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Timothy R Walsh

Publications and source records attributed to Timothy R Walsh.

At least 19 recordsLinked to original sources
PubMed2006

A multicenter study evaluating the current strategies for isolating Staphylococcus aureus strains with reduced susceptibility to glycopeptides.

Glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA) strains are notoriously difficult to detect in the diagnostic laboratory. The clinical importance of GISA, and particularly hGISA, will only be obvious when a definitive detection method is available. A few novel GISA and hGISA detection methods have been proposed; however, their validity has never been tested on a significant scale and in different laboratories. This study compares three screening methods for detecting GISA and hGISA strains in 12 laboratories, using a blind panel of 48 strains with known glycopeptide susceptibilities. The three screening methods used were brain heart infusion agar with 6 mg/liter vancomycin (BHIA6V) (CDC/CLSI), Mueller-Hinton agar with 5 mg/liter teicoplanin (MHA5T) (European Antimicrobial Resistance Surveillance System [EARSS]), and the macrodilution method Etest (MET) (EARSS), with population analysis profile-area under the curve analysis as the gold standard. Sensitivity and specificity were highest for MHA5T and MET, which identified 82.5% and 85.9% of strains, respectively. BHIA6V had poor sensitivity, particularly for hGISA (11.5% of strains were detected), and gave the largest interlaboratory variation in performance. MET exhibited the least interlaboratory variation. It is essential that laboratories use appropriate methods to detect GISA/hGISA strains so that the prevalence and clinical importance of these strains can be assessed properly.

Agar↗
PubMed2006

Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway.

Consecutive clinical isolates of Escherichia coli (n = 87) and Klebsiella pneumoniae (n = 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined for bla(TEM/SHV/CTX-M) extended-spectrum-beta-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-beta-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n = 23) and CTX-M-9-like (n = 15) ESBLs dominated among the 50 ESBL-positive E. coli isolates. SHV-5-like (n = 9) and SHV-2-like (n = 4) ESBLs were the most prevalent in 19 ESBL-positive K. pneumoniae isolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in three E. coli strains. The relatively high proportion of ESBL-producing E. coli organisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.

Drug Resistance, Multiple, Bacterial↗
PubMed2006

Comparative bactericidal activities of daptomycin and vancomycin against glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA isolates.

Staphylococcus aureus strains from the U.S. SENTRY Antimicrobial Surveillance Program, 2002-2003, glycopeptide-intermediate S. aureus (GISA) strains, and heterogeneous GISA (hGISA) strains were used to compare bactericidal activities of daptomycin and vancomycin using MICs and minimum bactericidal concentrations. Glycopeptide-susceptible S. aureus and hGISA strains were further studied by using time-kill curves. For all isolates, the daptomycin MIC50 and MIC90 are four times lower and the log drops in viable counts at 6 h and 24 h are significantly greater than those for vancomycin.

Anti-Bacterial Agents↗
PubMed2006

Integrons containing the VIM-2 metallo-beta-lactamase gene among imipenem-resistant Pseudomonas aeruginosa strains from different Chinese hospitals.

A total of 140 nonrepetitive strains of imipenem-resistant Pseudomonas aeruginosa were isolated from five different Chinese hospitals. Fourteen isolates were confirmed to contain the VIM-2 metallo-beta-lactamase gene. Twelve isolates harbored two kinds of class 1 integron, containing both VIM-2- and aminoglycoside-resistant genes.

Conjugation, Genetic↗
PubMed2006

Combinatorial genetic evolution of multiresistance.

The explosion in genetic information, whilst extending our knowledge, might not necessary increase our conceptual understanding on the complexities of bacterial genetics, or why some antibiotic resistant genotypes such as blaCTX-M-15 and blaVIM-2 appear to dominate. However, the information we have thus far suggests that clinical isolates have 'hijacked' plasmids, primarily built of backbone-DNA originating from environmental bacteria. Additionally, the combinatorial presence of other elements such as transposons, integrons, insertion sequence (IS) elements and the 'new' ISCR (IS common region) elements have also contributed to the increase in antibiotic resistance - an antibiotic resistant cluster composing four or five genes has become commonplace. In some instances, the presence of antibiotics themselves, such as fluoroquinolones, can mediate a bacterial SOS cell response, subsequently amplifying and/or augmenting the transfer of large genetic entities therefore, potentially promoting long-term detrimental effects.

