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26.  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
27.  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
28.  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
29.  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
30.  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
31.  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
32.  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
33.  A novel, double mutation in DNA gyrase A of Escherichia coli conferring resistance to quinolone antibiotics. 
A spontaneous Escherichia coli mutant, named Q3, resistant to nalidixic acid was obtained from a previously described clinical isolate of E. coli, Q2, resistant to fluoroquinolones but susceptible to nalidixic acid (E. Cambau, F. Bordon, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 37:1247-1252, 1993). Q3 harbored the mutation Asp82Gly in addition to the Gly81Asp mutation of Q2. The different mutations leading to Gly81Asp, Asp82Gly, and Gly81AspAsp82Gly were introduced into the gyrA gene harbored on plasmid pJSW102, and the resulting plasmids were introduced into E. coli KNK453 (gyrAts) by transformation. The presence of Asp82Gly or Gly81Asp alone led to a low-level resistance to fluoroquinolones but not to nalidixic acid resistance. When both mutations were present, resistance to both nalidixic acid and fluoroquinolones was expressed. Purified gyrases of the different mutants showed similar rates of supercoiling. Dominance of the various gyrA mutant alleles harbored on plasmids was examined. The susceptibility to quinolones associated with wild-type gyrA was always dominant. The susceptibility to nalidixic acid expressed by the Gly81Asp mutant was dominant, while that expressed by the Asp82Gly mutant was recessive. From these results, we hypothesize that some amino acids within the quinolone resistance-determining region of gyrase A are more important for the association of subunits rather than for the activity of the holoenzyme.
PMCID: PMC163665  PMID: 8980760
34.  In vitro modulation of hippocampal pyramidal cell response by quinolones: effects of HA 966 and gamma-hydroxybutyric acid. 
Antimicrobial Agents and Chemotherapy  1996;40(11):2573-2576.
The influence of quinolones on electrically evoked pyramidal cell activity in the rat hippocampus in vitro was studied by using the slice technique. We hoped to learn more about the possible mechanisms for the development of side effects of different quinolones and to find a possible treatment. As reported earlier (W. Dimpfel, M. Spüler, A. Dalhoff, W. Hofmann, and G. Schlüter, Antimicrob. Agents Chemother. 35:1142-1146, 1991), the amplitude of the population spike increased in the presence of ciprofloxacin, lomefloxacin, or ofloxacin about twofold in comparison with reference values. This increase could be prevented in a concentration-dependent manner by the concomitant presence of 3-amino-1-hydroxy-2-pyrrolidone (HA 966), a compound acting at the so-called glycine site of the N-methyl-D-aspartate (NMDA) receptor, but not in the presence of aminophosphonovaleric acid (APV), which acts at a different recognition site of the NMDA receptor. Another tool, 6,7-dinitroquinoxaline-2,3-dione, an antagonist of the so-called AMPA receptor (named after the binding of L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] to this site), could not antagonize the effect induced by the quinolones. Activation of the glycine site of the NMDA receptor induced by the presence of D-serine in the superfusion medium also resulted in a concentration-dependent increase in the population spike amplitude. This response remained unchanged in the presence of ciprofloxacin, whereas lomefloxacin and ofloxacin led to further increases in the amplitude, especially in the presence of higher concentrations of D-serine. These results also point to an involvement of the glycine site of the central NMDA receptor in the development of side effects by different quinolones. A complete attenuation of the quinolone-induced effects was obtained in the presence of 2.5 microM gamma-hydroxybutyric acid (GHB), a physiological neuromodulator which is marketed in some countries of Europe as a sedative. It is therefore concluded that the excitatory adverse effects of quinolones might be treated by the administration of GHB.
PMCID: PMC163578  PMID: 8913467
35.  Hydrophilicity of quinolones is not an exclusive factor for decreased activity in efflux-mediated resistant mutants of Staphylococcus aureus. 
