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1.  In Vitro Resistance of Staphylococcus aureus to Thrombin-Induced Platelet Microbicidal Protein Is Associated with Alterations in Cytoplasmic Membrane Fluidity 
Infection and Immunity  2000;68(6):3548-3553.
Platelet microbicidal proteins (PMPs) are small, cationic peptides which possess potent microbicidal activities against common bloodstream pathogens, such as Staphylococcus aureus. We previously showed that S. aureus strains exhibiting resistance to thrombin-induced PMP (tPMP-1) in vitro have an enhanced capacity to cause human and experimental endocarditis (T. Wu, M. R. Yeaman, and A. S. Bayer, Antimicrob. Agents Chemother. 38:729–732, 1994; A. S. Bayer et al., Antimicrob. Agents Chemother. 42:3169–3172, 1998; V. K. Dhawan et al., Infect. Immun. 65:3293–3299, 1997). However, the mechanisms mediating tPMP-1 resistance in S. aureus are not fully delineated. The S. aureus cell membrane appears to be a principal target for the action of tPMP-1. To gain insight into the basis of tPMP-1 resistance, we compared several parameters of membrane structure and function in three tPMP-1-resistant (tPMP-1r) strains and their genetically related, tPMP-1-susceptible (tPMP-1s) counterpart strains. The tPMP-1r strains were derived by three distinct methods: transposon mutagenesis, serial passage in the presence of tPMP-1 in vitro, or carriage of a naturally occurring multiresistance plasmid (pSK1). All tPMP-1r strains were found to possess elevated levels of longer-chain, unsaturated membrane lipids, in comparison to their tPMP-1s counterparts. This was reflected in corresponding differences in cell membrane fluidity in the strain pairs, with tPMP-1r strains exhibiting significantly higher degrees of fluidity as assessed by fluorescence polarization. These data provide further support for the concept that specific alterations in the cytoplasmic membrane of S. aureus strains are associated with tPMP-1 resistance in vitro.
PMCID: PMC97641  PMID: 10816510
2.  Mutations in the tetA(B) gene that cause a change in substrate specificity of the tetracycline efflux pump. 
The tetA(B) gene from transposon Tn10 fails to mediate resistance to the novel tetracycline analog 9-(dimethylglycylamido)minocycline (DMG-Mino) (P. E. Sum, V. J. Lee, R. T. Testa, J. J. Hlavka, G. A. Ellestad, J. D. Bloom, Y. Gluzman, and F. P. Tally, J. Med. Chem. 37:184-188, 1994; R. T. Testa, P. Petersen, N. V. Jacobus, P. E. Sum, V. J. Lee, and F. P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). Mutations in either of two codons of tetA(B) that resulted in increased resistance to DMG-Mino also caused diminished resistance to tetracycline, identifying amino acid residues critical for the recognition of tetracycline.
PMCID: PMC284555  PMID: 8031059
3.  Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. 
The distribution of strains producing metallo-beta-lactamase among 105 strains of Serratia marcescens was investigated. All of these strains were isolated in seven general hospitals located in Aichi Prefecture, Japan, from April to May 1993. Southern hybridization analysis suggested that four S. marcescens strains, AK9373, AK9374, AK9385, and AK9391, had a metallo-beta-lactamase genes similar to the blaIMP gene found by our laboratory (E. Osano, Y. Arakawa, R. Wacharotayankun, M. Ohta, T. Horii, H. Ito, F. Yoshimura, and N. Kato, Antimicrob. Agents Chemother. 38:71-78, 1994), and these four strains showed resistance to carbapenems as well as to the other broad-spectrum beta-lactams. In particular, strains AK9373, AK9374, and AK9391 showed an extraordinarily high-level resistance to imipenem (MICs, > or = 64 micrograms/ml), whereas strain AK9385 demonstrated moderate imipenem resistance (MIC, 8 micrograms/ml). The imipenem resistance of AK9373 was transferred to Escherichia coli CSH2 by conjugation with a frequency of 10(-5). The DNA probe of the blaIMP gene hybridized to a large plasmid (approximately 120 kb) transferred into the E. coli transconjugant as well as to the large plasmids harbored by AK9373. On the other hand, although we failed in the conjugational transfer of imipenem resistance from strains AK9374, AK9385, and AK9391 to E. coli CSH2, imipenem resistance was transferred from these strains to E. coli HB101 by transformation. A plasmid (approximately 25 kb) was observed in each transformant which acquired imipenem resistance. The amino acid sequence at the N terminus of the enzyme purified from strain AK9373 was identical to that of the metallo-beta-lactamase IMP-1. In contrast, strains ES9348, AK9386, and AK93101, which were moderately resistant to imipenem (MICs, > or = 4 to < or = 8 micrograms/ml), had no detectable blaIMP gene. As a conclusion, 19% of clinically isolated S. marcescens strains in Aichi Prefecture, Japan, in 1993 were resistant to imipenem (MICs, > or = 2 micrograms/ml), and strains which showed high-level imipenem resistance because of acquisition of a plasmid-mediated blaIMP-like metallo-beta-lactamase gene had already proliferated as nosocomial infections, at least in a general hospital.
