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Timothy R Walsh

Publications and source records attributed to Timothy R Walsh.

47 records · Page 3Linked to original sources
PubMed2003

Salmonella bloodstream infections: report from the SENTRY Antimicrobial Surveillance Program (1997-2001).

Salmonella spp. are significant bloodstream pathogens and are routinely monitored for antimicrobial resistance by the SENTRY Antimicrobial Surveillance Program. Six hundred and one bloodstream infection (BSI) isolates of Salmonella spp., collected over a 5-year period (1997-2001) were tested for their susceptibility against 20 antimicrobial agents, comparing year and geographical region. Salmonella enterica serotype Typhi was the most frequently identified 'species' (43% of identified strains), although 'unspeciated' strains predominated overall (54.2%). The rank order for six selected drugs tested by their MIC(90) values and percentage susceptibility was: ceftriaxone (< or =0.25 mg/l; 99.5% susceptible)>ciprofloxacin (0.12 mg/l; 99.3%)> trimethoprim/sulphamethoxazole (< or =0.5 mg/l; 92.7%)>amoxycillin/clavulanate (16 mg/l; 89.7%)>ampicillin (>16 mg/l; 81.0%)>tetracycline (>8 mg/l; 79.4%). Most isolates remained highly susceptible to all 20 agents examined, with the exception of Salmonella Typhimurium (only 35.3% susceptible to tetracycline, 41.2% to ampicillin, and 61.8% to amoxycillin/clavulanate). DT104 resistance phenotypes were noted in 3.4 and nearly 60.0% of unspeciated Salmonella and S. Typhimurium, respectively. Unexpectedly, the highest overall susceptibility rates were recorded in Latin America. Fluoroquinolone resistance was observed and nalidixic acid screening MICs (< or =8 mg/l) predicted full susceptibility to ciprofloxacin. Five-year results from the SENTRY Program show no clear trend toward greater resistances in Salmonella spp. BSIs for the commonly used antimicrobial classes. With the exception of S. Typhimurium DT104, most Salmonella spp. remain highly susceptible to the tested antimicrobials that maybe utilized for Salmonella BSI.

Bacteremia↗
PubMed2003

Activity of AZD2563, a novel oxazolidinone, against Staphylococcus aureus strains with reduced susceptibility to vancomycin or linezolid.

The susceptibilities of clinical vancomycin-intermediate Staphylococcus aureus (VISA), heterogeneous VISA, and laboratory-generated linezolid-resistant S. aureus strains to the new oxazolidinone AZD2563 were assessed by agar dilution MIC determination. All clinical strains were susceptible to linezolid, and the linezolid MICs for them were equal to or twofold higher than those of AZD2563. Cross-resistance with linezolid was seen in laboratory-generated mutants, and for these strains the MIC of AZD2563 was twofold higher than that of linezolid.

Acetamides↗
PubMed2003

Biochemical characterization of the acquired metallo-beta-lactamase SPM-1 from Pseudomonas aeruginosa.

SPM-1 is a new metallo-beta-lactamase recently identified in Pseudomonas aeruginosa strain 48-1997A, isolated in Sao Paulo, Brazil. Kinetic analysis demonstrated that SPM-1 has a broad hydrolytic profile across a wide range of beta-lactam antibiotics. Considerable variation was observed within the penicillin, cephalosporin, and carbapenem subfamilies; however, on the whole, SPM-1 appears to preferentially hydrolyze cephalosporins. The highest k(cat/)K(m) ratios (in micromolar per second) overall were observed for this subgroup. The hydrolytic profile of SPM-1 bears the most similarity to that of the metallo-beta-lactamase IMP-1, yet for the most part, SPM-1 has k(cat)/K(m) values higher than those of IMP-1. Zinc chelator studies established that progressive inhibition of SPM-1 by EDTA, dipicolinic acid, and 1-10-o-phenanthroline demonstrated a biexponential pattern in which none of the chelators completely inhibited SPM-1. A homology model of SPM-1 was developed on the basis of the IMP-1 crystal structure, which showed the protein folding and active-site structure characteristic of metallo-beta-lactamases and which provides an explanation for the kinetic profiles observed.

