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N Woodford

Publications and source records attributed to N Woodford.

At least 19 recordsLinked to original sources
PubMed2007

The emergence of antibiotic resistance by mutation.

The emergence of mutations in nucleic acids is one of the major factors underlying evolution, providing the working material for natural selection. Most bacteria are haploid for the vast majority of their genes and, coupled with typically short generation times, this allows mutations to emerge and accumulate rapidly, and to effect significant phenotypic changes in what is perceived to be real-time. Not least among these phenotypic changes are those associated with antibiotic resistance. Mechanisms of horizontal gene spread among bacterial strains or species are often considered to be the main mediators of antibiotic resistance. However, mutational resistance has been invaluable in studies of bacterial genetics, and also has primary clinical importance in certain bacterial species, such as Mycobacterium tuberculosis and Helicobacter pylori, or when considering resistance to particular antibiotics, especially to synthetic agents such as fluoroquinolones and oxazolidinones. In addition, mutation is essential for the continued evolution of acquired resistance genes and has, e.g., given rise to over 100 variants of the TEM family of beta-lactamases. Hypermutator strains of bacteria, which have mutations in genes affecting DNA repair and replication fidelity, have elevated mutation rates. Mutational resistance emerges de novo more readily in these hypermutable strains, and they also provide a suitable host background for the evolution of acquired resistance genes in vitro. In the clinical setting, hypermutator strains of Pseudomonas aeruginosa have been isolated from the lungs of cystic fibrosis patients, but a more general role for hypermutators in the emergence of clinically relevant antibiotic resistance in a wider variety of bacterial pathogens has not yet been proven.

Anti-Bacterial Agents↗
PubMed2006

Molecular characterization of plasmids encoding CTX-M-15 beta-lactamases from Escherichia coli strains in the United Kingdom.

OBJECTIVES: The UK, like other countries worldwide, has a growing problem with CTX-M beta-lactamase-producing Escherichia coli. Five major clonally related strains have been identified among CTX-M-15 producers. We characterize here the plasmids from clonal strains A and D. METHODS: Plasmids were extracted and transformed into E. coli DH5alpha; conjugative mating was attempted on agar. MICs were determined by agar dilution. beta-Lactamases were typed by isoelectric focusing; antibiotic resistance genes and integrons were identified by PCR and sequenced. Plasmid incompatibility groups were determined by replicon PCR. RESULTS: bla(CTX-M-15) was carried by a 150 kb plasmid in strain A and a 70 kb plasmid in strain D. Conjugative transfer of cefotaxime resistance was only achieved from strain D; plasmids from both strains were transferred by transformation. The plasmid from strain A additionally carried bla(TEM-1) (variably), bla(OXA-1), aac(6')-Ib-cr and tet(A), as well as a class 1 integron with the gene cassettes aadA5 and dfr(17); the plasmid from strain D carried bla(TEM-1) consistently, also bla(OXA-1), aac(6')-Ib-cr, aac3-IIa and tet(A). Both plasmids belonged to incompatibility group FII. CONCLUSIONS: bla(CTX-M-15) was plasmid-mediated in both strains A and D and was linked to other antibiotic resistance genes including aac(6')-Ib-cr, which encodes an acetyltransferase, not previously found in Europe, acting on both aminoglycosides and some fluoroquinolones. Although the plasmids from the two strains differed in size, both were related and conferred similar multi-drug resistance phenotypes, suggesting that they may share a similar genetic scaffold. Both shared features with plasmids encoding CTX-M-15 beta-lactamases in E. coli from Canada and India.

Anti-Bacterial Agents↗
PubMed2006

Antimicrobial susceptibility and molecular analysis of Enterococcus faecalis originating from endodontic infections in Finland and Lithuania.

BACKGROUND/AIMS: Enterococcus faecalis strains with multiple antibiotic resistances can cause infections that are difficult to treat. The microbial flora in treatment-resistant apical periodontitis is dominated by E. faecalis, and is a potential source of infections at other sites. MATERIAL AND METHODS: Sensitivities to a range of antibiotics were determined for 59 endodontic E. faecalis isolates from Finland and Lithuania. The DNA sequence of the gene responsible for the species' intrinsic quinupristin-dalfopristin resistance, lsa, was determined from two isolates with diminished resistance. Four pairs of isolates from the same root canal were typed by pulsed-field gel electrophoresis. RESULTS: A high prevalence of resistance to rifampicin was found, whereas all isolates were susceptible or showed intermediate susceptibility to penicillin and ampicillin and four isolates were unusually susceptible to cefotaxime. No vancomycin or high-level gentamicin resistance was detected. Nine of 59 isolates were susceptible to quinupristin-dalfopristin. A fully quinupristin-dalfopristin-susceptible isolate also susceptible to clindamycin produced a truncated Lsa polypeptide, and an isolate with borderline quinupristin-dalfopristin-susceptibility had mutations proximal to the predicted ribosomal binding site. Pulsed-field gel electrophoresis showed that the same root canal could harbor two different strains of E. faecalis during the course of the same infection. CONCLUSION: Despite the differing antibiotic usage in Finland and Lithuania, E. faecalis from endodontic infections in these countries showed similar susceptibility patterns with levels of resistance considered typical for the species, and decreased resistance to clindamycin and quinupristin-dalfopristin as well as lesions in the lsa gene which were similar to those described in other clinical isolates.