PMCID: PMC162636  PMID: 7785978
4.  Effects of Net Charge and the Number of Positively Charged Residues on the Biological Activity of Amphipathic α-Helical Cationic Antimicrobial Peptides 
Biopolymers  2008;90(3):369-383.
In our previous study, we utilized a 26-residue amphipathic α-helical antimicrobial peptide L-V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398−1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L-V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self-association). The net charge of V13K analogs at pH 7 varied between −5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram-negative and gram-positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32-fold increase in hemolytic activity (250 μg/ml to <7.8 μg/ml, respectively).
PMCID: PMC2761230  PMID: 18098173
antimicrobial peptide; antimicrobial activity; hemolytic activity; hydrophobicity; amphipathicity; helicity; peptide self-association
5.  Genetic Identification of the Bacteriocins Produced by Enterococcus faecium IT62 and Evidence that Bacteriocin 32 Is Identical to Enterocin IT▿  
Enterococcus faecium IT62, a strain isolated from ryegrass in Japan, produces three bacteriocins (enterocins L50A, L50B, and IT) that have been previously purified and the primary structures of which have been determined by amino acid sequencing (E. Izquierdo, A. Bednarczyk, C. Schaeffer, Y. Cai, E. Marchioni, A. Van Dorsselaer, and S. Ennahar, Antimicrob. Agents Chemother., 52:1917-1923, 2008). Genetic analysis showed that the bacteriocins of E. faecium IT62 are plasmid encoded, but with the structural genes specifying enterocin L50A and enterocin L50B being carried by a plasmid (pTAB1) that is separate from the one (pTIT1) carrying the structural gene of enterocin IT. Sequencing analysis of a 1,475-bp region from pTAB1 identified two consecutive open reading frames corresponding, with the exception of 2 bp, to the genes entL50A and entL50B, encoding EntL50A and EntL50B, respectively. Both bacteriocins are synthesized without N-terminal leader sequences. Genetic analysis of a sequenced 1,380-bp pTIT1 fragment showed that the genes entIT and entIM, encoding enterocin IT and its immunity protein, respectively, were both found in E. faecium VRE200 for bacteriocin 32. Enterocin IT, a 6,390-Da peptide made up of 54 amino acids, has been previously shown to be identical to the C-terminal part of bacteriocin 32, a 7,998-Da bacteriocin produced by E. faecium VRE200 whose structure was deduced from its structural gene (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). By combining the biochemical and genetic data on enterocin IT, it was concluded that bacteriocin 32 is in fact identical to enterocin IT, both being encoded by the same plasmid-borne gene, and that the N-terminal leader peptide for this bacteriocin is 35 amino acids long and not 19 amino acids long as previously reported.
PMCID: PMC2681497  PMID: 19273675
6.  Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to beta-lactam resistance. 
