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26.  Macrolide Resistance Gene mreA of Streptococcus agalactiae Encodes a Flavokinase 
The mreA gene from Streptococcus agalactiae COH31 γ/δ, resistant to macrolides and clindamycin by active efflux, has recently been cloned in Escherichia coli, where it was reported to confer macrolide resistance (J. Clancy, F. Dib-Hajj, J. W. Petitpas, and W. Yuan, Antimicrob. Agents Chemother. 41:2719–2723, 1997). Cumulative data suggested that the mreA gene was located on the chromosome of S. agalactiae COH31 γ/δ. Analysis of the deduced amino acid sequence of mreA revealed significant homology with several bifunctional flavokinases/(flavin adenine dinucleotide (FAD) synthetases, which convert riboflavin to flavin mononucleotide (FMN) and FMN to FAD, respectively. High-performance liquid chromatography experiments showed that the mreA gene product had a monofunctional flavokinase activity, similar to that of RibR from Bacillus subtilis. Sequences identical to those of the mreA gene and of a 121-bp upstream region containing a putative promoter were detected in strains of S. agalactiae UCN4, UCN5, and UCN6 susceptible to macrolides. mreA and its allele from S. agalactiae UCN4 were cloned on the shuttle vector pAT28. Both constructs were introduced into E. coli, where they conferred a similar two- to fourfold increase in the MICs of erythromycin, spiramycin, and clindamycin. The MICs of a variety of other molecules, including crystal violet, acriflavin, sodium dodecyl sulfate, and antibiotics, such as certain cephalosporins, chloramphenicol, doxycycline, nalidixic acid, novobiocin, and rifampin, were also increased. In contrast, resistance to these compounds was not detected when the constructs were introduced into E. faecalis JH2–2. In conclusion, the mreA gene was probably resident in S. agalactiae and may encode a metabolic function. We could not provide any evidence that it was responsible for macrolide resistance in S. agalactiae COH31 γ/δ; broad-spectrum resistance conferred by the gene in E. coli could involve multidrug efflux pumps by a mechanism that remains to be elucidated.
PMCID: PMC90643  PMID: 11451686
27.  Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata 
Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.
PMCID: PMC90441  PMID: 11257032
28.  Antiviral Activity of β-l-2′,3′-Dideoxy-2′,3′-Didehydro-5-Fluorocytidine in Woodchucks Chronically Infected with Woodchuck Hepatitis Virus 
The l-nucleoside analog β-l-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (β-l-Fd4C) was first shown to exhibit potent activity against hepatitis B virus (HBV) in tissue culture and then to significantly inhibit viral spread during acute infection in the duck HBV model (F. Le Guerhier et al., Antimicrob. Agents Chemother. 44:111–122, 2000). We have therefore examined its antiviral activity in a mammalian model of chronic HBV infection, the woodchuck chronically infected with woodchuck hepatitis virus (WHV). Side-by-side comparison of β-l-Fd4C and lamivudine administered intraperitoneally during short-term and long-term protocols demonstrated a more profound inhibition of viremia in β-l-Fd4C-treated groups. Moreover, β-l-Fd4C induced a marked inhibition of intrahepatic viral DNA synthesis compared with that induced by lamivudine. Nevertheless, covalently closed circular (CCC) DNA persistence explained the lack of clearance of infected hepatocytes expressing viral antigens and the relapse of WHV replication after drug withdrawal. Liver histology showed a decrease in the inflammatory activity of chronic hepatitis in woodchucks receiving β-l-Fd4C. An electron microscopy study showed the absence of ultrastructural changes of hepatic mitochondria, biliary canaliculi, and bile ducts. However, a loss of weight was observed in all animals, whatever the treatment, as was a transient skin pigmentation in all woodchucks during β-l-Fd4C treatment. There was no evidence that lamivudine or β-l-Fd4C could prevent the development of hepatocellular carcinoma with the protocols used. These results indicate that β-l-Fd4C exhibits a more potent antiviral effect than lamivudine in the WHV model but was not able to eradicate CCC DNA and infected cells from the liver at the dosage and with the protocol used.
