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Timothy R Walsh

Publications and source records attributed to Timothy R Walsh.

At least 37 records · Page 2Linked to original sources
PubMed2005

Metallo-beta-lactamases: the quiet before the storm?

The ascendancy of metallo-beta-lactamases within the clinical sector, while not ubiquitous, has nonetheless been dramatic; some reports indicate that nearly 30% of imipenem-resistant Pseudomonas aeruginosa strains possess a metallo-beta-lactamase. Acquisition of a metallo-beta-lactamase gene will invariably mediate broad-spectrum beta-lactam resistance in P. aeruginosa, but the level of in vitro resistance in Acinetobacter spp. and Enterobacteriaceae is less dependable. Their clinical significance is further embellished by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. The genes encoding metallo-beta-lactamases are often procured by class 1 (sometimes class 3) integrons, which, in turn, are embedded in transposons, resulting in a highly transmissible genetic apparatus. Moreover, other gene cassettes within the integrons often confer resistance to aminoglycosides, precluding their use as an alternative treatment. Thus far, the metallo-beta-lactamases encoded on transferable genes include IMP, VIM, SPM, and GIM and have been reported from 28 countries. Their rapid dissemination is worrisome and necessitates the implementation of not just surveillance studies but also metallo-beta-lactamase inhibitor studies securing the longevity of important anti-infectives.

Gram-Negative Bacteria↗
PubMed2004

Italian metallo-beta-lactamases: a national problem? Report from the SENTRY Antimicrobial Surveillance Programme.

OBJECTIVES: As part of the SENTRY Antimicrobial Surveillance Programme, 383 non-replicative randomly collected Pseudomonas aeruginosa isolates were collected during 1999-2002. These strains originated from three geographically distinct hospitals within Italy: Genoa (Northern Italy); Rome and Catania (Sicily), and were further studied to identify the prevalence of metallo-beta-lactamase (MbetaL) alleles across Italy and to determine their genetic details. METHODS: Multidrug-resistant (MDR) strains were identified by MIC analysis followed by genotyping and PCR-based strategies. RESULTS: Initial MIC analysis identified 31 MDR isolates that displayed an Etest MbetaL-positive phenotype. Of these, 25 produced either the MbetaL VIM-1 or IMP-13 as detected by PCR and sequencing. VIM-1-producing isolates were found at all sites, whereas IMP-13-producing isolates were only found in Rome. MbetaL-producing isolates were found at all Italian SENTRY sites and together amounted to 6.5% of all P. aeruginosa isolates. Genetic analysis indicated that many strains contained multiple integrons and identified two novel MbetaL integrons, one from the site in Genoa and one from Sicily. Integrons identical in structure and sequence to In70, the first identified and characterized bla(VIM)-containing integron from Verona, were found in isolates with distinct ribotypes at the Roman and Sicilian sites indicating that this integron has recently disseminated across Italy. All 25 MbetaL-producing isolates were genetically linked in that all isolates contained Tn5051 sequences and all harboured the insertion sequence IsPa7 which may be involved in the mobilization of these resistance alleles. CONCLUSIONS: Taken together, these results indicate that Italy has a nationwide problem of MDR P. aeruginosa produced by mobile MbetaL genes.

Anti-Bacterial Agents↗
PubMed2004

Analysis of AmpC beta-lactamase expression and sequence in biochemically atypical ceftazidime-resistant Enterobacteriaceae from paediatric patients.

OBJECTIVES: To analyse the variation of ampC beta-lactamase gene sequence and expression in biochemically atypical Enterobacteriaceae isolates, and to identify them definitively. METHODS: beta-Lactamase gene-containing recombinant plasmids transformed into Escherichia coli were selected using ampicillin. PCR analysis was used to locate specific ampC and 16S rRNA genes, and the amplicons were sequenced. Random amplified polymorphic DNA PCR was used to group isolates and API 20E biochemical profiling was used to identify them putatively. RESULTS: Of 50 ceftazidime-resistant clinical Enterobacteriaceae isolates, 36 were identified (>95% confidence)-using API 20E test strips-as being organisms known to express inducible class C beta-lactamases (Citrobacter freundii, Enterobacter cloacae, Morganella morganii or Hafnia alvei). The rest were biochemically atypical. Of these, isolate I113, putatively identified as E. coli, possesses a chromosomally encoded ampC which differs by 15% from C. freundii OS60 ampC and by >30% from E. coli ampC. A related ampC gene was found in another seven of the atypical isolates. The use of various identification methods, including ampC sequence analysis, revealed that these I113-like ampC-positive isolates represent Citrobacter murliniae and Citrobacter youngae. CONCLUSIONS: We report sequences for two new Citrobacter spp. ampC genes, and provide evidence that ampC sequencing is a discriminatory method for identifying atypical Citrobacter spp. isolates.