Wild-type strains of Pseudomonas aeruginosa are more resistant to various beta-lactam antibiotics as well as other agents than most enteric bacteria. Although resistance to compounds of earlier generations is explained by the synergism between the outer membrane barrier and the inducible beta-lactamase, it was puzzling to see significant levels of resistance to compounds that do not act as inducers or are not hydrolyzed rapidly by the chromosomally encoded enzyme. This intrinsic-resistance phenotype becomes enhanced in those strains with the so-called intrinsic carbenicillin resistance. In the accompanying paper (X.-Z. Li, D. M. Livermore, and H. Nikaido, Antimicrob. Agents Chemother. 38:1732-1741, 1994), we showed that active efflux played a role in the resistance, to various non-beta-lactam agents, of P. aeruginosa strains in general and that the efflux was enhanced in intrinsically carbenicillin-resistant strains. We show in this paper that, in comparison with the drug-hypersusceptible mutant K799/61, less benzylpenicillin was accumulated in wild-type strains of P. aeruginosa and that the accumulation levels were even lower in intrinsically carbenicillin-resistant strains. Deenergization by the addition of a proton conductor increased the accumulation level to that expected for equilibration across the cytoplasmic membrane. In intrinsically carbenicillin-resistant isolates, there was no evidence that either nonspecific or specific permeation rates of beta-lactams across the outer membrane were lowered in comparison with those of the more susceptible isolates. Furthermore, these carbenicillin-resistant isolates were previously shown to have no alteration in the level or the inducibility of beta-lactamase and in the affinity of penicillin-binding proteins. These data together suggest the involvement of an active efflux mechanism also in the resistance to beta-lactams. Hydrophilic beta-lactams with more than one charged group did not cross the cytoplasmic membrane readily. Yet one such compound, ceftriaxone, appeared to be extruded from the cells of more-resistant strains, although with this compound effects of proton conductors could not be shown. We postulate that wild-type strains of P. aeruginosa pump out such hydrophilic beta-lactams either from the periplasm or from the outer leaflet of the lipid bilayer of the cytoplasmic membrane, in a manner analogous to that hypothesized for multidrug resistance protein of human cancer cells (M.M. Gottesman and I. Pastan, Annu. Rev. Biochem. 62:385-427, 1993).
PMCID: PMC284631  PMID: 7986004
7.  Bacterial biofilms and the bioelectric effect. 
Bacterial biofilms are acknowledged to be a major factor in problems of ineffective sterilization often encountered in clinics, hospitals, and industrial processes. There have been indications that the addition of a relatively small direct current electric field with the sterilant used to combat the biofilm greatly increases the efficacy of the sterilization process. The results of the experiments reported in this paper support the concept of the "bioelectric effect" as reported by J.W. Costerton, B. Ellis, K. Lam, F. Johnson, and A.E. Khoury (Antimicrob. Agents Chemother, 38:2803-2809, 1994). With a current of 1 mA flowing through the chamber containing the biofilm, an increase in the killing of the bacteria of about 8 log orders was observed at the end of 24 h (compared with the control with the same amount of antibacterial agent but no current). We also confirmed that the current alone does not affect the biofilm and that there appear to be optimum levels of both the current and the sterilant that are needed to obtain the maximum effect.
PMCID: PMC163464  PMID: 8878572
8.  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
9.  Surface action of gentamicin on Pseudomonas aeruginosa. 
Journal of Bacteriology  1993;175(18):5798-5805.
The mode of action of gentamicin has traditionally been considered to be at the 30S ribosomal level. However, the inhibition of bacterial protein synthesis alone appears to be insufficient to entirely explain the bactericidal effects. Bacteriolysis is also mediated through perturbation of the cell surface by gentamicin (J.L. Kadurugamuwa, J.S. Lam, and T.J. Beveridge, Antimicrob. Agents Chemother. 37:715-721, 1993). In order to separate the surface effect from protein synthesis in Pseudomonas aeruginosa PAO1, we chemically conjugated bovine serum albumin (BSA) to gentamicin, making the antibiotic too large to penetrate through the cell envelope to interact with the ribosomes of the cytoplasm. Furthermore, this BSA-gentamicin conjugate was also used to coat colloidal gold particles as a probe for electron microscopy to study the surface effect during antibiotic exposure. High-performance liquid chromatography confirmed the conjugation of the protein to the antibiotic. The conjugated gentamicin and BSA retained bactericidal activity and inhibited protein synthesis on isolated ribosomes in vitro but not on intact cells in vivo because of its exclusion from the cytoplasm. When reacted against the bacteria, numerous gentamicin-BSA-gold particles were clearly seen on the cell surfaces of whole mounts and thin sections of cells, while the cytoplasm was devoid of such particles. Disruption of the cell envelope was also observed since gentamicin-BSA and gentamicin-BSA-gold destabilized the outer membrane, evolved outer membrane blebs and vesicles, and formed holes in the cell surface. The morphological evidence suggests that the initial binding of the antibiotic disrupts the packing order of lipopolysaccharide of the outer membrane, which ultimately forms holes in the cell envelope and can lead to cell lysis. It is apparent that gentamicin has two potentially lethal effects on gram-negative cells, that resulting from inhibition of protein synthesis and that resulting from surface perturbation; the two effects in concert make aminoglycoside drugs particularly effective antibiotics.
PMCID: PMC206658  PMID: 8376327
10.  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
11.  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
12.  A novel glycylcycline, 9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10-encoded metal-tetracycline/H+ antiporter. 