Anti-Bacterial Agents↗
PubMed2006

Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons.

The ability of bacteria to procure, sometimes rearrange, and evince acquired DNA continues to impress us-even more so if this genetic plasticity involves the sequestering of antibiotic resistance genes. The acquisition of genes in bacteria is often facilitated by transposons, integrons and archetype insertion elements. Recently however, a new element, 'orf513', has been increasingly associated with class 1 integrons. Moreover, these 'complex' class 1 integrons can potentially mediate resistance to chloramphenicol, trimethoprim, aminoglycosides and tetracycline and may carry a range of beta-lactamase genes as well as the qnrA gene. Elements such as 'orf513' demonstrate IS91-like characteristics and will mobilize adjacent DNA via a process called rolling circle replication, and thus we have renamed them 'insertion sequence CRs' (ISCRs) to appropriately reflect their structure-function properties. In this article, we provide a brief description of these new and clinically important mobile elements, and how they are able to mobilize antibiotic resistance genes.

Anti-Bacterial Agents↗
PubMed2006

Characterization of fluoroquinolone-resistant beta-hemolytic Streptococcus spp. isolated in North America and Europe including the first report of fluoroquinolone-resistant Streptococcus dysgalactiae subspecies equisimilis: report from the SENTRY Antimicrobial Surveillance Program (1997-2004).

Beta-hemolytic streptococci are common bacterial pathogens that can cause serious invasive disease, and although this group of species has remained susceptible to the fluoroquinolone class, resistant strains have been reported. This multicenter investigation determined the rate of fluoroquinolone-resistant beta-hemolytic streptococci using the SENTRY Antimicrobial Surveillance Program network data (1997-2004). Forty-seven surveillance culture isolates of beta-hemolytic streptococci from North America and Europe with elevated levofloxacin MIC results (2 to >32) microg/mL were tested for susceptibility to other fluoroquinolones including ciprofloxacin, garenoxacin, gatifloxacin, gemifloxacin, and moxifloxacin using reference broth microdilution and Etest (BIODISK, Solna, Sweden) methods. Strains were characterized using polymerase chain reaction and sequencing to detect mutations in the quinolone-resistance determining region (QRDR). The beta-hemolytic streptococci isolates with reduced fluoroquinolone susceptibility included the following Lancefield groups: A (Streptococcus pyogenes; 9 strains), B (Streptococcus agalactiae; 24 strains), C and G (14 strains). Vitek and API 20 strep (bioMerieux, Hazelwood, MO) identification systems, as well as conventional biochemical methods and colony morphology, were used to confirm the organism identifications. The overall potency (MIC90 in microg/mL) for the fluoroquinolones against all tested beta-hemolytic streptococci showed the following rank order: gemifloxacin (0.06) > garenoxacin (0.12) > moxifloxacin (0.25) > gatifloxacin (0.5) > levofloxacin = ciprofloxacin (1). The rate of levofloxacin-resistant beta-hemolytic streptococci in the SENTRY program was 0.14% (Europe) and 0.51% (North America) during the study period. All levofloxacin-resistant strains tested by molecular methods had significant mutations in either parC (position 79 or 83) and/or gyrA (position 81 or 85). All but 2 isolates with high-level resistance to levofloxacin (>32 microg/mL) had gyrA mutations. Strains with lower MIC values to levofloxacin (2-4 microg/mL) only had mutations in parC. The increasing rate of fluoroquinolone-resistant streptococci including Streptococcus pneumoniae, viridans group streptococci, and the more recently reported beta-hemolytic streptococci, is becoming a clinical concern due to the morbidity and mortality caused by these pathogens.

Adolescent↗
PubMed2006

Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases.