Bacterial Proteins↗
PubMed2004

Penicillin-derived inhibitors that simultaneously target both metallo- and serine-beta-lactamases.

The synthesis and beta-lactamase inhibitory activity of four 6-(mercaptomethyl)penicillinates and the four corresponding 6-(hydroxymethyl)penicillinates are described. These penicillins include both C6 stereoisomers as well as the sulfide and sulfone oxidation states of the penam thiazolidine sulfur. All compounds were evaluated as inhibitors of representative metallo- and serine-beta-lactamases enzymes. Selected (mercaptomethyl)penicillinates are shown to inactivate both metallo- and serine-beta-lactamases and to display synergism with piperacillin against beta-lactamase producing strains.

Drug Delivery Systems↗
PubMed2004

Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001).

OBJECTIVES: During 1997-2001, 151 isolates of imipenem-resistant Pseudomonas aeruginosa were obtained from clinical specimens taken from children hospitalized in Warsaw, Poland. These strains were investigated further to determine the mechanism of resistance. METHODS: The strains were analysed by a combination of genotyping and PCR-based strategies. RESULTS: Eleven of these strains were found to contain the metallo-beta-lactamase (M beta L) gene bla(VIM-4). The first strain appeared in 1998, and P. aeruginosa strains harbouring this M beta L have become endemic in this hospital since then. All P. aeruginosa strains belonged to serotype O:6, and PFGE analysis revealed four different patterns and three sub-types. All 11 M beta L-producing strains contained an identical class 1 integron with the usual 5' and 3' conserved sequences. The integron included two resistance cassettes, aacA4 in the first position and the bla(VIM-4) cassette in the second position. The bla(VIM-4) gene included an unusual direct repeat of 169 bp of the 3' portion of the bla(VIM-4) gene. CONCLUSIONS: An unusual bla(VIM-4) M beta L has become endemic in P. aeruginosa isolates infecting Polish children hospitalized on surgical wards. The formation of this unusual bla(VIM-4) gene cassette could be explained by a mechanism involving deletion of a segment of an ancestral tandem repeat of bla(VIM-4) via slipped strand replication, mediated by a combination of polymerase and integrase.

Child↗
PubMed2004

Role of the 'cre/blr-tag' DNA sequence in regulation of gene expression by the Aeromonas hydrophila beta-lactamase regulator, BlrA.

OBJECTIVES: To further understand the mechanisms used to regulate expression of the blr regulon of Aeromonas hydrophila T429125, including three unlinked beta-lactamase genes, ampH, cepH and imiH, and to examine the role of the 'cre/blr-tag' DNA sequence (TTCAC) in transcriptional control exerted by the two-component system, BlrAB. METHODS: Genes linked to blrAB-ampH were cloned using standard methods; gene expression was measured by RT-PCR or beta-lactamase assays; transcription start sites were determined by reversed-transcript analysis; cepH promoter probe reporter constructs including cre/blr-tag deletions were generated by PCR; and BlrA was overexpressed in Escherichia coli using the pBAD plasmid. RESULTS: The blrD gene, encoding a putative inner membrane protein, was found to be located downstream of blrAB-ampH. RT-PCR analysis showed that blrD is part of the A. hydrophila blr regulon, and transcript start-point determinations revealed that blr-regulon promoters (including that of blrD) are preceded by at least one cre/blr-tag. Targeted deletion of the 16 bp cepH cre/blr-tag dimer blocked BlrA-induced overproduction of cepH in E. coli. CONCLUSIONS: This is the first report of non-beta-lactamase genes being co-ordinately regulated with a normally co-resident beta-lactamase gene, and the first direct evidence for a role of the cre/blr-tag sequence in the regulation of transcription by BlrA.

Aeromonas hydrophila↗
PubMed2004

Over-expression, purification, and characterization of metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria.