PMCID: PMC90426  PMID: 11257017
29.  Efficacy of 2-Amino-7-(1,3-Dihydroxy-2-Propoxymethyl)Purine for Treatment of Vaccinia Virus (Orthopoxvirus) Infections in Mice 
We have previously shown that the N-7 substituted acyclic nucleoside analog 2-amino-7-[1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) is, both in vitro and in animal models, a potent inhibitor of the replication of several herpesviruses (Neyts et al., Antimicrob. Agents Chemother. 39:56–60, 1995). Here we report on the potent and selective antiviral activity of S2242 against vaccinia virus (VV), an orthopoxvirus. The 50% effective concentrations for inhibition of VV-induced cytopathic effect and viral DNA synthesis in cell culture were 2.4 and 0.2 μg/ml, respectively. We next studied the efficacy of S2242 in VV-infected mice. Immunocompetent NMRI mice that had been inoculated intravenously with VV developed tail lesions. Mice that had been treated for 5 consecutive days via the subcutaneous (s.c.) route with 100 mg of the diacetate ester prodrug of S2242 (compound H961) per kg of body weight did not develop any lesions and demonstrated no adverse effects. Severe combined immunodeficient (SCID) mice that had been inoculated intraperitoneally with VV became sick and died within 1 month after infection. Following treatment with H961 at 100 mg/kg for 10 consecutive days (either via oral gavage or s.c. injection) VV-inoculated SCID mice were completely protected, for at least 3 months, against virus-induced morbidity and mortality. At that time, no virus could be recovered from the organs of these mice (as assessed by titration for infectious virus, a DNA hybridization assay, and a PCR for VV-specific sequences). Compound S2242 and its oral prodrug H961 could be useful in treatment of orthopoxvirus infections.
PMCID: PMC90244  PMID: 11120949
30.  Inducible Azole Resistance Associated with a Heterogeneous Phenotype in Candida albicans 
The development of azole resistance in Candida albicans is most problematic in patients with AIDS who receive long courses of drug for therapy or prevention of oral candidiasis. Recently, the rapid development of resistance was noted in other immunosuppressed patients who developed disseminated candidiasis despite fluconazole prophylaxis. One of these series of C. albicans isolates became resistant, with an associated increase in mRNA specific for a CDR ATP-binding cassette transporter efflux pump (K. A. Marr, C. N. Lyons, T. R. Rustad, R. A. Bowden, and T. C. White, Antimicrob. Agents Chemother. 42:2584–2589, 1998). Here we study this series of C. albicans isolates further and examine the mechanism of azole resistance in a second series of C. albicans isolates that caused disseminated infection in a recipient of bone marrow transplantation. The susceptible isolates in both series become resistant to fluconazole after serial growth in the presence of drug, while the resistant isolates in both series become susceptible after serial transfer in the absence of drug. Population analysis of the inducible, transiently resistant isolates reveals a heterogeneous population of fluconazole-susceptible and -resistant cells. We conclude that the rapid development of azole resistance occurs by a mechanism that involves selection of a resistant clone from a heterogeneous population of cells.
PMCID: PMC90239  PMID: 11120944
31.  Cloning and Characterization of SmeDEF, a Novel Multidrug Efflux Pump from Stenotrophomonas maltophilia 
Antimicrobial Agents and Chemotherapy  2000;44(11):3079-3086.
Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous multidrug-resistant S. maltophilia D457R mutant (A. Alonso and J. L. Martinez, Antimicrob. Agents Chemother. 41:1140–1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli ΔacrAB strain. Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E. coli ΔacrAB strain. We identified three open reading frames, smeD, smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively. Drug susceptibility assays indicated that the SmeDEF system cloned in E. coli mediates resistance to a wide range of antibiotics. Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force. The presence of high levels of smeDEF mRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain. In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain. Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.
PMCID: PMC101606  PMID: 11036026
32.  In Vitro and In Vivo Potentiation of Artemisinin and Synthetic Endoperoxide Antimalarial Drugs by Metalloporphyrins 
Antimicrobial Agents and Chemotherapy  2000;44(10):2836-2841.