Amino Acid Sequence↗
PubMed2003

Molecular and biochemical characterization of OXA-45, an extended-spectrum class 2d' beta-lactamase in Pseudomonas aeruginosa.

As part of the CANCER Antimicrobial Surveillance Program in North America, a clinical strain of Pseudomonas aeruginosa, strain 07-406, isolated in Texas was found to be resistant to all antimicrobials except polymyxin B. Genetic analysis of this isolate identified two unique extended-spectrum beta-lactamase genes. One, bla(VIM-7), encoded a metallo-beta-lactamase (unpublished data), and the other, bla(OXA-45), described here, encoded a class D extended-spectrum beta-lactamase. bla(OXA-45) was isolated on a Sau3A1 genomic fragment of 1.8 kb and encodes a protein of 264 amino acids with the highest identities to OXA-18 (65.9%), OXA-9 (42.8%), OXA-22 (40.2%), OXA-12 (38.6%), and OXA-29 (35.2%) but weak identities with other class D beta-lactamases. bla(OXA-45) was found to be harbored on a 24-kb plasmid in a region that displays high identities with a section of the 43-kb genomic island of Salmonella enterica serovar Typhimurium DT104. Biochemically OXA-45 is most similar to OXA-18 in its substrate profile and inhibition by clavulanic acid and is a member of the 2d' class of beta-lactamases.

Amino Acid Sequence↗
PubMed2002

Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis.

The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.

Amino Acid Sequence↗
PubMed2002

The prevalence and mechanisms of vancomycin resistance in Staphylococcus aureus.

The emergence of Staphylococcus aureus resistant to vancomycin has caused considerable concern. Such strains are currently rare, although they have been isolated from many areas of the world. Considerable controversy surrounds strains of S. aureus displaying heterogeneous resistance to vancomycin regarding their definition and methods for detection. This has led to considerable variance in estimates of prevalence (0-1.3%-20% in Japan) and has hindered efforts to define the clinical relevance of these strains. The mechanism of resistance involves a complex reorganization of cell wall metabolism, leading to a grossly thickened cell wall with reduced peptidoglycan cross-linking. There may be many different ways in which strains achieve this endpoint. Current knowledge and theories are summarized.

Anti-Bacterial Agents↗
PubMed2002

Preliminary analysis of the genetic basis for vancomycin resistance in Staphylococcus aureus strain Mu50.

Glycopeptides, such as vancomycin, are frequently the antibiotics of choice for treatment of infections caused by the now common methicillin-resistant Staphylococcus aureus (MRSA). Incidences of vancomycin resistance in S. aureus (VRSA) have been increasing worldwide for the last 5 years. Complex mechanisms producing changes in cell wall content and composition generate the VRSA phenotype, but the genetic basis of these changes has not yet been determined. To facilitate the genetic investigation, entire genome sequences of the archetypal VRSA (Mu50), and vancomycin-susceptible MRSA strains N315, EMRSA 16 and COL were compared. The in silico analysis revealed several loss-of-function mutations in Mu50, affecting important cell wall biosynthesis and intermediary metabolism genes, not previously reported. The new findings provide further evidence for the hypothesis that vancomycin resistance in Mu50 is due to fundamental changes, important to metabolic pathways that impinge on peptidoglycan biosynthesis. These observations will inform targeted experiments aimed at a complete understanding of the mechanism(s) of vancomycin resistance in S. aureus Mu50 and other VRSA strains.

Humans↗
PubMed2002

Differential regulation of L1 and L2 beta-lactamase expression in Stenotrophomonas maltophilia.