The elevated expression of the norA gene is responsible for efflux-mediated resistance to quinolones in Staphylococcus aureus (E.Y.W. Ng, M. Trucksis, and D.C. Hooper, Antimicrob. Agents Chemother. 38:1345-1355, 1994). For S. aureus transformed with a plasmid containing the cloned norA gene, SA113(pTUS20) (H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, and M. Konno, J. Bacteriol. 172:6942-6949, 1990), and an overexpressed mutant, SA-1199B (G.W. Kaatz, S.M. Seo, and C.A. Ruble, J. Infect. Dis. 163:1080-1086, 1991), the MICs of norfloxacin increased 16 and 64 times compared with its MICs for the recipient and wild-type strains, SA113 and SA-1199, respectively. MICs of CS-940, however, increased only two and eight times, even though these two fluoroquinolones are similarly hydrophilic (apparent logPs of approximately -1). No good correlation was found, among 15 developed and developing quinolones, between the increment ratio in MICs and hydrophobicity (r = 0.61). Analysis of the quantitative structure-activity relationship among 40 fluoroquinolones revealed that the MIC increment ratio was significantly correlated with the bulkiness of the C-7 substituent and bulkiness and hydrophobicity of the C-8 substituent of fluoroquinolones (r = 0.87) and not with its molecular hydrophobicity (r = 0.47). Cellular accumulation of norfloxacin in SA-1199B was significantly lower than that in SA-1199, and it was increased by addition of carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, accumulations of CS-940 in these strains were nearly identical, and they were not affected by addition of the protonophore.
PMCID: PMC163426  PMID: 8843290
36.  Accumulation of trimethoprim, sulfamethoxazole, and N-acetylsulfamethoxazole in fish and shrimp fed medicated Artemia franciscana. 
In a previous paper (H.J. Nelis, P. Léger, P. Sorgeloos, and A. P. De Leenheer, Antimicrob. Agents Chemother. 35:2486-2489, 1991) it was reported that two selected antibacterial agents, i.e., trimethoprim and sulfamethoxazole, can be efficiently bioencapsulated in nauplii of the brine shrimp Artemia franciscana for administration to fish. This follow-up study showed that larvae of the sea bass and the turbot as well as postlarvae of the white shrimp accumulate the therapeutic agents in high quantities when fed medicated A. franciscana. To monitor their levels as a function of time, the liquid chromatographic method originally developed for the analysis of A. franciscana was modified with respect to chromatography, internal standardization, and sample pretreatment. The levels of trimethoprim ranged from 1 to 7 micrograms/g (sea bass), 1 to 13 micrograms/g (turbot), and 4 to 38 micrograms/g (white shrimp). The corresponding values for sulfamethoxazole were 0.3 to 4 micrograms/g (sea bass), 1 to 42 micrograms/g (turbot), and 4 to 35 micrograms/g (white shrimp). Only the two fish species, unlike the shrimp, metabolized the latter to N-acetylsulfamethoxazole (concentration range, 1 to 10 micrograms/g). These data suggest the potential of the bioencapsulation of therapeutic agents in live food as a tool to control infectious diseases in aquaculture. A preliminary challenge test also confirmed the in vivo efficacy of this approach.
PMCID: PMC163389  PMID: 8807056
37.  Characterization of IS1272, an insertion sequence-like element from Staphylococcus haemolyticus. 
We have previously shown (G. L. Archer, D. M. Niemeyer, J. A. Thanassi, and M. J. Pucci, Antimicrob. Agents Chemother. 38:447-454, 1994) that some methicillin-resistant staphylococcal isolates contain a partial deletion of the genes (mecR1 and mecI) that regulate the transcription of the methicillin resistance structural gene (mecA). When a fragment of DNA inserted at the point of the mecR1 deletion was used as a probe, hybridization with multiple bands was detected for Staphylococcus haemolyticus genomic DNA. In the present study, DNA sequencing of four unique clones recovered from a lambda library of S. haemolyticus revealed identical 1,934-bp elements. Each element, designated IS1272, contained 16-bp terminal inverted repeats (sequence identity, 15 of 16 bp) and two open reading frames of 819 and 687 bp; there were no flanking target site duplications. Database searches yielded amino acid homology with proteins predicted to be encoded by open reading frames from a putative insertion sequence element from Enterococcus hirae. DNA probes from each end and the middle of IS1272 were hybridized with restriction endonuclease-digested genomic DNA from clinical S. haemolyticus, Staphylococcus epidermidis, and Staphylococcus aureus isolates. Each of the 20 or more copies of the element found in S. haemolyticus isolates was intact, and copies were found in most chromosomal SmaI fragments. S. aureus and S. epidermidis isolates contained mostly incomplete fragments of the element, and there were many more hybridizing fragments in methicillin-resistant than in methicillin-susceptible isolates. IS1272, which appears to be primarily resident in S. haemolyticus, has disseminated to multiple staphylococcal species and is prevalent in multiresistant isolates.
