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1.  Pyrosequencing Using the Single-Nucleotide Polymorphism Protocol for Rapid Determination of TEM- and SHV-Type Extended-Spectrum β-Lactamases in Clinical Isolates and Identification of the Novel β-Lactamase Genes blaSHV-48, blaSHV-105, and blaTEM-155▿  
TEM- and SHV-type extended-spectrum β-lactamases (ESBLs) are the most common ESBLs found in the United States and are prevalent throughout the world. Amino acid substitutions at a number of positions in TEM-1 lead to the ESBL phenotype, although substitutions at residues 104 (E to K), 164 (R to S or H), 238 (G to S), and 240 (E to K) appear to be particularly important in modifying the spectrum of activity of the enzyme. The SHV-1-derived ESBLs are a less diverse collection of enzymes; however, the majority of amino acid substitutions resulting in an ESBL mirror those seen in the TEM-1-derived enzymes. Pyrosequencing by use of the single-nucleotide polymorphism (SNP) protocol was applied to provide sequence data at positions critical for the ESBL phenotype spanning the blaTEM and blaSHV genes. Three novel β-lactamases are described: the ESBLs TEM-155 (Q39K, R164S, E240K) and SHV-105 (I8F, R43S, G156D, G238S, E240K) and a non-ESBL, SHV-48 (V119I). The ceftazidime, ceftriaxone, and aztreonam MICs for an Escherichia coli isolate expressing blaSHV-105 were >128, 128, and >128 μg/ml, respectively. Likewise, the ceftazidime, ceftriaxone, and aztreonam MICs for an E. coli isolate expressing blaTEM-155 were >128, 64, and > 128 μg/ml, respectively. Pyrosequence analysis determined the true identity of the β-lactamase on plasmid R1010 to be SHV-11 rather than SHV-1, as previously reported. Pyrosequencing is a real-time sequencing-by-synthesis approach that was applied to SNP detection for TEM- and SHV-type ESBL identification and represents a robust tool for rapid sequence determination that may have a place in the clinical setting.
PMCID: PMC2650538  PMID: 19075050
2.  Characterization and Sequence Analysis of Extended-Spectrum-β-Lactamase-Encoding Genes from Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis Isolates Collected during Tigecycline Phase 3 Clinical Trials▿  
In concert with the development of novel β-lactams and broad-spectrum cephalosporins, bacterially encoded β-lactamases have evolved to accommodate the new agents. This study was designed to identify, at the sequence level, the genes responsible for the extended-spectrum-β-lactamase (ESBL) phenotypes of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates collected during the global tigecycline phase 3 clinical trials. PCR assays were developed to identify and clone the blaTEM, blaSHV, blaOXA, and blaCTX genes from clinical strains. Isolates were also screened for AmpC genes of the blaCMY, blaACT, blaFOX, and blaDHA families as well as the blaKPC genes encoding class A carbapenemases. E. coli, K. pneumoniae, and P. mirabilis isolates with ceftazidime MICs of ≥2 μg/ml were designated possible ESBL-producing pathogens and were then subjected to a confirmatory test for ESBLs by use of Etest. Of 272 unique patient isolates, 239 were confirmed by PCR and sequencing to carry the genes for at least one ESBL, with 44% of the positive isolates harboring the genes for multiple ESBLs. In agreement with current trends for ESBL distribution, blaCTX-M-type β-lactamase genes were found in 83% and 71% of the ESBL-positive E. coli and K. pneumoniae isolates, respectively, whereas blaSHV genes were found in 41% and 28% of the ESBL-positive K. pneumoniae and E. coli isolates, respectively. Ninety-seven percent of the E. coli and K. pneumoniae isolates were tigecycline susceptible (MIC90 = 2 μg/ml), warranting further studies to define the therapeutic utility of tigecycline against strains producing ESBLs in a clinical setting.
PMCID: PMC2630642  PMID: 19015360
3.  Real-Time PCR and Statistical Analyses of acrAB and ramA Expression in Clinical Isolates of Klebsiella pneumoniae▿  
Clinical isolates of Klebsiella pneumoniae were tested for a correlation between tigecycline MIC and expression of ramA by using real-time PCR. At MICs of 4 and 8 μg/ml, the expression of ramA was statistically significantly different from MICs of 2 μg/ml or less, supporting the tigecycline susceptibility breakpoint of ≤2 μg/ml for K. pneumoniae.