ATP-Binding Cassette Transporters↗
PubMed2006

Development of high-level ceftazidime resistance via single-base substitutions of blaCTX-M-3 in hyper-mutable Escherichia coli.

Mutations can increase the ceftazidimase activity of CTX-M-3 beta-lactamase, as seen with its widespread variant CTX-M-15. This study compared the frequencies of emerging ceftazidime resistance in isogenic wild-type and hyper-mutable mutS CTX-M-3-producing Escherichia coli strains, and sequenced the mutant bla(CTX-M) alleles selected. Ceftazidime resistance emerged more readily in the hyper-mutable background than in the wild-type strain. All selected CTX-M mutants, in both the wild-type and the mutS derivatives, had single amino-acid changes at position 167, including a novel Pro167Gln substitution. These data emphasise the potential for further diversification of CTX-M enzymes.

Anti-Bacterial Agents↗
PubMed2006

Development of extended-spectrum activity in TEM beta-lactamases in hyper-mutable, mutS Escherichia coli.

TEM-1 and TEM(pUC19)beta-lactamases can gain activity against ceftazidime and other expanded-spectrum cephalosporins via point mutation. The frequency of emergent resistance to ceftazidime at 4 x MIC was elevated >or= 250-fold in hyper-mutable, MutS-deficient Escherichia coli harbouring these beta-lactamase genes on high- or low-copy plasmids. Moreover, although ceftazidime-resistant mutants, or those with reduced susceptibility, were selected in both the wild-type and mutS hosts, many more mutants in the mutS host showed ceftazidimase-type extended-spectrum beta-lactamase (ESBL) activity. This correlated with a G-A point mutation at position 484 in the bla(TEM-1) and bla(TEM-pUC19) genes, conferring the Arg164His amino-acid substitution found in the TEM-29 ESBL. Non-ESBL mutants lacked changes in bla(TEM).

Ceftazidime↗
PubMed2005

Biological counterstrike: antibiotic resistance mechanisms of Gram-positive cocci.

The development of antibiotic resistance by bacteria is an evolutionary inevitability, a convincing demonstration of their ability to adapt to adverse environmental conditions. Since the emergence of penicillinase-producing Staphylococcus aureus in the 1940s, staphylococci, enterococci and streptococci have proved themselves adept at developing or acquiring mechanisms that confer resistance to all clinically available antibacterial classes. The increasing problems of methicillin-resistant S. aureus and coagulase-negative staphylococci (MRSA and MRCoNS), glycopeptide-resistant enterococci and penicillin-resistant pneumococci in the 1980s, and recognition of glycopeptide-intermediate S. aureus in the 1990s and, most recently, of fully vancomycin-resistant isolates of S. aureus have emphasised our need for new anti-Gram-positive agents. Antibiotic resistance is one of the major public health concerns for the beginning of the 21st century. The pharmaceutical industry has responded with the development of oxazolidinones, lipopeptides, injectable streptogramins, ketolides, glycylcyclines, second-generation glycopeptides and novel fluoroquinolones. However, clinical use of these novel agents will cause new selective pressures and will continue to drive the development of resistance. This review describes the various antibiotic resistance mechanisms identified in isolates of staphylococci, enterococci and streptococci, including mechanisms of resistance to recently introduced anti-Gram-positive agents.

Aminoglycosides↗
PubMed2004

Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum beta-lactamases in the UK.

OBJECTIVES: During 2003, the Health Protection Agency's Antibiotic Resistance Monitoring and Reference Laboratory began to receive isolates of Escherichia coli for confirmation of extended-spectrum beta-lactamase production with a phenotype implying a CTX-M-type beta-lactamase, i.e. MICs of cefotaxime > or = 8-fold higher than MICs of ceftazidime. Many were referred as being from community patients. We examined 291 CTX-M-producing isolates from the UK and investigated the genetic basis of their phenotype. METHODS: PCR was used to detect alleles encoding CTX-M enzymes and to assign these to their blaCTX-M phylogenetic groups. Selected alleles were sequenced. Producers were compared by analysis of banding patterns generated by pulsed-field gel electrophoresis of XbaI-digested genomic DNA. MICs were determined by an agar dilution method or by Etest. RESULTS: Of 291 CTX-M-producing E. coli isolates studied from 42 UK centres, 70 (24%) were reportedly from community patients, many of whom had only limited recent hospital contact. Community isolates were referred by 12 centres. Two hundred and seventy-nine (95.9%) producers contained genes encoding group 1 CTX-M enzymes and 12 contained blaCTX-M-9-like alleles. An epidemic CTX-M-15-producing strain was identified, with 110 community and inpatient isolates referred from six centres. Representatives of four other major strains also produced CTX-M-15, as did several sporadic isolates examined. Most producers were multi-resistant to fluoroquinolones, trimethoprim, tetracycline and aminoglycosides as well as to non-carbapenem beta-lactams. CONCLUSIONS: CTX-M-producing E. coli are a rapidly developing problem in the UK, with CTX-M-15 particularly common. The diversity of producers and geographical scatter of referring laboratories indicates wide dissemination of blaCTX-M genes. Because of the public health implications, including for the treatment of community-acquired urinary tract infections, the spread of these strains--and CTX-M-15 beta-lactamase in particular--merits close monitoring.

