused on one narrow group of * Corresponding author.

1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

Abstract: 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

Protegrin-1: a broad-spectrum, rapidly microbicidal peptide with in vivo activity.

Abstract: Protegrin-1 (PG-1) is a cysteine-rich, 18-residue beta-sheet peptide isolated from porcine leukocytes with antimicrobial activity against a broad range of microorganisms. The MICs of PG-1 against representative gram-positive and gram-negative bacteria ranged from 0.12 to 2 microg/ml. At these levels, PG-1 was rapidly bactericidal in vitro, reducing the number of viable CFU of either methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa by more than three log units in less than 15 min. Resistance to PG-1 did not develop after 11 subculturings of P. aeruginosa or 18 subcultures of MRSA in Mueller-Hinton broth containing PG-1 at one-half the MIC. Under similar conditions of serial passage, the MICs of norfloxacin and gentamicin against P. aeruginosa increased 10 and 190 times, respectively. Similarly, the MIC of norfloxacin against MRSA increased 85 times. Immunocompetent mice inoculated intraperitoneally (i.p.) with P. aeruginosa or S. aureus exhibited 93 to 100% mortality in the vehicle control group compared with 0 to 27% mortality in animals that received a single i.p. injection of PG-1 (0.5 mg/kg of body weight). Mice inoculated with S. aureus by intravenous (i.v.) injection and dosed 0 to 60 min later with a single i.v. injection of PG-1 (5 mg/kg) had a mortality of 7 to 33%, compared to a mortality of 73 to 93% in the vehicle controls. In leukopenic mice inoculated i.v. with vancomycin-resistant Enterococcus faecium, mortality was 87% in the vehicle control group and 33% in animals that received a single i.v. injection of PG-1 (2.5 mg/kg). Taken together, these data indicate that PG-1 has potential for use as an antimicrobial agent in the treatment of local or systemic infections caused by clinically relevant pathogens.

A multicenter, randomized study comparing the efficacy and safety of intravenous and/or oral levofloxacin versus ceftriaxone and/or cefuroxime axetil in treatment of adults with community-acquired pneumonia.

Abstract: Five hundred ninety patients were enrolled in a prospective, multicenter, randomized trial comparing the efficacy and safety of 7 to 14 days of levofloxacin treatment with that of ceftriaxone and/or cefuroxime axetil in the management of community-acquired pneumonia in adults. Patients received either intravenous and/or oral levofloxacin (500 mg once daily) or the comparative agents, parenteral ceftriaxone (1 to 2 g once to twice daily) and/or oral cefuroxime axetil (500 mg twice daily). Erythromycin or doxycycline could be added to the comparator arm at the investigator9s discretion. The decision to use an intravenous or oral antimicrobial agent for initial therapy was made by the investigator. Clinical and microbiological evaluations were completed at the baseline, during treatment, 5 to 7 days posttherapy, and 3 to 4 weeks posttherapy. Four hundred fifty-six patients (226 given levofloxacin and 230 administered ceftriaxone and/or cefuroxime axetil) were evaluable for clinical efficacy. Streptococcus pneumoniae and Haemophilus influenzae were isolated in 15 and 12%, respectively, of clinically evaluable patients. One hundred fifty atypical pathogens were identified: 101 were Chlamydia pneumoniae, 41 were Mycoplasma pneumoniae, and 8 were Legionella pneumophila. Clinical success at 5 to 7 days posttherapy was superior for the levofloxacin group (96%) compared with the ceftriaxone and/or cefuroxime axetil group (90%) (95% confidence interval [CI] of -10.7 to -1.3). Among patients with typical respiratory pathogens who were evaluable for microbiological efficacy, the overall bacteriologic eradication rates were superior for levofloxacin (98%) compared with the ceftriaxone and/or cefuroxime axetil group (85%) (95% CI of -21.6 to -4.8). Levofloxacin eradicated 100% of the most frequently reported respiratory pathogens (i.e., H. influenzae and S. pneumoniae) and provided a >98% clinical success rate in patients with atypical pathogens. Both levofloxacin and ceftriaxone-cefuroxime axetil eradicated 100% of the S. pneumoniae cells detected in blood culture. Drug-related adverse events were reported in 5.8% of patients receiving levofloxacin and in 8.5% of patients administered ceftriaxone and/or cefuroxime axetil. Gastrointestinal and central and peripheral nervous system adverse events were the most common events reported in each treatment group. In conclusion, these results demonstrate that treatment with levofloxacin is superior to ceftriaxone and/or cefuroxime axetil therapy in the management of community-acquired pneumonia in adults.