The in vitro potentiation of artemisinin by synthetic manganese porphyrin complexes has been recently reported (F. Benoit-Vical, A. Robert, and B. Meunier, Antimicrob. Agents Chemother. 43:2555–2558, 1999). Since the activity of artemisinin and synthetic antimalarial endoperoxides is related to their interaction with heme (S. R. Meshnick, A. Thomas, A. Ranz, C. M. Xu, and H. Z. Pan, Mol. Biochem. Parasitol. 49:181–190, 1991), an improvement of their efficiency may be expected in the presence of a synthetic metalloporphyrin having the same activating role as endogenous heme. With the aim to boost the activity of antimalarial endoperoxide drugs, we were thus led to evaluate the in vitro and in vivo potentiation of natural and synthetic drugs of this family by a nontoxic and cheap metalloporphyrin. The potentiation of artemisinin, β-artemether, and arteflene (Ro 42-1611) by synthetic heme models is reported. In vitro studies on the chloroquine-resistant Plasmodium falciparum FcB1-Columbia strain indicate a synergistic effect of the manganese complex of meso-tetrakis(4-sulfonatophenylporphyrin) (Mn-TPPS) on the activity of artemisinin or β-artemether, whereas this heme model has no influence on the activity of arteflene. A significant synergistic effect on rodent malaria was also observed in vivo between artemisinin and Mn-TPPS using Plasmodium vinckei petteri strain.
PMCID: PMC90158  PMID: 10991867
33.  Functional Analysis of the Active Site of a Metallo-β-Lactamase Proliferating in Japan 
An R-plasmid-mediated metallo-β-lactamase was found in Klebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the β-lactamase termed DK4 was identical to the IMP-1 metallo-β-lactamase which was mediated by a chromosomal gene of Serratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71–78, 1994). The dose effect of DK4 β-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the β-lactams except monobactams. The enzymatic characteristics of the DK4 β-lactamase and its kinetic parameters for nine β-lactams were examined. The DK4 β-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.
PMCID: PMC90062  PMID: 10952572
34.  Antibiotic Resistance in the ECOR Collection: Integrons and Identification of a Novel aad Gene 
The 72 Escherichia coli strains of the ECOR collection were examined for resistance to 10 different antimicrobial agents including ampicillin, tetracycline, mercury, trimethoprim, and sulfonamides. Eighteen strains were resistant to at least one of the antibiotics tested, and nearly 20% (14 of 72) were resistant to two or more. Several of the resistance determinants were shown to be carried on conjugative elements. The collection was screened for the presence of the three classes of integrons and for the sul1 gene, which is generally associated with class 1 integrons. The four strains found to carry a class 1 integron also had Tn21-encoded mercury resistance. One of the integrons encoded a novel streptomycin resistance gene, aadA7, with an attC site (or 59-base element) nearly identical to the attC site associated with the qacF gene cassette found in In40 (M.-C. Ploy, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 42:2557–2563, 1998). The conservation of associated attC sites among unrelated resistance cassettes is similar to arrangements found in the Vibrio cholerae superintegrons (D. Mazel, B. Dychinco, V. A. Webb, and J. Davies, Science 280:605–608, 1998) and supports the hypothesis that resistance cassettes are picked up from superintegron pools and independently assembled from unrelated genes and related attC sites.
PMCID: PMC89914  PMID: 10817710
35.  The Legionella (Fluoribacter) gormanii Metallo-β-Lactamase: a New Member of the Highly Divergent Lineage of Molecular-Subclass B3 β-Lactamases 
A metallo-β-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297T constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5α, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B β-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a β-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925–926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the blaFEZ-1 gene in E. coli, based on the T7 phage promoter, was also developed.
PMCID: PMC89909  PMID: 10817705
36.  Accumulation of 3-Ketosteroids Induced by Itraconazole in Azole-Resistant Clinical Candida albicans Isolates 
Antimicrobial Agents and Chemotherapy  1999;43(11):2663-2670.