PMCID: PMC2533484  PMID: 18625776
4.  Functional, Biophysical, and Structural Bases for Antibacterial Activity of Tigecycline 
Tigecycline is a novel glycylcycline antibiotic that possesses broad-spectrum activity against many clinically relevant species of bacterial pathogens. The mechanism of action of tigecycline was delineated using functional, biophysical, and molecular modeling experiments in this study. Functional assays showed that tigecycline specifically inhibits bacterial protein synthesis with potency 3- and 20-fold greater than that of minocycline and tetracycline, respectively. Biophysical analyses demonstrated that isolated ribosomes bind tigecycline, minocycline, and tetracycline with dissociation constant values of 10−8, 10−7, and >10−6 M, respectively. A molecular model of tigecycline bound to the ribosome was generated with the aid of a 3.40-angstrom resolution X-ray diffraction structure of the 30S ribosomal subunit from Thermus thermophilus. This model places tigecycline in the A site of the 30S subunit and involves substantial interactions with residues of H34 of the ribosomal subunit. These interactions were not observed in a model of tetracycline binding. Modeling data were consistent with the biochemical and biophysical data generated in this and other recent studies and suggested that tigecycline binds to bacterial ribosomes in a novel way that allows it to overcome tetracycline resistance due to ribosomal protection.
PMCID: PMC1479133  PMID: 16723578
5.  A Novel MATE Family Efflux Pump Contributes to the Reduced Susceptibility of Laboratory-Derived Staphylococcus aureus Mutants to Tigecycline 
Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.
PMCID: PMC1087644  PMID: 15855508
6.  Influence of Transcriptional Activator RamA on Expression of Multidrug Efflux Pump AcrAB and Tigecycline Susceptibility in Klebsiella pneumoniae 
Tigecycline is an expanded broad-spectrum antibacterial agent that is active against many clinically relevant species of bacterial pathogens, including Klebsiella pneumoniae. The majority of K. pneumoniae isolates are fully susceptible to tigecycline; however, a few strains that have decreased susceptibility have been isolated. One isolate, G340 (for which the tigecycline MIC is 4 μg/ml and which displays a multidrug resistance [MDR] phenotype), was selected for analysis of the mechanism for this decreased susceptibility by use of transposon mutagenesis with IS903φkan. A tigecycline-susceptible mutant of G340, GC7535, was obtained (tigecycline MIC, 0.25 μg/ml). Analysis of the transposon insertion mapped it to ramA, a gene that was previously identified to be involved in MDR in K. pneumoniae. For GC7535, the disruption of ramA led to a 16-fold decrease in the MIC of tigecycline and also a suppression of MDR. Trans-complementation with plasmid-borne ramA restored the original parental phenotype of decreased susceptibility to tigecycline. Northern blot analysis revealed a constitutive overexpression of ramA that correlated with an increased expression of the AcrAB transporter in G340 compared to that in tigecycline-susceptible strains. Laboratory mutants of K. pneumoniae with decreased susceptibility to tigecycline could be selected at a frequency of approximately 4 × 10−8. These results suggest that ramA is associated with decreased tigecycline susceptibility in K. pneumoniae due to its role in the expression of the AcrAB multidrug efflux pump.
PMCID: PMC549240  PMID: 15728897
7.  AcrAB Efflux Pump Plays a Role in Decreased Susceptibility to Tigecycline in Morganella morganii 
Transposon mutagenesis of a clinical isolate of Morganella morganii, G1492 (tigecycline MIC of 4 μg/ml), yielded two insertion knockout mutants for which tigecycline MICs were 0.03 μg/ml. Transposon insertions mapped to acrA, which is constitutively overexpressed in G1492, suggesting a role of the AcrAB efflux pump in decreased susceptibility to tigecycline in M. morganii.