PMCID: PMC163232  PMID: 8849253
38.  Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium. 
High-level ampicillin resistance in Enterococcus faecium has been shown to be associated with the synthesis of a modified penicillin-binding protein 5 (PBP 5) which had apparently lost its penicillin-binding capability (R. Fontana, M. Aldegheri, M. Ligozzi, H. Lopez, A. Sucari, and G. Satta. Antimicrob. Agents Chemother. 38:1980-1983, 1994). The pbp5 gene of the highly resistant strain E. faecium 9439 was cloned and sequenced. The deduced amino acid sequence showed 77 and 54% homologies with the PBPs 5 of Enterococcus hirae and Enterococcus faecalis, respectively. A gene fragment coding for the C-terminal part of PBP 5 containing the penicillin-binding domain was also cloned from several E. faecium strains with different levels of ampicillin resistance. Sequence comparison revealed a few point mutations, some of which resulted in amino acid substitutions between SDN and KTG motifs in PBPs 5 of highly resistant strains. One of these converted a polar residue (the T residue at position 562 or 574) of PBP 5 produced by susceptible and moderately resistant strains into a nonpolar one (A or I). This alteration could be responsible for the altered phenotype of PBP 5 in highly resistant strains.
PMCID: PMC163115  PMID: 8834879
39.  Growth inhibition of Ureaplasma urealyticum by the proton pump inhibitor lansoprazole: direct attribution to inhibition by lansoprazole of urease activity and urea-induced ATP synthesis in U. urealyticum. 
Antimicrobial Agents and Chemotherapy  1995;39(10):2187-2192.
The proton pump inhibitors (PPIs) omeprazole and lansoprazole and the acid-activated analog of lansoprazole AG-2000, which potently inhibit the urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), also inhibited the urease activities of cell-free extracts as well as intact cells of Ureaplasma urealyticum. The 50% inhibitory concentrations were between 1 and 25 microM. These compounds also inhibited the ATP synthesis induced by urea in ureaplasma cells. The 50% inhibitory concentrations for ATP synthesis were close to those for urease activity, but they were lower than those of urease inhibitors, such as acetohydroxamic acid, hydroxyurea, and thiourea. In addition, one of the metabolites of lansoprazole found in human urine, M-VI, also inhibited ureaplasmal urease activity and the ATP synthesis induced by urea at almost the same concentrations as those of lansoprazole. The inhibition of PPIs against ureaplasma urease was very similar to those against H. pylori urease, suggesting that the inhibitory mechanism against these ureases was due to the blockage of the SH residues on the cysteine of the enzyme. Omeprazole, lansoprazole, AG-2000, and M-VI inhibited the growth of U. urealyticum. Since ureaplasma urease is thought to be involved in the pathogenicity of this organism in the urogenital tract, PPIs and their analogs may be useful as chemotherapeutic agents against diseases caused by U. urealyticum.
PMCID: PMC162911  PMID: 8619564
40.  Identification of daptomycin-binding proteins in the membrane of Enterococcus hirae. 
Daptomycin, a lipopeptide antibiotic active against gram-positive bacteria, was preliminarily shown to inhibit lipoteichoic acid (LTA) synthesis as a consequence of membrane binding in the presence of Ca2+ (P. Canepari, M. Boaretti, M. M. Lleó, and G. Satta, Antimicrob. Agents Chemother. 34:1220-1226, 1990). In the present study, it is shown that, along with binding bacterial-membrane components, daptomycin binds the protein fraction with a noncovalent bond, as suggested by the instability of the bond in the presence of ionic detergents such as sodium dodecyl sulfate. Analysis of membrane proteins by isoelectric focusing electrophoresis reveals that five bands with isoelectric points ranging from 5.9 to 6.2 bind radioactive daptomycin. These proteins are therefore called daptomycin-binding proteins. In an attempt to correlate these proteins to the main inhibition observed during LTA synthesis, two-dimensional thin-layer chromatography of lipids synthesized during daptomycin treatment was performed. A threefold increase in diglucosyl diacylglycerol is demonstrated, while the compounds phosphatidyl-alpha-kojibiosyldiacylglycerol, glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol, and glycerophospho-kojibiosyldiacylglycerol, which follow diglucosyl diacylglycerol in LTA synthesis, decrease progressively with time during the course of daptomycin treatment.