The effects of itraconazole on ergosterol biosynthesis were investigated in a series of 16 matched clinical Candida albicans isolates which had been previously analyzed for mechanisms of resistance to azoles (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother., 39:2378–2386, 1995). Under control conditions, all isolates contained ergosterol as the predominant sterol, except two strains (C48 and C56). In isolates C48 and C56, both less susceptible to azoles than their parent, C43, substantial concentrations (20 to 30%) of 14α-methyl-ergosta-8,24(28)-diene-3β,6α-diol (3,6-diol) were found. Itraconazole treatment of C43 resulted in a dose-dependent inhibition of ergosterol biosynthesis (50% inhibitory concentration, 2 nM) and accumulation of 3,6-diol (up to 60% of the total sterols) together with eburicol, lanosterol, obtusifoliol, 14α-methyl-ergosta-5,7,22,24(28)-tetraene-3βol, and 14α-methyl-fecosterol. In strains C48 and C56, no further increase of 3,6-diol was observed after exposure to itraconazole. Ergosterol synthesis was less sensitive to itraconazole inhibition, as was expected for these azole-resistant isolates which overexpress ATP-binding cassette transporter genes CDR1 and CDR2. In addition to 3,6-diol, substantial amounts of obtusifolione were found after exposure to itraconazole. This toxic 3-ketosteroid was demonstrated previously to accumulate after itraconazole treatment in Cryptococcus neoformans and Histoplasma capsulatum but has not been reported in Candida isolates. Accumulation of obtusifolione correlated with nearly complete growth inhibition in these azole-resistant strains compared to that found in the susceptible parent strain, although the onset of growth inhibition only occurred at higher concentrations of itraconazole. ERG25 and ERG26 are the only genes assigned to the 4-demethylation process, of which the 3-ketoreductase is part. To verify whether mutations in these ERG25 genes contributed to obtusifolione accumulation, their nucleotide sequences were determined in all three related isolates. No mutations in ERG25 alleles of isolates C48 and C56 were found, suggesting that this gene is not involved in obtusifolione accumulation. The molecular basis for the accumulation of this sterol in these two strains remains to be established.
PMCID: PMC89540  PMID: 10543744
37.  Involvement of an Active Efflux System in the Natural Resistance of Pseudomonas aeruginosa to Aminoglycosides 
Antimicrobial Agents and Chemotherapy  1999;43(11):2624-2628.
A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415–417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.
PMCID: PMC89534  PMID: 10543738
38.  Comparative Efficacies of Liposomal Amikacin (MiKasome) plus Oxacillin versus Conventional Amikacin plus Oxacillin in Experimental Endocarditis Induced by Staphylococcus aureus: Microbiological and Echocardiographic Analyses 
Optimal treatment strategies for serious infections caused by Staphylococcus aureus have not been fully characterized. The combination of a β-lactam plus an aminoglycoside can act synergistically against S. aureus in vitro and in vivo. MiKasome, a new liposome-encapsulated formulation of conventional amikacin, significantly prolongs serum half-life (t1/2) and increases the area under the concentration-time curve (AUC) compared to free amikacin. Microbiologic efficacy and left ventricular function, as assessed by echocardiography, were compared in animals administered either oxacillin alone or oxacillin in combination with conventional amikacin or MiKasome in a rabbit model of experimental endocarditis due to S. aureus. In vitro, oxacillin, combined with either free amikacin or MiKasome, prevented the bacterial regrowth observed with aminoglycosides alone at 24 h of incubation. Rabbits with S. aureus endocarditis were treated with either oxacillin alone (50 mg/kg, given intramuscularly three times daily), oxacillin plus daily amikacin (27 mg/kg, given intravenously twice daily), or oxacillin plus intermittent MiKasome (160 mg/kg, given intravenously, a single dose on days 1 and 4). The oxacillin-alone dosage represents a subtherapeutic regimen against the infecting strain in the endocarditis model (L. Hirano and A. S. Bayer, Antimicrob. Agents Chemother. 35:685–690, 1991), thus allowing recognition of any enhanced bactericidal effects between oxacillin and either aminoglycoside formulation. Treatment was administered for either 3 or 6 days, and animals were sacrificed after each of these time points or at 5 days after a 6-day treatment course (to evaluate for posttherapy relapse). Left ventricular function was analyzed by utilizing serial transthoracic echocardiography during treatment and posttherapy by measurement of left ventricular fractional shortening. At all sacrifice times, both combination regimens significantly reduced S. aureus vegetation counts versus control counts (P < 0.05). In contrast, oxacillin alone did not significantly reduce S. aureus vegetation counts after 3 days of therapy. Furthermore, at this time point, the two combinations were significantly more effective than oxacillin alone (P < 0.05). All three regimens were effective in significantly decreasing bacterial counts in the myocardium during and after therapy compared to controls (P < 0.05). In kidney and spleen abscesses, all regimens significantly reduced bacterial counts during therapy (P < 0.0001); however, only the combination regimens prevented bacteriologic relapse in these organs posttherapy. By echocardiographic analysis, both combination regimens yielded a significant physiological benefit by maintaining normal left ventricular function during treatment and posttherapy compared with oxacillin alone (P < 0.001). These results suggest that the use of intermittent MiKasome (similar to daily conventional amikacin) enhances the in vivo bactericidal effects of oxacillin in a severe S. aureus infection model and preserves selected physiological functions in target end organs.