Mechanism of Action of Oxazolidinones: Effects of Linezolid and Eperezolid on Translation Reactions

Abstract: The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

Carbapenem-hydrolyzing beta-lactamases.

Abstract: Carbapenem-hydrolyzing b-lactamases include some of the more recently described b-lactamases in the repertoire of penicillin-interactive proteins. These enzymes are responsible for conferring resistance to the carbapenems, the b-lactam class with the broadest spectrum of antibacterial activity. The number of these enzymes compared to the number of other b-lactamases remains low, with only 17 carbapenem-hydrolyzing enzymes included in the recent compilation of 190 b-lactamases described as functionally or molecularly distinct enzymes (12). Although carbapenems have remained relatively unscathed by the hydrolytic action of many clinically relevant b-lactamases, an increasing number of carbapenem-hydrolyzing enzymes have been described in the past 4 years, especially from Japan, where the carbapenem class is the market leader among parenteral b-lactams, in contrast to other geographical areas, where carbapenems are used on a more restricted basis (7, 30, 50, 74). Several recent reviews have summarized the properties of “carbapenemases” (43, 44), or the specific class of carbapenem-hydrolyzing metallo-b-lactamases (57). However, as more of these enzymes have been described, their diversity has become increasingly evident, both genetically and biochemically. Most of these b-lactamases confer resistance not only to carbapenems, but also to other b-lactams. Thus, the term “carbapenemase” used by some authors is misleading, in that cephalosporins or penicillins may be hydrolyzed more efficiently than carbapenems. In this review the term “carbapenemase” will be reserved for that subgroup of metallo-b-lactamases that preferentially hydrolyze carbapenems. On a molecular level these enzymes can belong either to the class A group of b-lactamases that have serine at the active site or to the class B enzymes that represent the only metallo-blactamases that have been identified (2). Although some class C cephalosporinases (33) have been reported to hydrolyze imipenem at a measurable rate (15), these enzymes are usually not considered to be serious carbapenem-hydrolyzing enzymes, because carbapenems do not represent a major substrate in their hydrolytic profile. In this review production of the carbapenem-hydrolyzing enzymes in their natural isolates will be described, with particular reference to coproduction with other b-lactamases. Biochemical characteristics are summarized, with a new subgrouping of the metallo-b-lactamases proposed on the basis of functional characteristics. The molecular aspects of the chromosomal carbapenem-hydrolyzing enzymes are evaluated, and the regulation of these enzymes is discussed. The sequences of the known enzymes are presented, with three molecular classes proposed for the metalloenzymes on the basis of possible amino acid alignments.

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis.

Abstract: Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations.

Abstract: Ethambutol [(S,S')-2,2'-(ethylenediimino)di-1-butanol; EMB], is a first-line drug used to treat tuberculosis. To gain insight into the molecular basis of EMB resistance, we characterized the 10-kb embCAB locus in 16 EMB-resistant and 3 EMB-susceptible genetically distinct Mycobacterium tuberculosis strains from diverse localities by automated DNA sequencing and single-stranded conformation polymorphism analysis. All 19 organisms had virtually identical sequences for the entire 10-kb region. Eight EMB-resistant organisms had mutations located in codon 306 of embB that resulted in the replacement of the wild-type Met residue with Ile or Val. Automated sequence analysis of the 5' region (1,892 bp) of embB in an additional 69 EMB-resistant and 30 EMB-susceptible M. tuberculosis isolates from diverse geographic localities and representing 70 distinct IS6110 fingerprints confirmed the unique association of substitutions in amino acid residue 306 of EmbB with EMB resistance. Six other embB nucleotide substitutions resulting in four amino acid replacements were uniquely found in resistant strains. Sixty-nine percent of epidemiologically unassociated EMB-resistant organisms had an amino acid substitution not found in susceptible strains, and most (89%) replacements occurred at amino acid residue 306 of EmbB. For strains with the Met306Leu or Met306Val replacements EMB MICs were generally higher (40 microg/ml) than those for organisms with Met306Ile substitutions (20 microg/ml). The data are consistent with the idea that amino acid substitutions in EmbB alter the drug-protein interaction and thereby cause EMB resistance.

mefE Is Necessary for the Erythromycin-Resistant M Phenotype in Streptococcus pneumoniae

Abstract: Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors.