A novel tetracycline derivative, DMG-DMDOT [9-(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline] , is one of the glycylcyclines which have a broad antibacterial spectrum, including many tetracyclineresistant bacteria (R.T. Testa, P.J. Petersen, N.V. Jacobus, P.-E. Sum, V.J. Lee, and F.P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). The mechanism by which DMG-DMDOT overcomes efflux-based tetracycline resistance was investigated. Tetracycline-resistant Escherichia coli cells carrying an R plasmid encoding the tet(B) gene, which encodes the typical tetracycline efflux pump [TetA(B)] of gram-negative bacteria, were as susceptible to DMG-DMDOT as was the tetracycline-susceptible host. When mid-log-phase cells carrying the tet(B) gene were incubated with a subbactericidal concentration of DMG-DMDOT (0.5 micrograms/ml) for 2 h, a significant amount of the TetA(B) protein was detected in the cell membrane by Western blotting (immunoblotting) with an anti-carboxyl-terminal antibody, similar to the case in which tetracycline was used as the inducer, indicating that the tet repressor, TetR, can recognize DMG-DMDOT as an efficient inducer. Everted membrane vesicles prepared from cells producing the TetA(B) protein showed absolutely no transport activity for DMG-DMDOT. Furthermore, the presence of excess DMG-DMDOT had no effect on the tetracycline transport activity of the everted vesicles, indicating that DMG-DMDOT is not recognized as a substrate by the TetA(B) protein.
PMCID: PMC162518  PMID: 7695316
13.  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
14.  Evaluation of reverse transcriptase and protease inhibitors in two-drug combinations against human immunodeficiency virus replication. 
Current treatments for human immunodeficiency virus (HIV) include both reverse transcriptase and protease inhibitors. Results from in vitro and clinical studies suggest that combination therapy can be more effective than single drugs in reducing viral burden. To evaluate compounds for combination therapy, stavudine (d4T), didanosine (ddI), or BMS-186,318, an HIV protease inhibitor, were combined with other clinically relevant compounds and tested in a T-cell line (CEM-SS) that was infected with HIV-RF or in peripheral blood mononuclear cells infected with a clinical HIV isolate. The combined drug effects were analyzed by the methods described by Chou and Talalay (Adv. Enzyme Regul. 22:27-55, 1984) as well as by Prichard et al. (Antimicrob. Agents Chemother. 37:540-545, 1993). The results showed that combining two nucleoside analogs (d4T-ddI, d4T-zidovudine [AZT], and d4T-zalcitabine [ddC]), two HIV protease inhibitors (BMS-186,318-saquinavir, BMS-186,318-SC-52151, and BMS-186,318-MK-639) or a reverse transcriptase and a protease inhibitor (BMS-186,318-d4T, BMS-186,318-ddI, BMS-186,318-AZT, d4T-saquinavir, d4T-MK-639, and ddI-MK-639) yielded additive to synergistic antiviral effects. In general, analysis of data by either method gave consistent results. In addition, combined antiviral treatments involving nucleoside analogs gave slightly different outcomes in the two cell types, presumably because of a difference in phosphorylation patterns. Importantly, no strong antagonism was observed with the drug combinations studied. These data should provide useful information for the design of clinical trials of combined chemotherapy.
PMCID: PMC163329  PMID: 8725999
15.  Insertional activation of cepA leads to high-level beta-lactamase expression in Bacteroides fragilis clinical isolates. 
Journal of Bacteriology  1994;176(14):4376-4384.
Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including beta-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400, 1993). Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low- and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B. fragilis 638 cepA mutant. The results of beta-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high- or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low- and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.
PMCID: PMC205651  PMID: 7517394
16.  Anti-influenza virus activities of 4-substituted 2,4-dioxobutanoic acid inhibitors. 
We previously identified a series of compounds which specifically inhibited the transcription of influenza A and B viruses (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J. Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994). The compounds, 4-substituted 2,4-dioxobutanoic acids, selectively targeted the cap-dependent endonuclease activity of the transcriptase complex. Additionally, several of these compounds effectively inhibited the replication of influenza virus but not other viruses in cell culture assays. Here, we report on the anti-influenza virus activities of other potent derivatives of the series evaluated in both in vitro and in vivo infectivity assays. These compounds inhibited the replication of influenza virus in yield reduction assays, with 50% inhibitory concentrations ranging from 0.18 to 0.71 microM. These 50% inhibitory concentrations were similar to those observed for inhibition of in vitro transcription (0.32 to 0.54 microM). One selected compound also elicited a dose-dependent inhibition of influenza virus replication in mice following an upper respiratory tract challenge. These studies demonstrate the antiviral efficacy of this inhibitor class and thereby establish the utility of influenza virus endonuclease as a chemotherapeutic target.