PMCID: PMC89353  PMID: 10390232
39.  In Vivo Efficacies of Combinations of β-Lactams, β-Lactamase Inhibitors, and Rifampin against Acinetobacter baumannii in a Mouse Pneumonia Model 
The effects of various regimens containing combinations of β-lactams, β-lactam inhibitor(s), and rifampin were assessed in a recently described mouse model of Acinetobacter baumannii pneumonia (M. L. Joly-Guillou, M. Wolff, J. J. Pocidalo, F. Walker, and C. Carbon, Antimicrob. Agents Chemother. 41:345–351, 1997). Two aspects of the therapeutic response were studied: the kinetics of the bactericidal effect (treatment was initiated 3 h after intratracheal inoculation, and bacterial counts were determined over a 24-h period) and survival (treatment was initiated 8 h after inoculation, and the cumulative mortality rate was assessed on day 5). Two clinical strains were used: a cephalosporinase-producing strain (SAN-94040) and a multiresistant strain (RCH-69). For SAN-94040 and RCH-69, MICs and MBCs (milligrams per liter) were as follows: ticarcillin, 32, 64, 256, and >256, respectively; ticarcillin-clavulanate, 32, 64, and 512, and >512, respectively; imipenem, 0.5, 0.5, 8, and 32, respectively; sulbactam, 0.5, 0.5, 8, and 8, respectively; and rifampin, 8, 8, 4, and 4, respectively. Against SAN-94040, four regimens, i.e., imipenem, sulbactam, imipenem-rifampin, and ticarcillin-clavulanate (at a 25/1 ratio)-sulbactam produced a true bactericidal effect (≥3-log10 reduction of CFU/g of lung). The best survival rate (i.e., 93%) was obtained with the combination of ticarcillin-clavulanate-sulbactam, and regimens containing rifampin provided a survival rate of ≥65%. Against RCH-69, only regimens containing rifampin and the combination of imipenem-sulbactam had a true bactericidal effect. The best survival rates (≥80%) were obtained with regimens containing rifampin and sulbactam. These results suggest that nonclassical combinations of β-lactams, β-lactamase inhibitors, and rifampin should be considered for the treatment of nosocomial pneumonia due to A. baumannii.
PMCID: PMC89287  PMID: 10348761
40.  Mechanism of the Intracellular Killing and Modulation of Antibiotic Susceptibility of Listeria monocytogenes in THP-1 Macrophages Activated by Gamma Interferon 
Listeria monocytogenes, a facultative intracellular pathogen, readily enters cells and multiplies in the cytosol after escaping from phagosomal vacuoles. Macrophages exposed to gamma interferon, one of the main cellular host defenses against Listeria, become nonpermissive for bacterial growth while containing Listeria in the phagosomes. Using the human myelomonocytic cell line THP-1, we show that the combination of l-monomethyl arginine and catalase restores bacterial growth without affecting the phagosomal containment of Listeria. A previous report (B. Scorneaux, Y. Ouadrhiri, G. Anzalone, and P. M. Tulkens, Antimicrob. Agents Chemother. 40:1225–1230, 1996) showed that intracellular Listeria was almost equally sensitive to ampicillin, azithromycin, and sparfloxacin in control cells but became insensitive to ampicillin and more sensitive to azithromycin and sparfloxacin in gamma interferon-treated cells. We show here that these modulations of antibiotic activity are largely counteracted by l-monomethyl arginine and catalase. In parallel, we show that gamma interferon enhances the cellular accumulation of azithromycin and sparfloxacin, an effect which is not reversed by addition of l-monomethyl arginine and catalase and which therefore cannot account for the increased activity of these antibiotics in gamma interferon-treated cells. We conclude that (i) the control exerted by gamma interferon on intracellular multiplication of Listeria in THP-1 macrophages is dependent on the production of nitric oxide and hydrogen peroxide; (ii) intracellular Listeria may become insensitive to ampicillin in macrophages exposed to gamma interferon because the increase in reactive oxygen and nitrogen intermediates already controls bacterial growth; and (iii) azithromycin and still more sparfloxacin cooperate efficiently with gamma interferon, one of the main cellular host defenses in Listeria infection.
PMCID: PMC89140  PMID: 10223943
41.  Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of Pseudomonas aeruginosa 
We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parC genes for 30 clinical strains of Pseudomonas aeruginosa resistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248–2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC (codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrA allele. The remaining two strains harbored the first gyrB mutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e., gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation in gyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 μg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6, P < 0.01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrA mutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.