Metallo-beta-lactamases (mbetals) confer broad-spectrum resistance to beta-lactam antibiotics upon host bacteria and escape the action of existing beta-lactamase inhibitors. SPM-1 is a recently discovered mbetal that is distinguished from related enzymes by possession of a substantial central insertion and by sequence variation at positions that maintain active site structure. Biochemical data show SPM-1 to contain two Zn2+ sites of differing affinities, a phenomenon that is well documented amongst mbetals but for which a structural explanation has proved elusive. Here, we report the crystal structure of SPM-1 to 1.9 A resolution. The structure reveals SPM-1 to lack a mobile loop implicated in substrate binding by related mbetals and to accommodate the central insertion in an extended helical interdomain region. Deleting this had marginal effect upon binding and hydrolysis of a range of beta-lactams. These data suggest that the interactions of SPM-1 with substrates differ from those employed by other mbetals. SPM-1 as crystallised contains a single Zn2+. Both the active site hydrogen-bonding network and main-chain geometry at Asp120, a key component of the binding site for the second zinc ion, differ significantly from previous mbetal structures. We propose that variable interactions made by the Asp120 carbonyl group modulate affinity for a second Zn2+ equivalent in mbetals of the B1 subfamily. We further predict that SPM-1 possesses the capacity to evolve variants of enhanced catalytic activity by point mutations altering geometry and hydrogen bonding in the vicinity of the second Zn2+ site.

Binding Sites↗
PubMed2006

ISCR elements: novel gene-capturing systems of the 21st century?

"Common regions" (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5' sequences via misreading of the cognate terIS, i.e., "unchecked transposition." Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-beta-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via "unchecked RC transposition," as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their "genetic construction kit" to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.

Aminoglycosides↗
PubMed2005

Analysis of Salmonella spp. with resistance to extended-spectrum cephalosporins and fluoroquinolones isolated in North America and Latin America: report from the SENTRY Antimicrobial Surveillance Program (1997-2004).

Emerging antimicrobial-resistant Salmonella spp. requires increased efforts to appropriately test susceptibility. The SENTRY Antimicrobial Surveillance Program monitored Salmonella spp. and detected nalidixic acid-resistant strains with elevated fluoroquinolone minimum inhibitory concentration (MIC) results and strains with extended-spectrum beta-lactamase (ESBL) "phenotypes" over the last 8 years. A total of 786 stool and bloodstream isolates from North American and Latin American medical centers (2001-2003) were tested by reference broth microdilution methods. Genetic analysis was used to further characterize the resistance mechanisms. Twenty-one sites forwarded 89 (11.3%) nalidixic acid-resistant (MIC, > or =32 microg/mL) strains. Nineteen of these isolates were studied to determine mutations in the quinolone resistance-determining region (QRDR). Among the nalidixic acid-resistant Salmonella spp. isolates, fluoroquinolone MIC values were also elevated (8- to 32-fold) compared with "wild-type" strains. Ciprofloxacin and gatifloxacin (MIC(90), 0.5 microg/mL) were more potent than levofloxacin and garenoxacin (1 microg/mL) against nalidixic acid-resistant strains. Single gyrA mutations were responsible for elevated fluoroquinolone MIC values and included D87Y (5), S83F (7), D87N (5), and S83Y (2). During 2001, 9 sites contributed 11 (2.9%) strains that met ESBL screening criteria (> or =2 microg/mL) for aztreonam or ceftazidime or ceftriaxone. ESBL confirmation was evaluated by Etest (AB BIODISK, Solna, Sweden) ESBL strips and the enzymes were characterized by polymerase chain reaction and gene sequencing. The ESBL phenotype isolates had the following MIC patterns: ceftazidime (> or =16 microg/mL), aztreonam (4 to >16 microg/mL), and ceftriaxone (8-32 microg/mL). All strains were susceptible to cefepime, carbapenems, gentamicin, and fluoroquinolones. No strains were inhibited by clavulanic acid consistent with all isolates producing the identified CMY-2, AmpC-like enzyme. Fluoroquinolones may be compromised among isolates with QRDR mutations detected using nalidixic acid as a screening agent. Salmonella spp. with ESBL phenotypes were likely to harbor CMY-2 (not an ESBL) and remain susceptible to cefepime, carbapenems, and fluoroquinolones, which can be used for serious invasive Salmonella spp. infections. Compared with the stool culture isolates, the blood culture isolates had higher QRDR mutations, but remained susceptible to the fluoroquinolones. The blood culture isolates were more susceptible to penicillins (ampicillin and ticarcillin) and not significantly different for ceftriaxone or trimethoprim/sulfamethoxazole susceptibility patterns. No QRDR trends over time were detected in North America, but increased resistance was observed in Latin America.