Alleles↗
PubMed2004

Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-beta-lactamase, in a tertiary care center in Cali, Colombia.

The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and bla(IMP) and bla(VIM) genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 microg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with bla(VIM)-specific primers, and sequencing of DNA from a representative isolate revealed bla(VIM-8), a novel allele with three polymorphisms compared with the sequence of bla(VIM-2). Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried bla(VIM) alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, < or =32 microg/ml) which was not significantly reduced by EDTA. No bla(IMP) alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with bla(VIM) were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.

Adult↗
PubMed2002

Emerging linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolated from two Austrian patients in the same intensive care unit.

Of two patients in the same intensive care unit who were treated with linezolid, one yielded linezolid-resistant Enterococcus faecalis, whereas the other yielded linezolid-resistant Enterococcus faecium. In each case, molecular typing indicated that the resistant isolates were related to linezolid-susceptible isolates from the same patient, but differed from them by the same G2576U ribosomal RNA mutation. This is the first clinical case report of emerging resistance to linezolid among Enterococcus faecalis and also the first report of resistance involving vancomycin-susceptible rather than vancomycin-resistant enterococci. The linezolid-resistant isolates showed cross-resistance to the experimental oxazolidinone AZD2563, suggesting that oxazolidinone resistance might be a class effect.

Acetamides↗
PubMed2002

Endemic carbapenem resistance associated with OXA-40 carbapenemase among Acinetobacter baumannii isolates from a hospital in northern Spain.

Eighty-two carbapenem-resistant isolates of Acinetobacter baumannii from a single hospital in Bilbao were typed into two major clusters and several subclusters. Disk synergy tests and PCR indicated the production of a zinc-independent OXA-class carbapenemase. Sequencing identified this enzyme, OXA-40, as a variant of the OXA-24-OXA-25-OXA-26 cluster.

Acinetobacter Infections↗
PubMed2001

Analysis of the expression pattern of Ebp1, an ErbB-3-binding protein.

Ebp1, a member of the PA2G4 family, was isolated as an ErbB-3-binding protein in our laboratory using yeast two hybrid analysis. Although Ebp1 mRNA is ubiquitously expressed, little is known about either the expression of Ebp1 protein in vivo or its translation initiation site. Western blotting analysis of a wide range of cell lines and primary tissue indicated that in the majority of cases Ebp1 is expressed as a single protein which migrates at 48 kDa in SDS-polyacrylamide gels. We show using epitope-tagged expression constructs that the second, not the first, in-frame ATG is used for the initiation of translation of the endogenous protein, encoding a protein predicted to be 41.5 kDa. The molecular mass of endogenous Ebp1 protein derived from mouse liver and brain was determined by mass spectrometry and the data confirm that translation of endogenous Ebp1 in tissues is initiated from the second in-frame ATG.

Adaptor Proteins, Signal Transducing↗
PubMed2001

A rapid molecular assay for the detection of antibiotic resistance determinants in causal agents of infective endocarditis.

AIMS: To develop and employ a PCR amplification system, directly from clinical specimens, for the rapid molecular detection of common antimicrobial resistance genes for streptococci, staphylococci and enterococci organisms causing infective endocarditis (IE). METHODS AND RESULTS: Eleven antibiotic resistance genes were targeted by PCR along with four identification-related loci. Blood culture and heart valve material from staphylococcal endocarditis patients were directly examined for methicillin resistance. PCR conditions were optimized for the following antibiotic resistance loci: staphylococci (mecA, aacA-aphD), streptococci (PBP 1A, PBP 2B, gyrB, parE) and enterococci (vanA, vanB, vanC-1, vanC-2, aacA-aphD, aphA3). The presence of methicillin resistance was confirmed in one of the eight IE patients examined. CONCLUSION: This study presents a PCR amplification system for the detection of antibiotic resistance genes. Detection of such genes may indicate susceptibility of the causal agents of IE to commonly prescribed antimicrobial agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid detection of antibiotic resistant organisms may reduce the use of inappropriate antibiotic agents or enable the use of the most appropriate combinations of antibiotics, other than those that would normally be prescribed empirically for IE. Such a method may be particularly valuable in cases of culture-negative endocarditis. Detection of antibiotic resistance genes by molecular-based techniques, namely PCR, will allow more directed antibiotic therapy and may also provide opportunities for earlier identification of resistant organisms.

Drug Resistance, Microbial↗