PMCID: PMC89021  PMID: 9869566
42.  Close Association between Clearance of Recombinant Human Granulocyte Colony-Stimulating Factor (G-CSF) and G-CSF Receptor on Neutrophils in Cancer Patients 
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is used to counter chemotherapy-induced neutropenia. Our previous study showed an inverse correlation between serum rhG-CSF levels and the number of circulating neutrophils in cancer patients (H. Takatani, H. Soda, M. Fukuda, M. Watanabe, A. Kinoshita, T. Nakamura, and M. Oka, Antimicrob. Agents Chemother. 40:988–991, 1996). The aim of this study was to clarify the relationship between rhG-CSF clearance and G-CSF receptors on circulating neutrophils. In five cancer patients receiving chemotherapy, a bolus dose of rhG-CSF (5 μg/kg) was injected intravenously during defined phases of posttreatment neutropenia and neutrophilia. Serum rhG-CSF levels were measured by a chemiluminescence enzyme immunoassay and analyzed by moment analysis. G-CSF receptors on neutrophils were detected by flow cytometry with biotinylated rhG-CSF. rhG-CSF clearance was significantly higher at neutrophilia than at neutropenia (1,497 ± 132 versus 995 ± 266 ml/h; P < 0.01). The percentage of G-CSF receptor-positive neutrophils, reflecting the number of G-CSF receptors per cell, was low at neutropenia without rhG-CSF therapy (44.5% ± 22.1%) and high at neutrophilia with rhG-CSF therapy (73.0% ± 11.4%; P < 0.01). rhG-CSF clearance closely correlated with the percentage of G-CSF receptor-positive neutrophils (r2 = 0.91; P < 0.0001) and neutrophil count (r2 = 0.72; P < 0.005). Our results indicate that, in cancer patients receiving chemotherapy, rhG-CSF increases the number of G-CSF receptors per cell as well as circulating neutrophil counts, resulting in modulation of its own clearance.
PMCID: PMC89014  PMID: 9869559
43.  In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein among Clinical Bacteremic Isolates of Staphylococcus aureus Correlates with an Endovascular Infectious Source 
Antimicrobial Agents and Chemotherapy  1998;42(12):3169-3172.
Platelet microbicidal proteins (PMPs), small cationic peptides released at sites of endovascular damage, kill common bloodstream pathogens in vitro. Our group previously showed that in vitro resistance of clinical staphylococcal and viridans group streptococcal bacteremic strains to PMPs correlated with the diagnosis of infective endocarditis (IE) (Wu et al., Antimicrob. Agents Chemother. 38:729–732, 1994). However, that study was limited by (i) the small number of Staphylococcus aureus isolates from IE patients, (ii) the retrospective nature of the case definitions, and (iii) the diverse geographic sources of strains. The present study evaluated the in vitro PMP susceptibility phenotype of a large number of staphylococcemic isolates (n = 60), collected at a single medical center and categorized by defined and validated clinical criteria. A significantly higher proportion of staphylococcemic strains from patients with IE was PMP resistant in vitro than the proportion of strains from patients with soft tissue sepsis (83% and 33%, respectively; P < 0.01). Moreover, the levels of PMP resistance (mean percent survival of strains after 2-h exposure to PMP in vitro) were significantly higher for isolates from patients with IE and with vascular catheter sepsis than for strains from patients with abscess sepsis (P < 0.005 and P < 0.01, respectively). These data further support the concept that bloodstream pathogens that exhibit innate or acquired PMP resistance have a survival advantage with respect to either the induction or progression of endovascular infections.
PMCID: PMC106018  PMID: 9835510
44.  Gonococcal Resistance to β-Lactams and Tetracycline Involves Mutation in Loop 3 of the Porin Encoded at the penB Locus 
Antimicrobial Agents and Chemotherapy  1998;42(11):2799-2803.