The gene from Aeromonas veronii bv. sobria encoding the metallo-beta-lactamase ImiS was subcloned into pET-26b, and ImiS was over-expressed in BL21(DE3) Escherichia coli and purified using SP-Sepharose chromatography. This protocol yielded over 5 mg of ImiS per liter of growth culture under optimum conditions. The biochemical properties of recombinant ImiS were compared with those of native ImiS. Recombinant and native ImiS have the same N-terminus of A-G-M-S-L, and CD spectroscopy was used to show that the enzymes have similar secondary structures. Gel filtration chromatography revealed that both enzymes exist as monomers in solution. MALDI-TOF mass spectra showed that the enzymes have a molecular mass of 25,247 Da, and metal analyses demonstrated that both as-isolated enzymes bind ca. 0.7 mol of Zn(II). Metal titrations demonstrate that the maximum activity of recombinant ImiS occurs when the enzyme binds one equivalent of zinc. Steady-state kinetic studies reveal that recombinant ImiS is a carbapenemase like native ImiS and that the recombinant enzyme exhibits similar kcat and K(m) values for the substrates tested, as compared to the native enzyme. This over-expression protocol now allows for detailed spectroscopic and mechanistic studies on ImiS as well as site-directed mutants of ImiS to be prepared for future structure/function studies.

Bacterial Proteins↗
PubMed2004

blaVIM-7, an evolutionarily distinct metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States.

As part of the CANCER Antimicrobial Surveillance Program in North America, a Pseudomonas aeruginosa isolate, strain 07-406, was shown to possess a metallo-beta-lactamase, designated VIM-7. bla(VIM-7) is located on a 24-kb plasmid which can be readily transferred into Enterobacteriaceae and other pseudomonads. This is the first report of a mobile metallo-beta-lactamase gene, bla(VIM-7), being detected within the United States.

Amino Acid Sequence↗
PubMed2004

Molecular characterization of a beta-lactamase gene, blaGIM-1, encoding a new subclass of metallo-beta-lactamase.

As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant Pseudomonas aeruginosa clinical isolates were detected with metallo-beta-lactamase (MbetaL) activity. The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany. The resistant determinant was isolated amplifying the region between the integrase and the aacA4 gene cassette. Sequencing revealed a novel MbetaL gene, designated bla(GIM-1). Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively. The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MbetaL family. Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-beta-lactamase inhibitors. bla(GIM-1) was found on a 22-kb nontransferable plasmid. The new MbetaL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an aacA4 cassette downstream of the MbetaL gene that appeared to be truncated with bla(GIM-1). The aacA4 was followed by an aadA1 gene cassette that was interrupted by a copy of the IS1394. This integron also carried an oxacillinase gene, bla(OXA-2), before the 3'-CS region. GIM-1 appears to be a unique MbetaL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MbetaL enzymes to be characterized.

Amino Acid Sequence↗
PubMed2004

Integron carrying a novel metallo-beta-lactamase gene, blaIMP-16, and a fused form of aminoglycoside-resistant gene aac(6')-30/aac(6')-Ib': report from the SENTRY Antimicrobial Surveillance Program.

Since January 2002 Pseudomonas sp. strains resistant to carbapenems and ceftazidime have been routinely screened as part of the SENTRY Antimicrobial Surveillance Program for metallo-beta-lactamase production, and their resistance determinants have been analyzed. Pseudomonas aeruginosa index strain 101-4704, which harbors a novel bla(IMP) variant, bla(IMP-16), was isolated in April 2002 from a 60-year-old man in Brasilia, Brazil. bla(IMP-16) was found on the chromosome of the P. aeruginosa index strain, and the deduced amino acid sequence (IMP-16) showed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%). Sequence analysis revealed that bla(IMP-16) was associated with a class 1 integron, which also encoded aminoglycoside-modifying enzymes. Downstream of bla(IMP-16) resided an open reading frame, which consisted of a new aminoglycoside-modifying gene, namely, aac(6')-30, which was fused with aac(6')-Ib'. The amino acid sequence of the aac(6')-30 putative protein showed the most identity (52.7%) to the sequence of AAC(6')-29b described previously. The fourth gene cassette constituted aadA1. The steady-state kinetics of IMP-16 demonstrated that the enzyme preferred cephalosporins and carbapenems to penicillins. The main functional difference observed among the kinetic values for IMP-16 compared to those for other IMPs was a lack of cefoxitin hydrolysis and a lower kcat/Km value for imipenem (0.36 microM(-1) . s(-1)). This report further emphasizes the spread of metallo-beta-lactamase genes and their close association with various aminoglycoside resistance genes.

Amino Acid Sequence↗
PubMed2004

Antimicrobial susceptibility and epidemiology of a worldwide collection of Chryseobacterium spp: report from the SENTRY Antimicrobial Surveillance Program (1997-2001).