PMCID: PMC162882  PMID: 8540717
41.  In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii. 
In order to characterize the delayed effect of clindamycin and macrolide antibiotics against Toxoplasma gondii tachyzoites (E. R. Pfefferkorn and S. E. Borotz, Antimicrob. Agents Chemother. 38:31-37, 1994), we have carefully examined the replication of parasites as a function of time after drug addition. Intracellular tachyzoites treated with up to 20 microM clindamycin (> 1,000 times the 50% inhibitory concentration) exhibit doubling times indistinguishable from those of controls (approximately 7 h). Drug-treated parasites emerge from infected cells and establish parasitophorous vacuoles inside new host cells as efficiently as untreated controls, but replication within the second vacuole is dramatically slowed. Growth inhibition in the second vacuole does not require continued presence of drug, but it is dependent solely on the concentration and duration of drug treatment in the first (previous) vacuole. The susceptibility of intracellular parasites to nanomolar concentrations of clindamycin contrasts with that of extracellular tachyzoites, which are completely resistant to treatment, even through several cycles of subsequent intracellular replication. This peculiar phenotype, in which drug effects are observed only in the second infectious cycle, also characterizes azithromycin and chloramphenicol treatment, but not treatment with cycloheximide, tetracycline, or anisomycin. These findings provide new insights into the mode of clindamycin and macrolide action against T. gondii, although the relevant target for their action remains unknown.
PMCID: PMC162776  PMID: 7492099
42.  Molecular characterization of the OXA-7 beta-lactamase gene. 
The OXA-7 gene, which encodes an oxacillinase, was cloned from plasmid pMG202 of Escherichia coli isolate 7181 (A. A. Medeiros, M. Cohenford, and G. A. Jacoby, Antimicrob. Agents Chemother. 27:715-719, 1985) and sequenced. The nucleotide sequence of the OXA-7 gene was closely related to that of the OXA-10 (PSE-2) gene, with a derived amino acid sequence of the OXA-7 enzyme showing greater than 95% homology with those of OXA-10 and OXA-11.
PMCID: PMC162747  PMID: 7574536
43.  Characterization of an LysR family protein, SmeR from Serratia marcescens S6, its effect on expression of the carbapenem-hydrolyzing beta-lactamase Sme-1, and comparison of this regulator with other beta-lactamase regulators. 
Serratia marcescens S6 produces a chromosomally encoded carbapenem-hydrolyzing class A beta-lactamase, Sme-1 (T. Naas, L. Vandel, W. Sougakoff, D. M. Livermore, and P. Nordmann, Antimicrob. Agents Chemother. 38:1262-1270, 1994). Upstream from smeA we identified a second open reading frame (EMBL accession number Z30237). This encodes a 33.1-kDa protein, SmeR, which has a high degree of homology with NmcR, the LysR regulatory protein of the only other sequenced carbapenem-hydrolyzing class A beta-lactamase, NmcA from Enterobacter cloacae NOR-1. It is weakly related to AmpR of the chromosomal cephalosporinase regulatory systems described in E. cloacae, Yersinia enterocolitica, Citrobacter freundii, and Pseudomonas aeruginosa and is very weakly related to other LysR-type regulators of class A beta-lactamases. SmeR is a weakly positive regulator for Sme-1 expression in the absence of or in the presence of beta-lactam inducers. The -35 and -10 regions of smeR are in the opposite orientations and are face-to-face relative to the smeA promoter. SmeR acts similarly to NmcR and not as the AmpR regulators described for class C beta-lactamase systems. SmeR is a weak inducer in the absence or presence of beta-lactams. As was found for the AmpC-AmpR and NmcA-NmcR systems, a putative SmeR-binding site was present upstream from the beta-lactamase gene promoter regions. beta-Galactosidase activity from a smeR-lacZ translational fusion was expressed constitutively and decreased in the presence of SmeR from a coresident plasmid, suggesting that SmeR is autogeneously controlled. Finally, beta-lactams did not affect the expression of SmeR, which is the second regulator of a class A carbapenem-hydrolyzing beta-lactamase to be identified.
