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Biomedical subjects

T R Walsh

Publications and source records attributed to T R Walsh.

At least 19 recordsLinked to original sources
PubMed2003

Cefuroxime resistance in non-beta-lactamase Haemophilus influenzae is linked to mutations in ftsI.

The penicillin binding protein (PBP) genes dacA, dacB and ftsI from 14 cefuroxime-resistant (CXM(R)) isolates and three clinical isolates with low CXM MIC for non-beta-lactamase-producing Haemophilus influenzae type b were molecularly characterized. One strain, 5788, was used to transform H. influenzae Rd to CXM(R) for direct comparison of the pbps in the same genetic background. No obvious mutations in the dacA and dacB gene products could be associated with CXM(R). One amino acid substitution in the ftsI gene product in particular, S357N, could give rise to CXM(R). Sequence analysis from the CXM(R) transformants also implicated FtsI; in this case, the substitutions were V511A and R517H. To verify S357N substitution, the protein sequence of H. influenzae FtsI was threaded through the S. pneumoniae PBP 2X structure giving an average root mean square deviation of the alpha-carbon chains of 0.5 A. The S357N substitution alters both the residue size and charge. One explanation for the contribution of S357N to CXM(R) is that the asparagine side-chain produces unfavourable steric hindrance with the side chain of Val-362 changing the torsion angles of the asparagine residue, which in turn may influence the position of the loop V362-P366 adjacent to the active site. Whilst other groups have examined the contribution of H. influenzae PBPs in ampicillin resistance, this is the first report analysing their role in CXM(R).

Anti-Bacterial Agents↗
PubMed2002

Randomized clinical trial of magnesium, diazepam, or both after out-of-hospital cardiac arrest.

OBJECTIVE: To evaluate the feasibility, safety, and efficacy of interventions aimed at improving neurologic outcome after cardiac arrest. METHODS: The authors conducted a double-blind, placebo-controlled, randomized clinical trial with factorial design to see if magnesium, diazepam, or both, when given immediately following resuscitation from out-of-hospital cardiac arrest, would increase the proportion of patients awakening, defined as following commands or having comprehensible speech. If the patient regained a systolic blood pressure of at least 90 mm Hg and had not awakened, paramedics injected IV two syringes stored in a sealed kit. The first always contained either 2 g magnesium sulfate (M) or placebo (P); the second contained either 10 mg diazepam (D) or P. Awakening at any time by 3 months was determined by record review, and independence at 3 months was determined by telephone calls. Over 30 months, 300 patients were randomized in balanced blocks of 4, 75 each to MD, MP, PD, or PP. The study was conducted under waiver of consent. RESULTS: Despite the design, the four treatment groups differed on baseline variables collected before randomization. Percent awake by 3 months for each group were: MD, 29.3%; MP, 46.7%; PD, 30.7%; PP, 37.3%. Percent independent at 3 months were: MD, 17.3%; MP, 34.7%; PD, 17.3%; PP, 25.3%. Significant interactions were lacking. After adjusting for baseline imbalances, none of these differences was significant, and no adverse effects were identified. CONCLUSIONS: Neither magnesium nor diazepam significantly improved neurologic outcome from cardiac arrest.

Activities of Daily Living↗
PubMed2002

[Endemic heteroresistant glycopeptide intermediate Staphylcoccus aureus (hGISA) comprising unrelated clonal types and not associated with vancomycin therapy].

The detection of methicillin-resistant S. aureus (SA) (MRSA) refractory to glycopeptides is a serious clinical issue. The prevalence of hetero-resistant GISA (hGISA) strains at H. Maréchal Joffre, France is reported.858 non-repeat SA were isolated during 1999. 367 (43%) of these, from 257 patients, were MRSA (mean incidence 11.9/1000 admissions). All MSRA detected during 1999 were screened for vancomycin (VAN) resistance (BHI+4 mg/l VAN). Isolates recovered were retested using Etest strips (2 McFarland inoculum on BHI) and population analysis profile/area under the curve (PAP-AUC) analysis with hGISA SA Mu3 as a comparator. 58 selected strains were screened for teicoplanin resistance(TEI) using SFM recommended screen (2 McFarland inoculum on MH+5 mg/L TEI) and MIC (0.5 MF inoculum swabbed on MH agar) methods. 188 (51.3%) grew on VAN screen agar (6.1/1000 admissions). 58 strains (7.6%) possessed Etest VAN MIC > 8 mg/l all others being VAN < 8 mg/l. Of these 58 isolates, 10 were stably heterogeneously resistant to both VAN and teicoplanin (MIC > 8 mg/l). PAP-AUC showed 12 strains to have PAP-AUC ratios > 0.95 but < 1.5 (ie. hGISA, not GISA). All 7 isolates defined as hGISA by both Etest and PAP-AUC comprised 1 PFGE clone (< 3 bands difference). Additionally 2 distinct PFGE types were detected among the other 5 hGISA identified PAP-AUC. The 12 hGISAs, were derived from 12 patients with severe underlying disease. None were on glycopeptide therapy prior to hGISA isolation. This is the first report of endemic hGISA, comprising 3 clonal types. The isolation of hVISA seems not to be associated with patient-specific glycopeptide therapies.