Limited data are available on Chryseobacterium spp. leading to an evaluation of the patient demographics and susceptibility patterns for Chryseobacterium spp. collected in the first 5 years of the SENTRY Antimicrobial Surveillance Program (1997 to 2001). Fifty isolates (24 Chryseobacterium meningosepticum, 20 Chryseobacterium indologenes, two Chryseobacterium gleum, and 4 Chryseobacterium spp. isolates) were collected. The highest Chryseobacterium prevalence was detected among the elderly. The most active antimicrobials were the newer quinolones (garenoxacin, gatifloxacin, and levofloxacin, each with a MIC at which 90 percent of the isolates are inhibited [MIC(90)] of 1 micro g/ml and 98.0% susceptibility) followed by rifampin (MIC(90), 2 microg/ml and 85.7% susceptibility). Trimethoprim-sulfamethoxazole, ciprofloxacin, and piperacillin-tazobactam also showed reasonable activity; vancomycin showed poor potency.

Anti-Infective Agents↗
PubMed2004

Vancomycin susceptibility within methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) with reduced vancomycin susceptibility vancomycin-intermediate S. aureus (VISA) has been reported from many countries. Whether resistance is evolving regularly in different genetic backgrounds or in a single clone with a genetic predisposition, as early results suggest, is unclear. We have studied 101 MRSA with reduced vancomycin susceptibility from nine countries by multilocus sequence typing (MLST), characterization of SCCmec (staphylococcal chromosomal cassette mec), and agr (accessory gene regulator). We found nine genotypes by MLST, with isolates within all five major hospital MRSA lineages. Most isolates (88/101) belonged to two of the earliest MRSA clones that have global prevalence. Our results show that reduced susceptibility to vancomycin has emerged in many successful epidemic lineages with no clear clonal disposition. Increasing antimicrobial resistance in genetically distinct pandemic clones may lead to MRSA infections that will become increasingly difficult to treat.

Anti-Bacterial Agents↗
PubMed2003

Genetic characterization of a novel metallo-beta-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme.

OBJECTIVE: In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86-14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection. The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA. The resistance determinant was investigated. METHODS: The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach. RESULTS: Sequence analysis revealed the presence of a novel IMP-type metallo-beta-lactamase (MBL) gene, blaIMP-13. This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy). The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8. The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron. A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative. This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York. Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland. CONCLUSIONS: The resistance determinant of P. aeruginosa 86-14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.

Amino Acid Sequence↗
PubMed2003

Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program.

As part of the SENTRY Antimicrobial Surveillance Program, an imipenem-resistant Pseudomonas aeruginosa strain (81-11963A) was isolated from the blood culture of a female neonate institutionalized at the local children's hospital in Warsaw, Poland. Cloning of an imipenem resistance determinant revealed it to be a VIM-2 metallo-beta-lactamase, but sequence analysis of DNA adjacent to blaVIM-2 revealed it to have a unique gene context. Downstream of the blaVIM-2 gene resides an aacA4 gene encoding the AAC(6')-Ib aminoglycoside acetyltransferase. The integron containing blaVIM-2 shows high similarity to that reported from In58 in France but was novel in that it possessed a gene cassette with a 59 truncated base element only 19 base pairs (bp) long, consisting of a conserved core site and an inverse core site separated by only 5 bp. This appears to be the first report of a metallo-beta-lactamase gene arising from a pathogenic strain in Eastern Europe.

Base Sequence↗
PubMed2003

Genetic linkage of the penicillinase gene, amp, and blrAB, encoding the regulator of beta-lactamase expression in Aeromonas spp.

Aeromonas hydrophila T429125, a human clinical isolate, possesses three coordinately inducible beta-lactamases encoded by ampH (class D beta-lactamase), cepH (class C beta-lactamase) and imiH (class B beta-lactamase). We report that upstream of ampH there are two genes, blrA and blrB, encoding a putative two-component regulatory system. PCR studies revealed the same blrAB-amp gene arrangement in all Aeromonas spp. isolates tested; namely, Aeromonas veronii bv. sobria, Aeromonas jandaei, Aeromonas mediae, Aeromonas salmonicida and Aeromonas trota. A dominant mutation in the predicted BlrB kinase domain results in beta-lactamase overexpression in A. hydrophila T429125, but in other beta-lactamase-overexpressing mutants blrAB remains intact. Relative to the parent strain, A. hydrophila T429125, beta-lactamase- overexpressing mutants show a clear hierarchy of increased beta-lactamase expression: ImiH > CepH > AmpH. The same hierarchy is seen following beta-lactam challenge of A. hydrophila T429125, and correlates with the number of blr-tag sequences (TTCAC) found upstream of each beta-lactamase gene: ampH (one), cepH (two) and imiH (three).

Aeromonas↗