Anti-Bacterial Agents↗
PubMed2005

Reduced expression of the atl autolysin gene and susceptibility to autolysis in clinical heterogeneous glycopeptide-intermediate Staphylococcus aureus (hGISA) and GISA strains.

OBJECTIVES: To assess a link between resistance to Triton X-100 induced autolysis (TIA) and lowered atl expression in a collection of clinical glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA). METHODS: Nine clinical GISA, 11 hGISA and 11 glycopeptide-susceptible S. aureus (GSSA), including three pairs of related isolates, were analysed using TIA assays. Lysostaphin MICs were determined by a broth microdilution technique and reverse transcriptase PCR was used to compare atl expression levels in all isolates. RESULTS: Eight of nine clinical GISA and six of 11 hGISA exhibited lower susceptibility to TIA and higher MICs of lysostaphin than GSSA. Eight of nine GISA and all hGISA strains had lowered atl expression levels compared with GSSA. CONCLUSIONS: The majority of GISA and hGISA isolates exhibited lowered susceptibility to TIA and lysostaphin and reduced atl expression when compared with GSSA isolates. These factors could contribute to, or predispose to the development of, a thickened cell wall and glycopeptide-intermediate resistance.

Anti-Bacterial Agents↗
PubMed2005

Emerging metallo-beta-lactamase-mediated resistances: a summary report from the worldwide SENTRY antimicrobial surveillance program.

The rates of occurrence of metallo-beta-lactamase-mediated resistances in Pseudomonas aeruginosa, Acinetobacter species, and Serratia marcescens, among other gram-negative bacilli, have escalated since 2000, severely limiting treatment options in Asia, Europe, and Latin America to non-beta-lactam antimicrobial classes. Clinical isolates harboring metallo-beta-lactamases have also recently been reported in western Canada and in Texas, signaling the need for development of accurate diagnostic tests by clinical laboratories to detect their presence and for new, and more potent, antimicrobial agents.

Acinetobacter↗
PubMed2005

Bulgecin A: a novel inhibitor of binuclear metallo-beta-lactamases.

Bulgecin A, a sulphonated N-acetyl-D-glucosamine unit linked to a 4-hydroxy-5-hydroxymethylproline ring by a beta-glycosidic linkage, is a novel type of inhibitor for binuclear metallo-beta-lactamases. Using steady-state kinetic analysis with nitrocefin as the beta-lactam substrate, bulgecin A competitively inhibited the metallo-beta-lactamase BceII from Bacillus cereus in its two-zinc form, but failed to inhibit when the enzyme was in the single-zinc form. The competitive inhibition was restored by restoring the second zinc ion. The single-zinc metallo-beta-lactamase from Aeromonas veronii bv. sobria, ImiS, was not inhibited by bulgecin A. The tetrameric L1 metallo-beta-lactamase from Stenotrophomonas maltophilia was subject to partial non-competitive inhibition, which is consistent with a kinetic model in which the enzyme bound to inhibitor retains catalytic activity. Docking experiments support the conclusion that bulgecin A co-ordinates to the zinc II site in metallo-beta-lactamases via the terminal sulphonate group on the sugar moiety.

Acetylglucosamine↗
PubMed2005

Evolution and dissemination of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae: epidemiology and molecular report from the SENTRY Antimicrobial Surveillance Program (1997-2003).