Anti-Bacterial Agents↗
PubMed2001

A novel metallo-beta-lactamase, Mbl1b, produced by the environmental bacterium Caulobacter crescentus.

Caulobacter crescentus 101123 possesses a gene (Mbl1b) encoding a metallo-beta-lactamase with 32% amino acid identity to the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia. The gene was cloned into an expression vector and the enzyme, Mbl1b, was expressed in Escherichia coli. Mbl1b was purified. Catalytic properties for several antibiotics were determined. The enzyme exhibits Michaelis-Menten kinetics for imipenem, meropenem and nitrocefin but substrate inhibition kinetics with cefoxitin, cefaloridine, penicillin G and ampicillin. A homology model predicts Mbl1b has the same structural fold as other metallo-beta-lactamases with a detailed structure very similar to L1 but whereas L1 is a homotetramer, Mbl1b is monomeric. The main differences between Mbl1 and L1 are in the N-terminal region.

Amino Acid Sequence↗
PubMed2001

Novel mechanism of hydrolysis of therapeutic beta-lactams by Stenotrophomonas maltophilia L1 metallo-beta-lactamase.

Stopped-flow tryptophan fluorescence under single turnover and pseudo-first-order conditions has been used to investigate the kinetic mechanism of beta-lactam hydrolysis by the Stenotrophomonas maltophilia L1 metallo-beta-lactamase. For the cephalosporin substrates nitrocefin and cefaclor and the carbapenem meropenem, a substantial quench of fluorescence is observed on association of substrate with enzyme. We have assigned this to a rearrangement event subsequent to formation of an initial collision complex. For the colorimetric compound nitrocefin, decay of this dark inter- mediate represents the overall rate-determining step for the reaction and is equivalent to decay of a previously observed state in which the beta-lactam amide bond has already been cleaved. For both cefaclor and meropenem, the rate-determining step for hydrolysis is loss of a second, less quenched state, in which, however, the beta-lactam amide bond remains intact. We suggest, therefore, that the mechanism of hydrolysis of nitrocefin by binuclear metallo-beta-lactamases may be atypical and that cleavage of the beta-lactam amide bond is the rate-determining step for breakdown of the majority of beta-lactam substrates by the L1 enzyme.

Catalysis↗
PubMed2001

Escherichia coli CreBC is a global regulator of gene expression that responds to growth in minimal media.

We have identified nine genes, the expression of which are regulated by the CreBC two-component system: the first members of the cre regulon. They are divided into eight transcriptional units, each having a promoter-proximal TTCACnnnnnnTTCAC "cre-tag" motif. The cre regulon genes are: the ackA/pta operon, the products of which collectively catalyze the conversion of acetyl-CoA into acetate and ATP; talA, which encodes an enzyme involved in the mobilization of glyceraldehyde-3-phosphate into the pentose phosphate pathway; radC, which encodes a RecG-like DNA recombination/repair function; malE, which is the first gene in the malEFG maltose transporter operon; trgB, which encodes an ADP-ribose pyrophosphorylase; and three other genes, creD, yidS and yieI, the products of which have not been assigned a function. Expression of each of these cre regulon genes is induced via CreBC during growth in minimal media, with the exception of malE, which is more tightly repressed. The diverse functions encoded by the cre regulon suggest that CreBC is a global regulator that sits right at the heart of metabolic control in Escherichia coli.

Aeromonas↗
PubMed2001

pUb6060: a broad-host-range, DNA polymerase-I-independent ColE2-like plasmid.