Abstract: Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

In vitro preclinical evaluation studies with the echinocandin antifungal MK-0991 (L-743,872).

Abstract: The echinocandin MK-0991, formerly L-743,872, is a water-soluble lipopeptide that has been demonstrated in preclinical studies to have potent activity against Candida spp., Aspergillus fumigatus, and Pneumocystis carinii. An extensive in vitro biological evaluation of MK-0991 was performed to better define the potential activities of this novel compound. Susceptibility testing with MK-0991 against approximately 200 clinical isolates of Candida, Cryptococcus neoformans, and Aspergillus isolates was conducted to determine MICs and minimum fungicidal concentrations MF(s). The MFC at which 90% of isolates are inhibited for 40 C. albicans clinical isolates was 0.5 microg/ml. Susceptibility testing with panels of antifungal agent-resistant species of Candida and C. neoformans isolates indicated that the MK-0991 MFCs for these isolates are comparable to those obtained for susceptible isolates. Growth kinetic studies of MK-0991 against Candida albicans and Candida tropicalis isolates showed that the compound exhibited fungicidal activity (i.e., a 99% reduction in viability) within 3 to 7 h at concentrations ranging from 0.06 to 1 microg/ml (0.25 to 4 times the MIC). Drug combination studies with MK-0991 plus amphotericin B found that this combination was not antagonistic against C. albicans, C. neoformans, or A. fumigatus in vitro. Studies with 0 to 50% pooled human or mouse serum established that fungal susceptibility to MK-0991 was not significantly influenced by the presence of human or mouse serum. Results from resistance induction studies suggested that the susceptibility of C. albicans was not altered by repeated exposure (40 passages) to MK-0991. Erythrocyte hemolysis studies with MK-0991 with washed and unwashed human or mouse erythrocytes indicated minimal hemolytic potential with this compound. These favorable results of preclinical studies support further studies with MK-0991 with humans.

Monotherapy with Meropenem versus Combination Therapy with Ceftazidime plus Amikacin as Empiric Therapy for Fever in Granulocytopenic Patients with Cancer

Abstract: Early empiric antibiotic therapy is common practice in granulocytopenic cancer patients who develop fever. For the past two decades combinations of beta-lactams and aminoglycosides have been standard therapy for suspected infections in granulocytopenic patients, especially in those with profound long-lasting granulocytopenia. The rationale for using aminoglycoside-containing combinations was based on the high incidence of gram-negative bacteremias, which represented 70% of the single organism bacteremic episodes. The usefulness of aminoglycosides in neutropenic patients with gram-negative bacteremia was emphasised in previous studies which reported outcome improved with combinations of antibiotics synergistic in vitro against the offending gram-negative bacteria. However, over the past 10 years there has been a continuous shift in the type of micro-organisms recovered from the blood of granulocytopenic cancer patients, with a considerable reduction in gram-negative bacteremic episodes, including those due to Pseudomonas aeruginosa and a significant increase in gram-positive isolates. Thus the need for the aminoglycoside-containing combination must be reassessed in view of this striking epidemiological change.

Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of beta-lactamase-positive strains resistant to amoxicillin-clavulanate: results of a national multicenter surveillance study.

Abstract: A total of 1,537 clinical isolates of Haemophilus influenzae were recovered in 30 U.S. medical center laboratories between 1 November 1994 and 30 April 1995 and were characterized in a central laboratory with respect to serotype and beta-lactamase production and the in vitro activities of 15 oral antimicrobial agents. Overall, 36.4% of the isolates were found to produce beta-lactamase. The rank order of activity of six cephalosporins on the basis of MICs was cefixime > cefpodoxime > cefuroxime > loracarbef > or = cefaclor > cefprozil. On the basis of current National Committee for Clinical Laboratory Standards (NCCLS) breakpoints ages of isolates found to be resistant or intermediate to these agents were as follows: 0.1, 0.3, 6.4, 16.3, 18.3, and 29.8, respectively (National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. M7-A4, 1995). Azithromycin was, on a weight basis, the most potent of the macrolides tested in this study, followed by erythromycin and then clarithromycin. Azithromycin was typically fourfold more active than erythromycin, which was, in turn, slightly more active than clarithromycin. However, when compared on the basis of the frequency of resistance determined by using current NCCLS breakpoints, there was essentially no difference between azithromycin and clarithromycin, i.e., 0.5 and 1.9%, respectively (P = 0.086). Interpretive breakpoints for erythromycin MIC tests versus H. influenzae have not been developed. Resistance to other non- beta-lactam agents was variable, as follows: trimethoprim-sulfamethoxazole, 9.0%; chloramphenicol, 0.2%; tetracycline, 1.3%; and rifampin, 0.3%. Two conspicuous findings in this study were the identification of 39 strains H. influenzae that were beta-lactamase negative but ampicillin intermediate or resistant (BLNAR) and, even more surprisingly, 17 beta-lactamase-positive isolates that were resistant to amoxicillin-clavulanate (BLPACR). Strains of H. influenzae in the first group have heretofore been very uncommon; organisms in the second group have not previously been described in the literature. The percentages of all study isolates comprised of BLNAR and BLPACR organisms were 2.5 and 1.1, respectively. Overall resistance to ampicillin was thus 38.9%, and that to amoxicillin-clavulanate was 4.5%.