penB is a chromosomal mutation that confers resistance to β-lactams and tetracyclines and reduced susceptibility to quinolones in Neisseria gonorrhoeae. It is linked to the porin gene (por) and requires the increased expression of an efflux pump due to mtr. Transformation of a susceptible gonococcus (strain H1) with chromosomal DNA from strain FA140 (penA mtr penB; porin serovar IB1) and conjugal transfer of a β-lactamase-expressing plasmid was used to produce isogenic strains for determination of equilibrium periplasmic penicillin concentrations by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368–372, 1977). In transformants with the Mtr and PenB phenotypes, equilibrium concentrations of penicillin were reduced. DNA sequence analysis of por from isogenic penB and penB+ transformants revealed 14 sequence differences; nine of these differences resulted in amino acid changes. Three amino acid changes were found in the putative gonococcal equivalent of the pore-constricting loop 3 of Escherichia coli OmpF. Two of these changes (Gly-101–Ala-102→Asp-Asp) result in an increased negative charge at this position in por loop 3. PCR products comprising the complete por gene from strain FA140 were transformed into strain H1-2 (penA mtr; porin serovar IB-3), with the resulting transformants having the antibiotic susceptibility phenotype associated with penB. penB-like mutations were found in loop 3 of clinical isolates of gonococci with chromosomally mediated resistance to penicillin. We conclude that penB is a mutation in loop 3 of por that reduces porin permeability to hydrophilic antibiotics and plays an important role in the development of chromosomally mediated resistance to penicillin and tetracycline in gonococci.
PMCID: PMC105946  PMID: 9797206
45.  Molecular Characterization of OXA-20, a Novel Class D β-Lactamase, and Its Integron from Pseudomonas aeruginosa 
The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum β-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188–2195, 1997). A second β-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 β-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D β-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar β-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of oxa20 revealed the presence of the following integron features: (i) a second antibiotic resistance gene, aacA4; (ii) an intI1 gene; and (iii) two 59-base elements, each associated with either oxa20 or aacA4. This integron is peculiar because it lacks the 3′ conserved region, and therefore is not a sul1-associated integron like most of them, and because its 3′ end is located within tnpR, a gene involved in the transposition of Tn5393, a gram-negative transposon. P. aeruginosa Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.
PMCID: PMC105865  PMID: 9687410
46.  Amino Acid Substitutions in the Cytochrome P-450 Lanosterol 14α-Demethylase (CYP51A1) from Azole-Resistant Candida albicans Clinical Isolates Contribute to Resistance to Azole Antifungal Agents 
The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.
PMCID: PMC105395  PMID: 9527767
47.  Mechanisms of quinolone resistance in Escherichia coli: characterization of nfxB and cfxB, two mutant resistance loci decreasing norfloxacin accumulation. 
Two genetic loci selected for norfloxacin (nfxB) and ciprofloxacin (cfxB) resistance were characterized. Both mutations have previously been shown to confer pleiotropic resistance to quinolones, chloramphenicol, and tetracycline and to decrease expression of porin outer-membrane protein OmpF. nfxB was shown to map at about 19 min and thus to be genetically distinct from ompF (21 min), and cfxB was shown to be very closely linked to marA (34 min). cfxB was dominant over cfxB+ in merodiploids, in contrast to other quinolone resistance mutations. The two loci appear to interact functionally, because nfxB was not expressed in the presence of marA::Tn5. Both nfxB and cfxB decreased the expression of ompF up to 50-fold at the posttranscriptional level as determined in strains containing ompF-lacZ operon and protein fusions. Both mutations also decreased norfloxacin accumulation in intact cells. This decrease in accumulation was abolished by energy inhibitors and by removal of the outer membrane. These findings, in conjunction with those of Cohen et al. (S. P. Cohen, D. C. Hooper, J. S. Wolfson, K. S. Souza, L. M. McMurry, and S. B. Levy, Antimicrob. Agents Chemother. 32:1187-1191, 1988), suggest a model for quinolone resistance by decreased permeation in which decreased diffusion through porin channels in the outer membrane interacts with a saturable drug efflux system at the inner membrane.