PMCID: PMC547285  PMID: 15673770
8.  Mechanism of Action of the Mannopeptimycins, a Novel Class of Glycopeptide Antibiotics Active against Vancomycin-Resistant Gram-Positive Bacteria 
The naturally occurring mannopeptimycins (formerly AC98-1 through AC98-5) are a novel class of glycopeptide antibiotics that are active against a wide variety of gram-positive bacteria. The structures of the mannopeptimycins suggested that they might act by targeting cell wall biosynthesis, similar to other known glycopeptide antibiotics; but the fact that the mannopeptimycins retain activity against vancomycin-resistant organisms suggested that they might have a unique mode of action. By using a radioactive mannopeptimycin derivative bearing a photoactivation ligand, it was shown that mannopeptimycins interact with the membrane-bound cell wall precursor lipid II [C55-MurNAc-(peptide)-GlcNAc] and that this interaction is different from the binding of other lipid II-binding antibiotics such as vancomycin and mersacidin. The antimicrobial activities of several mannopeptimycin derivatives correlated with their affinities toward lipid II, suggesting that the inhibition of cell wall biosynthesis was primarily through lipid II binding. In addition, it was shown that mannopeptimycins bind to lipoteichoic acid in a rather nonspecific interaction, which might facilitate the accumulation of antibiotic on the bacterial cell surface.
PMCID: PMC353120  PMID: 14982757
9.  Further Evidence that a Cell Wall Precursor [C55-MurNAc-(Peptide)-GlcNAc] Serves as an Acceptor in a Sorting Reaction 
Journal of Bacteriology  2002;184(8):2141-2147.
Previous studies suggested that a Gly-containing branch of cell wall precursor [C55-MurNAc-(peptide)-GlcNAc], which is often referred to as lipid II, might serve as a nucleophilic acceptor in sortase-catalyzed anchoring of surface proteins in Staphylococcus aureus. To test this hypothesis, we first simplified the procedure for in vitro biosynthesis of Gly-containing lipid II by using branched UDP-MurNAc-hexapeptide isolated from the cytoplasm of Streptomyces spp. Second, we designed a thin-layer chromatography-based assay in which the mobility of branched but not linear lipid II is shifted in the presence of both sortase and LPSTG-containing peptide. These results and those of additional experiments presented in this study further suggest that lipid II indeed serves as a natural substrate in a sorting reaction.
PMCID: PMC134952  PMID: 11914345
10.  Equivalence of Lauric Acid and Glycerol Monolaurate as Inhibitors of Signal Transduction in Staphylococcus aureus 
Journal of Bacteriology  2000;182(9):2668-2671.
Glycerol monolaurate (GML) inhibits the expression of virulence factors in Staphylococus aureus and the induction of vancomycin resistance in Enterococcus faecalis, presumably by blocking signal transduction. Although GML is rapidly hydrolyzed by bacteria, one of the products, lauric acid, has identical inhibitory activity and is metabolized much more slowly. At least four distinct GML-hydrolyzing activities are identified in S. aureus: the secreted Geh lipase, residual supernatant activity in a geh-null mutant strain, a novel membrane-bound esterase, and a cytoplasmic activity.
PMCID: PMC111339  PMID: 10762277
11.  Glycerol Monolaurate Inhibits Induction of Vancomycin Resistance in Enterococcus faecalis 
Journal of Bacteriology  1998;180(1):182-185.
Glycerol monolaurate (GML) is a surfactant that has been found to inhibit the post-exponential phase activation of virulence factor production and the induction of β-lactamase in Staphylococcus aureus. It has been suggested that signal transduction is the most probable target for GML (S. J. Projan, S. Brown-Skrobot, P. M. Schlievert, F. Vandenesch, and R. P. Novick, J. Bacteriol. 176:4204–4209, 1994). We found that GML suppresses growth of vancomycin-resistant Enterococcus faecalis on plates with vancomycin and blocks the induction of vancomycin resistance, which involves a membrane-associated signal transduction mechanism, either at or before initiation of transcription. Given the surfactant nature of GML and the results of previous experiments, we suggest that GML blocks signal transduction. In contrast, GML has no effect on the induction of erythromycin-inducible macrolide resistance in S. aureus, which does not involve signal transduction.
PMCID: PMC106868  PMID: 9422612

Results 1-11 (11)