It has been reported that Stenotrophomonas maltophilia L1 and L2 beta-lactamase expression is coordinated. We have isolated S. maltophilia mutants where (i) L1 is constitutively hyper-expressed and L2 is inducible; (ii) L2 is hyper-expressed and L1 is inducible; and (iii) L1 and L2 are constitutively hyper-expressed. The frequency of isolating type 1 and 2 mutants is c. 10(-7), indicating that promoter mutations are probably not involved and providing strong evidence that L1 and L2 expression is not, after all, coordinated. The frequency of isolating type 3 mutants is c. 10(-9), however, implying that there is a significant overlap between the regulatory mechanisms.

Gene Expression Regulation, Enzymologic↗
PubMed2002

Molecular characterization of SPM-1, a novel metallo-beta-lactamase isolated in Latin America: report from the SENTRY antimicrobial surveillance programme.

The gene encoding the metallo-beta-lactamase SPM-1 was cloned from a genomic library of Pseudomonas aeruginosa strain 48-1997 A. The insert carrying spm-1 possessed a GC content of 47%, indicating that it is of non-Pseudomonas origin. Upstream of spm-1 there is a small open reading frame (ORF), which is homologous to the LysR family of proteins (69% identity to the LysR protein from Salmonella enterica serovar Typhimurium). Downstream of spm-1 there is the start of an ORF, the product of which shows close homology with the GroEL-type proteins from Xanthomonas campestris. No transmissible element could be identified upstream or downstream of spm-1. The spm-1 gene is carried on a plasmid that can transform both Escherichia coli and P. aeruginosa to ceftazidime resistance. SPM-1 contains the classic metallo-beta-lactamase zinc-binding motif HXHXD and shows the highest identity (35.5%) to IMP-1. SPM-1 is a distinctly different metallo-beta-lactamase from VIM and IMP and, accordingly, represents a new subfamily of mobile metallo-beta-lactamases. The predicted molecular weight of the protein was 27 515 Da, significantly higher than that of IMP (25 041 Da) or VIM (25 322 Da). SPM-1 possesses a unique loop of 23 residues that accounts for the higher molecular mass.

Amino Acid Sequence↗
PubMed2002

Evaluation of a new Etest for detecting metallo-beta-lactamases in routine clinical testing.

Several Etest (AB BIODISK, Solna, Sweden) gradient formats were developed for detection of metallo-beta-lactamases based on the reduction of imipenem (IP) or ceftazidime (TZ) MICs in the presence of EDTA or 2-mercaptopropionic acid (MPA). The Etest metallo-beta-lactamase (Etest MBL) strips consisted of a double-sided seven-dilution range of IP or TZ (4 to 256 microg/ml) and IP or TZ (1 to 64 microg/ml) overlaid with a constant concentration of EDTA or MPA. The prototype strips were evaluated on several agar media (brain heart infusion agar, Isosensitest agar, nutrient agar, and Mueller-Hinton agar for aerobes and brucella blood agar for anaerobes) with 138 challenge strains: Acinetobacter spp. (n = 9), Aeromonas spp. (n = 8), Chryseobacterium spp. (n = 28), Escherichia coli (n = 1), Klebsiella pneumoniae (n = 4), Pseudomonas aeruginosa (n = 14), Proteus mirabilis (n = 3), Serratia spp. (n = 10), Stenotrophomonas maltophilia (n = 43), Sphingobacterium spp. (n = 3), and Bacteroides fragilis group (n = 15). PCR analysis using specific primers for IMP-1, L1, CcrA, and bla(B/C) confirmed the presence of the metallo-beta-lactamase genes. Enzyme assays were also performed with IP as an indicator substrate followed by EDTA inhibition profiles. EDTA was found to be a better inhibitor of metallo-beta-lactamases, especially for anaerobes. IP was a better than TZ. Mueller-Hinton agar was the preferred medium, particularly when compared to Isosensitest agar, which frequently produced falsely low MICs for IP. Etest IP plus IP-EDTA with Mueller-Hinton agar had a sensitivity of 94% (79 of 84) and specificity of 95% (124 of 130). The Etest MBL strip appears to be an acceptable diagnostic reagent to detect metallo-beta-lactamase phenotypes in the clinical microbiology laboratory.

Anti-Bacterial Agents↗