PMCID: PMC171480  PMID: 2658782
48.  Gentamicin uptake in Staphylococcus aureus possessing plasmid-encoded, aminoglycoside-modifying enzymes. 
[3H]gentamicin uptake and killing were studied in three strains of gentamicin-resistant Staphylococcus aureus possessing plasmid-encoded, gentamicin-modifying enzymes and in three isogenic, enzyme-free, gentamicin-susceptible derivatives. At low (less than or equal to 2.0 micrograms/ml) concentrations of gentamicin, uptake by resistant organisms was impaired compared with that of susceptible strains, and no killing was noted. In contrast, at higher (2.5 to 10.0 micrograms/ml) concentrations (which were below the MIC for the resistant strains), rapid gentamicin uptake similar to that seen in susceptible isolates was observed. Although growth inhibition at these concentrations was apparent, there was no loss of viability in resistant strains. Consistently, the membrane H+-ATPase inhibitor N,N'-dicyclohexyl carbodiimide caused resistant strains to take up low concentrations (1.0 microgram/ml) of gentamicin at rates comparable to those seen in susceptible organisms without causing an associated loss of viability. These studies show differences between gentamicin uptake in S. aureus and streptomycin uptake in Escherichia coli (Dickie et al., Antimicrob. Agents Chemother. 14:569-580, 1978) regarding the kinetics of uptake in resistant strains with plasmid-encoded aminoglycoside-modifying enzymes. Specifically, they suggest that for 2-deoxystreptamine compounds such as gentamicin, ribosomal binding followed by accelerated uptake and subsequent interference with cell growth may occur without invariably being associated with lethal effect.
PMCID: PMC179964  PMID: 6517546
49.  Conjugational transfer of gentamicin resistance plasmids intra- and interspecifically in Staphylococcus aureus and Staphylococcus epidermidis. 
We have previously reported the transfer of gentamicin resistance (Gmr) plasmids in a mixed culture inter- and intraspecifically between strains of Staphylococcus aureus and Staphylococcus epidermidis isolated at Michael Reese Hospital (Jaffe et al., Antimicrob. Agents Chemother. 21:773-779, 1982). We have now shown that representatives of these plasmids were transferred between apparently nonlysogenic strains of S. aureus either in mixed culture in broth or by filter-mating on agar medium. The mechanism of transfer appeared to be conjugation. A transferable Gmr plasmid (pSH8) mobilized or cotransferred a tetracycline R-plasmid and a chloramphenicol R-plasmid that were not independently transferable. The transfer of Gmr plasmids was accompanied by a high incidence of deletion mutations with varied loss of plasmid resistance determinants and, with some mutants, loss of the ability to effect self-transfer. Restriction endonuclease digestion of pSH8 and its deletion mutants made it possible to assign the property of self-transfer to a specific segment of the pSH8 genome and provided the basis for a physical and genetic map of that plasmid. Similar Gmr plasmids from S. aureus strains isolated in locations remote from Michael Reese Hospital had resistance determinants and transfer properties comparable to those of pSH8. Our observations provide evidence for the conjugal transfer of some staphylococcal plasmids, apparently independent of the presence of phage. This mechanism may be of significance in the intra- and interspecific dissemination of resistance to aminoglycosides and other antibiotics in Staphylococcus spp.
PMCID: PMC184633  PMID: 6219618
50.  Prevalence of Antimicrobial Resistance among Clinical Isolates of Bacteroides fragilis Group in Canada in 2010-2011: CANWARD Surveillance Study 
Clinical isolates of the Bacteroides fragilis group (n = 387) were collected from patients attending nine Canadian hospitals in 2010-2011 and tested for susceptibility to 10 antimicrobial agents using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. B. fragilis (59.9%), Bacteroides ovatus (16.3%), and Bacteroides thetaiotaomicron (12.7%) accounted for ∼90% of isolates collected. Overall rates of percent susceptibility were as follows: 99.7%, metronidazole; 99.5%, piperacillin-tazobactam; 99.2%, imipenem; 97.7%, ertapenem; 92.0%, doripenem; 87.3%, amoxicillin-clavulanate; 80.9%, tigecycline; 65.9%, cefoxitin; 55.6%, moxifloxacin; and 52.2%, clindamycin. Percent susceptibility to cefoxitin, clindamycin, and moxifloxacin was lowest for B. thetaiotaomicron (n = 49, 24.5%), Parabacteroides distasonis/P. merdae (n = 11, 9.1%), and B. ovatus (n = 63, 31.8%), respectively. One isolate (B. thetaiotaomicron) was resistant to metronidazole, and two isolates (both B. fragilis) were resistant to both piperacillin-tazobactam and imipenem. Since the last published surveillance study describing Canadian isolates of B. fragilis group almost 20 years ago (A.-M. Bourgault et al., Antimicrob. Agents Chemother. 36:343–347, 1992), rates of resistance have increased for amoxicillin-clavulanate, from 0.8% (1992) to 6.2% (2010-2011), and for clindamycin, from 9% (1992) to 34.1% (2010-2011).
PMCID: PMC3294939  PMID: 22203594

Results 26-50 (25696)