PMCID: PMC163316  PMID: 8723491
17.  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
18.  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
19.  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
20.  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
21.  Efficacies of cefotaxime and ceftriaxone in a mouse model of pneumonia induced by two penicillin- and cephalosporin-resistant strains of Streptococcus pneumoniae. 
Antimicrobial Agents and Chemotherapy  1996;40(12):2829-2834.
We previously demonstrated the efficacy of ceftriaxone (CRO), at 50 mg/kg of body weight every 12 h, against a highly penicillin-resistant (MIC, 4 micrograms/ml) Streptococcus pneumoniae strain with low-level resistance to CRO (MIC, 0.5 microgram/ml) in a leukopenic-mouse pneumonia model (P. Moine, E. Vallée, E. Azoulay-Dupuis, P. Bourget, J.-P. Bédos, J. Bauchet, and J.-J. Pocidalo, Antimicrob. Agents Chemother. 38:1953-1958, 1994). In the present study, we assessed the activity of CRO versus those of cefotaxime (CTX) and amoxicillin (AMO) against two highly penicillin- and cephalosporin-resistant S. pneumoniae strains (P40422 and P40984) (MICs of 2 and 8 for penicillin, 2 and 4 for AMO, and 4 and 8 for CRO or CTX, respectively). Against both strains, a greater than an 80% cumulative survival rate was observed with CRO at a dose of 100 or 200 mg/kg every 12 h (dose/MIC ratio, 25). With CTX, a high dosage of 400 mg/kg (dose/MIC ratio, 100 or 50) administered every 8 h (TID) was needed to protect 66 and 75% of the animals, respectively, with no statistically significant differences versus CRO. Against the P40422 strain, CRO (100 mg/kg) produced the greatest bactericidal effect, from the 8th to the 24th hour after a single injection (1.8-log-unit reduction over 24 h), and the fastest bacterial pulmonary clearance during treatment; with CTX, only multiple injections at a high dosage, i.e., 400 mg/kg TID, demonstrated a significant bactericidal effect. AMO in a high dosage, 400 mg/kg (dose/MIC ratio, 200) TID, showed good activity only against the P40422 strain. Despite the identical MICs of CTX and CRO, the longer time (3.6 to 4.6 h) that serum CRO concentrations remained above the MICs for the pathogens at a dose of 100 mg/kg resulted in greater efficacy versus CTX against highly penicillin- and cephalosporin-resistant S. pneumoniae strains.
PMCID: PMC163631  PMID: 9124850
22.  Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain. 
Two groups of recent clinical isolates of Candida albicans consisting of 101 isolates for which fluconazole MICs were < or = 0.5 microgram/ml (n = 50) and > or = 4.0 micrograms/ml (n = 51), respectively, were compared for their susceptibilities to fluconazole, clotrimazole, miconazole, ketoconazole, and itraconazole. Susceptibility tests were performed by a photometer-read broth microdilution method with an improved RPMI 1640 medium supplemented with 18 g of glucose per liter (RPMI-2% glucose; J. L. Rodríguez-Tudela and J. V. Martínez-Suárez, Antimicrob. Agents Chemother. 38:45-48, 1994). Preparation of drugs, basal medium, and inocula was done by the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h as the lowest drug concentration at which growth was just equal to or less than 20% of that in the positive control well (MIC 80%). In vitro susceptibility testing separated azole-susceptible strains from the strains with decreased susceptibilities to azoles if wide ranges of concentrations (20 doubling dilutions) were used for ketoconazole, miconazole, and clotrimazole. By comparison with isolates for which fluconazole MICs were < or = 0.5 microgram/ml, those isolates for which fluconazole MICs were > or = 4.0 micrograms/ml were in general less susceptible to other azole drugs, but different patterns of decreased susceptibility were found, including uniform increases in the MICs of all azole derivatives, higher MICs of several azoles but not others, and elevated MICs of fluconazole only. On the other hand, decreased susceptibility to any other azole drug was never found among strains for which MICs of fluconazole were lower.
PMCID: PMC162772  PMID: 7492095
23.  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
24.  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
25.  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

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