The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14alpha demethylase in Candida albicans.

Abstract: Azole resistance in the pathogenic yeast Candida albicans is an emerging problem in the human immunodeficiency virus (HIV)-infected population. The target enzyme of the azole drugs is lanosterol 14alpha demethylase (Erg16p), a cytochrome P-450 enzyme in the biosynthetic pathway of ergosterol. Biochemical analysis demonstrates that Erg16p became less susceptible to fluconazole in isolate 13 in a series of isolates from an HIV-infected patient. PCR-single-strand conformation polymorphism (PCR-SSCP) analysis was used to scan for genomic alterations of ERG16 in the isolates that would cause this change in the enzyme in isolate 13. Alterations near the 3' end of the gene that were identified by PCR-SSCP were confirmed by DNA sequencing. A single amino acid substitution (R467K) that occurred in isolate 13 was identified in both alleles of ERG16. Allelic differences within the ERG16 gene, in the ERG16 promoter, and in the downstream THR1 gene were eliminated in isolate 13. The loss of allelic variation in this region of the genome is most likely the result of mitotic recombination or gene conversion. The R467K mutation and loss of allelic variation that occur in isolate 13 are likely responsible for the azole-resistant enzyme activity seen in this and subsequent isolates. The description of R467K represents the first point mutation to be identified within ERG16 of a clinical isolate of C. albicans that alters the fluconazole sensitivity of the enzyme.

Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro.

Abstract: Candida dubliniensis is a recently described species of Candida associated with oral candidiasis in human immunodeficiency virus (HIV)-infected individuals. Nineteen oral isolates of C. dubliniensis recovered from 10 HIV-positive and 4 HIV-negative individuals and one vaginal isolate from an additional HIV-negative subject were assessed for fluconazole susceptibility by broth microdilution (BMD), hyphal elongation assessment, and Etest. The susceptibilities of these 20 isolates to itraconazole and amphotericin B and of 10 isolates to ketoconazole were also determined by BMD only. Sixteen of the C. dubliniensis isolates were susceptible to fluconazole (MIC range, 0.125 to 1.0 microgram ml-1), and four (recovered from two AIDS patients) were fluconazole resistant (MIC range, 8 to 32 micrograms ml-1). Fluconazole susceptibility data obtained by hyphal elongation assessment correlated well with results obtained by BMD, but the corresponding Etest MIC results were one to four times higher. All of the isolates tested were found to be sensitive to itraconazole, ketoconazole, and amphotericin B. Sequential exposure of two fluconazole-sensitive (MIC, 0.5 microgram ml-1) C. dubliniensis isolates to increasing concentrations of fluconazole in agar medium resulted in the recovery of derivatives which expressed a stable fluconazole-resistant phenotype (BMD-determined MIC range, 16 to 64 micrograms ml-1), even after a minimum of 10 consecutive subcultures on drug-free medium and following prolonged storage at -70 degrees C. The clonal relationship between the parental isolates and their respective fluconazole-resistant derivatives was confirmed by genomic DNA fingerprinting and karyotype analysis. The results of this study demonstrate that C. dubliniensis is inherently susceptible to commonly used antifungal drugs, that fluconazole resistance does occur in clinical isolates, and that stable fluconazole resistance can be readily induced in vitro following exposure to the drug.

Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia.