During 2001, occurrences of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were detected in a single medical center (Hospital A) from the SENTRY Antimicrobial Surveillance Program that became endemic in long-term acute care areas and in the intensive care unit in 2002-2003. Between 2001 and 2003, 123 patients were infected or colonized with ESBL-positive K. pneumoniae. Resistance profiles were determined by reference broth microdilution methods, and automated ribotyping and pulsed-field gel electrophoresis (PFGE) were performed. The ESBL-positive K. pneumoniae isolates were resistant to aztreonam, ceftazidime, aminoglycosides, and trimethoprim/sulfamethoxazole and susceptible to ciprofloxacin and tetracycline. In 1997, 1998, and 2000, 9 ESBL-producing K. pneumoniae strains from 2 New York City hospitals shared the same antibiograms and ribotype (204.2) as the strains from Hospital A. PFGE patterns divided Hospital A isolates into 2 subtypes (A and A1) and 3 New York City strains were similar to the Hospital A isolates (A2, A3, and A4). Isoelectric focusing studies of 1 New York City isolate (A4) revealed pIs at 5.4, 7.7, and 8.2. PCR and sequencing results from 1 strain of each Hospital A and 1 New York PFGE pattern determined that TEM-1 and SHV-5 (ESBL) were present in all strains. In addition, 2 New York isolates from 1998 (A3 and A4) also had an OXA-2 enzyme. ESBL-producing K. pneumoniae isolates with ribotype 204.2 from SENTRY Program sites have been recognized in New York only since 1997 and in Hospital A beginning in 2001. The similarities of the antibiogram and epidemiological patterns suggest that these isolates have persisted over time and may have evolved into different but genetically related endemic ESBL-positive K. pneumoniae clones that have the ability to cause sustained epidemic outbreaks in US medical centers.

Adolescent↗
PubMed2005

Dissemination and diversity of metallo-beta-lactamases in Latin America: report from the SENTRY Antimicrobial Surveillance Program.

Carbapenem resistance among Pseudomonas aeruginosa and Acinetobacter spp. is becoming a critical therapeutic problem worldwide. The SENTRY Antimicrobial Surveillance Program monitors pathogen frequency and antimicrobial resistance patterns of nosocomial and community-acquired infections through sentinel hospitals on five continents. Pseudomonas spp. and Acinetobacter spp. strains resistant to imipenem (MIC, >/=16 mg/l), meropenem (MIC, >/=16 mg/l), and ceftazidime (MIC, >/=32 mg/l) collected from January 2001 to December 2003 were routinely screened for antimicrobial resistance genes. Resistant isolates were initially tested for metallo-beta-lactamase (MbetaL) production by phenotypic tests (disk approximation or MbetaL Etest strip) and then characterization of the MbetaL (hydrolysis assays, PCR for bla(IMP), bla(VIM), bla(SPM), gene sequencing). Eighty-nine isolates (33 Acinetobacter spp., 54 Pseudomonas aeruginosa, and 2 P. fluorescens) had positive phenotypic screening tests. Among those, 34 isolates producing MbetaL were identified, including 7 Acinetobacter spp., 25 P. aeruginosa and 2 P. fluorescens. The MbetaLs identified were IMP-1, VIM-2 and two newly described enzymes: SPM-1 and IMP-16. The greatest concentration of MbetaL strains was in Brazil, where imipenem-resistant P. aeruginosa increased significantly in the time period evaluated by the SENTRY Program. MbetaL-producing P. aeruginosa was detected in São Paulo (SPM-1) and Brasilia (SPM-1 and IMP-16), Brazil and Caracas, Venezuela (VIM-2); while MbetaL-producing Acinetobacter spp. isolates were detected in São Paulo, Brazil (IMP-1). P. fluorescens isolates producing IMP-1 and VIM-2 were detected in São Paulo, Brazil and Santiago, Chile, respectively. The emergence and dissemination of mobile MbetaL-producing isolates represent an alarming factor for increasing resistance to carbapenems in several medical centres evaluated by the SENTRY Program in Latin America.

Acinetobacter↗