PMCID: PMC162596  PMID: 7793864
44.  Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope. 
Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.
PMCID: PMC162607  PMID: 7793875
45.  Inhibitory action of lansoprazole and its analogs against Helicobacter pylori: inhibition of growth is not related to inhibition of urease. 
The proton pump inhibitors omeprazole and lansoprazole and its acid-activated derivative AG-2000, which are potent and specific inhibitors of urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), inhibited the growth of H. pylori. The growth was inhibited not only in urease-positive clinical isolates but also in their urease-negative derivatives which had no urease polypeptides. AG-1789, a derivative of lansoprazole with no inhibitory activity against H. pylori urease, also inhibited the growth of both strains even more strongly than the urease inhibitors lansoprazole and AG-2000. Furthermore, the antibacterial activity of omeprazole and lansoprazole was not affected by glutathione or dithiothreitol, which completely abolished the inhibitory activity of lansoprazole against H. pylori urease. These results indicated that the inhibitory action of these compounds against the growth of H. pylori was independent from the inhibitory action against urease.
PMCID: PMC162584  PMID: 7726537
46.  Structure-activity relationships of carbapenems that determine their dependence on porin protein D2 for activity against Pseudomonas aeruginosa. 
A number of carbapenem derivatives were examined to determine the structure-activity relationships required for dependence on porin protein D2 for activity against Pseudomonas aeruginosa. As suggested by J. Trias and H. Nikaido (Antimicrob. Agents Chemother. 34:52-57, 1990), carbapenem derivatives, such as imipenem and meropenem, containing a sole basic group at position 2 of the molecule utilize the D2 channel for permeation through the outer membrane of pseudomonads; they are more active against D2-sufficient strains of P. aeruginosa. Our results indicated that carbapenems with a basic group at position 1 or 6 of the molecule did not depend on the D2 channel for activity; i.e. they were equally active against D2-sufficient and D2-deficient pseudomonal strains. However, addition of a basic group at position 1 or 6 of a carbapenem derivative already containing a basic group at position 2 resulted in its lack of dependency on the D2 pathway. Comparison between meropenem and its 1-guanidinoethyl derivative, BMY 45047, indicated that they differed in their dependence on D2; while meropenem required the D2 channel for uptake, BMY 45047 activity was independent of D2. Meropenem and BMY 45047 had similar affinities for the penicillin-binding proteins of P. aeruginosa. However, BMY 45047 and meropenem differed in the morphological changes that they induced in pseudomonal cells. While meropenem induced filamentation, BMY 45047 induced filaments only in BMS-181139-resistant mutants and not in imipenem-resistant mutants or in carbapenem-susceptible P. aeruginosa strains. These results suggested that in Mueller-Hinton medium the uptake of BMY 45047 through the non-D2 pathway is more rapid than that of meropenem through the D2 porin. In summary, the presence of a basic group at position 2 of a carbapenem is important for its preferential uptake by the D2 channel. However the addition of a basic group at position 1 or 6 of a carbapenem already containing a basic group at position 2 dissociates its necessity for porin protein D2 for activity.
PMCID: PMC162549  PMID: 7726504
47.  Multidrug resistance in Candida albicans: disruption of the BENr gene. 
The BENr gene of Candida albicans, which confers resistance on susceptible strains of Saccharomyces cerevisiae to six structurally and functionally unrelated drugs, was described recently (R. Ben-Yaacov, S. Knoller, G. Caldwell, J. M. Becker, and Y. Koltin, Antimicrob. Agents Chemother. 38:648-652, 1994). This gene bears similarity to membrane proteins encoding antibiotic resistance in prokaryotes and eukaryotes. The effect of disruption of this gene on viability and drug susceptibility was determined. The results indicate that the gene is not essential but its inactivation leads to susceptibility to three of the four drugs tested. Inactivation of this gene did not increase the susceptibility of the mutant to benomyl, suggesting that C. albicans has other mechanisms of resistance, some of which may be additional efflux pumps that confer resistance to this tubulin-destabilizing agent.