Abstract: Nine isolates of Escherichia coli were recovered from seven blood cultures over a period of 3 months from a 19-month-old female with aplastic anemia. Initial isolates were susceptible to extended-spectrum cephalosporins, including ceftazidime (MIC, or = 128 micrograms/ml) and other cephalosporins and the monobactam aztreonam. Molecular typing methods, including plasmid profile analysis, pulsed-field gel electrophoresis, and arbitrarily primed PCR, indicated that the nine isolates were derived from a common ancestor. Dot blot hybridization and PCR analysis of total bacterial DNA using blaSHV- and blaTEM-specific DNA probes and primers identified the presence of a blaTEM beta-lactamase gene in all of the isolates and a blaSHV gene in the isolates with elevated ceftazidime MICs. Isoelectric focusing analysis of crude lysates showed that all nine isolates contained an enzyme with a pI of 5.4 corresponding to the TEM-1 beta-lactamase, and those isolates containing an SHV-type beta-lactamase demonstrated an additional band with a pI of 7.6. The first of the ceftazidime-resistant isolates appeared to hyperproduce the SHV enzyme compared to the other resistant isolates. DNA sequencing revealed a blaSHV-1 gene in the first ceftazidime-resistant isolate and a novel blaSHV gene, blaSHV-8, with an Asp-to-Asn substitution at amino acid position 179 in the remaining four isolates. Three of the ceftazidime-resistant isolates also showed a change in porin profile. The patient had received multiple courses of antimicrobial agents during her illness, including multiple courses of ceftazidime. This collection of blood isolates from the same patient appears to represent the in vivo evolution of resistance under selective pressure of treatment with various cephalosporins.

In vitro activity of BAY 12-8039, a new fluoroquinolone.

Abstract: The in vitro activity of BAY 12-8039, a new fluoroquinolone, was studied in comparison with those of ciprofloxacin, trovafloxacin (CP 99,219), cefpodoxime, and amoxicillin-clavulanate against gram-negative, gram-positive, and anaerobic bacteria. Its activity against mycobacteria and chlamydia was also investigated. BAY 12-8039 was active against members of the family Enterobacteriaceae (MIC at which 90% of strains tested were inhibited [MIC90S] < or = 1 microgram/ml, except for Serratia spp. MIC90 2 microgram/ml), Neisseria spp. (MIC90S, 0.015 microgram/ml), Haemophilus influenzae (MIC90, 0.03 microgram/ml), and Moraxella catarrhalis (MIC90, 0.12 micrgram/ml), and these results were comparable to those obtained for ciprofloxacin and trovafloxacin. Against Pseudomonas aeruginosa, the quinolones were more active than the beta-lactam agents but BAY 12-8039 was less active than ciprofloxacin. Strains of Stenotrophomonas maltophilia were fourfold more susceptible to BAY 12-8039 and trovafloxacin (MIC90S, 2 micrograms/ml) than to ciprofloxacin. BAY 12-8039 was as active as trovafloxacin but more active than ciprofloxacin against Streptococcus pneumoniae (MIC90, 0.25 microgram/ml) and methicillin-susceptible Staphylococcus auerus (MIC90S, 0.12 micrograms/ml). The activity of BAY 12-8039 against methicillin-resistant S. aureus (MIC90, 2 micrograms/ml) was lower than that against methicillin-susceptible strains. BAY 12-8039 was active against anaerobes (MIC90S < or = 2 micrograms/ml), being three- to fourfold more active against Bacteroides fragilis, Prevotella spp., and Clostridium difficile than was ciprofloxacin. Against Mycobacterium tuberculosis, BAY 12-8039 exhibited activity comparable to that of rifampin (MICs < or = 0.5 micrograms/ml). Against Chlamydia trachomatis and Chlamydia pneumoniae BAY 12-8039 was more active (MICs < or = 0.12 microgram/ml) than either ciprofloxacin or erythromycin and exhibited a greater lethal effect than either to these two agents. The protein binding of BAY 12-8039 was determined at 1 and 5 micrograms/ml as 30 and 26.4%, respectively. The presence of human serum (at 20 or 70%) had no marked effect on the in vitro activity of BAY 12-8039.

Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

Abstract: BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.

Monotherapy versus beta-lactam-aminoglycoside combination treatment for gram-negative bacteremia: a prospective, observational study.

Abstract: The aim of the present study was to test whether the combination of a beta-lactam drug plus an aminoglycoside has advantage over monotherapy for severe gram-negative infections. Of 2,124 patients with gram-negative bacteremia surveyed prospectively, 670 were given inappropriate empirical antibiotic treatment and the mortality rate in this group was 34%, whereas the mortality rate was 18% for 1,454 patients given appropriate empirical antibiotic treatment (P = 0.0001). The mortality rates for patients given appropriate empirical antibiotic treatment were 17% for 789 patients given a single beta-lactam drug, 19% for 327 patients given combination treatment, 24% for 249 patients given a single aminoglycoside, and 29% for 89 patients given other antibiotics (P = 0.0001). When patients were stratified according to risk factors for mortality other than antibiotic treatment, combination therapy showed no advantage over treatment with a single beta-lactam drug except for neutropenic patients (odds ratio [OR] for mortality, 0.5; 95% confidence interval [95% CI], 0.2 to 1.3) and patients with Pseudomonas aeruginosa bacteremia (OR, 0.7; 95% CI, 0.3 to 1.8). On multivariable logistic regression analysis including all risk factors for mortality, combination therapy had no advantage over therapy with a single beta-lactam drug. The mortality rate for patients treated with a single appropriate aminoglycoside was higher than that for patients given a beta-lactam drug in all strata except for patients with urinary tract infections. When the results of blood cultures were known, 1,878 patients were available for follow-up. Of these, 816 patients were given a single beta-lactam drug, 442 were given combination treatment, and 193 were given a single aminoglycoside. The mortality rates were 13, 15, and 23%, respectively (P = 0.0001). Both on stratified and on multivariable logistic regression analyses, combination treatment showed a benefit over treatment with a single beta-lactam drug only for neutropenic patients (OR, 0.2; 95% CI, 0.05 to 0.7). In summary, combination treatment showed no advantage over treatment with an appropriate beta-lactam drug in nonneutropenic patients with gram-negative bacteremia.

Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene.

Abstract: Upon prolonged treatment with various antiretroviral nucleoside analogs such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, (-)- beta-L-2', 3'dideoxy-3'thiacytidine and 2',3'-didehydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) gene has been reported. We designed a reverse hybridization line probe assay (LiPA) for the rapid and simultaneous characterization of the following variations in the RT gene: M41 or L41; T69, N69, A69, or D69; K70 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), and Y215 (TAC or TAT) could be detected. In addition to the codons mentioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213, F214, and L214) were found, and specific probes were selected. In total, 48 probes were designed and applied to the LiPA test strips and optimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridization in an average of 92.7% of the samples for each individual codon. When combined with changes in viral load and CD4+ T-cell count, this LiPA approach proved to be useful in studying genetic resistance in follow-up samples from antiretroviral agent-treated HIV-1-infected individuals.

Widespread detection of PER-1-type extended-spectrum beta-lactamases among nosocomial Acinetobacter and Pseudomonas aeruginosa isolates in Turkey: a nationwide multicenter study.

Abstract: We studied the prevalence and molecular epidemiology of PER-1-type beta-lactamases among Acinetobacter, Klebsiella, and Pseudomonas aeruginosa strains isolated over a 3-month period in eight university hospitals from distinct regions of Turkey. A total of 72, 92, and 367 Acinetobacter, Klebsiella, and P. aeruginosa isolates were studied, respectively. The presence of blaPER was determined by the colony hybridization method and later confirmed by isoelectric focusing. We detected PER-1-type beta-lactamases in 46% (33/72) of Acinetobacter strains and in 11% (40/367) of P. aeruginosa strains but not in Klebsiella strains. PER-1-type enzyme producers were highly resistant to ceftazidime and gentamicin, intermediately resistant to amikacin, and susceptible or moderately susceptible to imipenem and meropenem. Among PER-1-type-beta-lactamase-positive isolates, five Acinetobacter isolates and six P. aeruginosa isolates from different hospitals were selected for ribosomal DNA fingerprinting with EcoRI and SalI. The EcoRI-digested DNAs were later hybridized with a digoxigenin-labelled PER-1 probe. The ribotypes and the lengths of blaPER-carrying fragments were identical in four Acinetobacter strains. A single isolate (Ac3) harbored a PER gene on a different fragment (approximately 4.2 kbp) than the others (approximately 3.4 kbp) and showed a clearly distinguishable ribotype. Ribotypes of P. aeruginosa strains obtained with EcoRI showed three patterns. Similarly, in Pseudomonas strains two different EcoRI fragments harbored blaPER (approximately 4.2 kbp in five isolates and 3.4 kbp in one isolate). PER-1-type beta-lactamases appear to be restricted to Turkey. However, their clonal diversity and high prevalence indicate a high spreading potential.