PMCID: PMC162553  PMID: 7726508
48.  Colistin-Resistant, Lipopolysaccharide-Deficient Acinetobacter baumannii Responds to Lipopolysaccharide Loss through Increased Expression of Genes Involved in the Synthesis and Transport of Lipoproteins, Phospholipids, and Poly-β-1,6-N-Acetylglucosamine 
We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971–4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-β-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.
doi:10.1128/AAC.05191-11
PMCID: PMC3256090  PMID: 22024825
49.  Impact of Granulocytes on the Antimicrobial Effect of Tedizolid in a Mouse Thigh Infection Model▿ 
Antimicrobial Agents and Chemotherapy  2011;55(11):5300-5305.
Tedizolid (TR-700, formerly torezolid) is the active component of the new oxazolidinone prodrug tedizolid phosphate (TR-701). We had previously demonstrated that tedizolid possessed potent antistaphylococcal activity superior to that of linezolid in a neutropenic mouse thigh infection model (A. Louie, W. Liu, R. Kulawy, and G. L. Drusano, Antimicrob. Agents Chemother. 55:3453-3460, 2011). In the current investigation, we used a mouse thigh infection model to delineate the effect of an interaction of TR-700 and granulocytes on staphylococcal cell killing. We compared the antistaphylococcal killing effect of doses of TR-701 equivalent to human exposures ranging from 200 to 3,200 mg/day in both granulocytopenic and normal mice. The mice were evaluated at 24, 48, and 72 h after therapy initiation. In granulocytopenic mice, a clear exposure response in which, depending on the time point of evaluation, stasis was achieved at “human-equivalent” doses of slightly below 2,300 mg/day (at 24 h) to slightly below 2,000 mg/day (at 72 h) was observed. In immune-normal animals, stasis was achieved at human-equivalent doses of slightly greater than 100 mg/day or less. The variance in bacterial cell killing results was attributable to the presence of granulocytes (without drug), the direct effect of TR-700 on Staphylococcus aureus, and the effect of the drug on Staphylococcus aureus mediated through granulocytes. The majority of the bacterial cell killing in normal animals was attributable to the effect of TR-700 mediated through granulocytes. Additional studies need to be undertaken to elucidate the mechanism underlying this observation.
doi:10.1128/AAC.00502-11
PMCID: PMC3195040  PMID: 21911576
50.  Mutation in the sdeS Gene Promotes Expression of the sdeAB Efflux Pump Genes and Multidrug Resistance in Serratia marcescens▿ 
Serratia marcescens gained resistance to both biocides and antibiotics on expressing the SdeAB efflux pump, following exposure to increasingly higher concentrations of a biocide (H. Maseda et al., Antimicrob. Agents Chemother. 53:5230–5235, 2009). To reveal the regulatory mechanism of sdeAB expression, wild-type cells were subjected to transposon-mediated random mutagenesis, and a mutant with antibiotic resistance, which mimicked the phenotype of the previous biocide-resistant cells, was obtained. The transposon element was found in the chromosomal DNA downstream of the sdeAB operon. Sequencing revealed the presence of an open reading frame (ORF) encoding a protein with 159 amino acid residues that is highly similar to the BadM-type transcriptional repressor, designated sdeS. The level of sdeB::xylE reporter gene expression, undetectable in the wild-type cells, appeared to be fully comparable to that in the biocide-resistant cells. Nucleotide sequencing of the mutant revealed sdeS to have a single G-to-A base substitution at position 269 that converted Trp90 to a stop codon. Introduction of a plasmid-borne intact sdeS into the mutant cells and the biocide-resistant cells resulted in a reduction in sdeB::xylE reporter activity to an undetectable level. These results suggested that SdeS functions as a repressor of the sdeAB operon. It was concluded that the original biocide-resistant cells had an impaired sdeS and, therefore, a derepressed level of the SdeAB efflux pump.
doi:10.1128/AAC.01755-10
PMCID: PMC3101466  PMID: 21422216

Results 26-50 (24769)