A ColE2-like, cryptic plasmid, pUB6060, of 5.8 kb has been found in a clinical isolate of Plesiomonas shigelloides. The complete sequence of pUB6060 has been determined and reveals a number of interesting features about the plasmid. The ColE2-like replication locus is linked to a functional ColE1-like mobilization locus. Replication is, unusually for ColE2 replicons, DNA polymerase-I-independent and may involve two, rather than the usual one, plasmid-encoded functions. Additionally, it carries two ORFs encoding products of unknown function. The pUB6060 replicon maintains a moderate plasmid copy number (10 per chromosome copy) and permits replication in diverse gram-negative bacteria.

Base Sequence↗
PubMed2001

The problem of a solvent exposable disulfide when preparing Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

In an effort to prepare Co(II)-substituted metallo-beta-lactamase L1 from Stenotrophomonas maltophilia for future spectroscopic and mechanistic studies, two methods for the preparation of Co(II)-L1 were tested. Method A involved adding CoCl2 directly to apo-L1 under anaerobic conditions. The resulting enzyme contained 1.9 moles of cobalt and exhibited very little activity, and UV-Vis, 1H NMR, and EPR studies indicated that most of the cobalt in this sample was Co(III). Method B involved reducing the single and unique disulfide bridge in L1 with tris(carboxyethyl)phosphine prior to adding CoCl2. The resulting enzyme was pink, contained 2.5 moles of cobalt per mole of enzyme, and exhibited kcat and Km values of 18+1 s(-1) and 10+/-1 microM, respectively, when using nitrocefin as the substrate. UV-Vis, 1H NMR, and EPR studies revealed that this enzyme sample contained high-spin Co(II). The UV-Vis spectra also provided evidence for Co(II) bound to one or both of the reduced cysteines. Efforts to block this non-specific Co(II) binding site using a chemical modification agent or site-directed mutagenesis were unsuccessful. The data presented here demonstrate the problem of solvent-exposed disulfides when preparing Co(II)-substituted enzymes and offers a convenient method to circumvent the problem.

Base Sequence↗
PubMed2001

Use of antimicrobial agents in veterinary medicine and food animal production.

Antimicrobial resistance is a growing area of concern in both human and veterinary medicine. This review presents an overview of the use of antimicrobial agents in animals for therapeutic, metaphylactic, prophylactic and growth promotion purposes. In addition, factors favouring resistance development and transfer of resistance genes between different bacteria, as well as transfer of resistant bacteria between different hosts, are described with particular reference to the role of animals as a reservoir of resistance genes or resistant bacterial pathogens which may cause diseases in humans.

Animal Diseases↗
PubMed2001

Resistance to antibiotics and biocides among non-fermenting Gram-negative bacteria.

OBJECTIVE: To investigate the antibiotic and biocide susceptibilities of clinical isolates of rarely encountered Gram-negative, non-fermenting bacteria. METHODS: Thirty Gram-negative non-fermenting bacterial strains were isolated from blood cultures of oncology patients. These were studied for their resistance to 11 antibiotics. Their susceptibilities to seven biocides used in hospitals were also examined. RESULTS: Isolates of Stenotrophomonas maltophilia and Ochrobactrum anthropi were generally resistant to at least five of the antibiotics, whereas isolates of Comamonas acidivorans, Flavobacterium oryzihabitans, Aeromonas hydrophila, Sphingobacterium spiritivorum, Acinetobacter junii and Acinetobacter lwoffi were generally sensitive to at least nine of the antibiotics. Trovafloxacin and trimethoprim-sulfamethoxazole were the most effective antibacterial agents tested, with 0% and 7%, respectively, of isolates being resistant, whereas 63% of isolates were resistant to aztreonam. Some isolates, sensitive to meropenem and/or ceftazidime in vitro, possessed very high MBC/MIC ratios for these beta-lactams. Two out of three biocides used in hospital pharmacies showed lethal activity towards all strains tested when used at less than one-third of their recommended in-use concentration. Proceine 40 failed to give a 5 log reduction in bacterial cell number for the isolates tested when used at its "in-use" concentration. A concentration of > 500 mg/L chlorhexidine was required to achieve a 5 log reduction for the same isolates. CONCLUSIONS: We have examined the antibiotic susceptibilities of non-fermenting Gram-negative bacterial strains isolated from immunocompromised patients. Despite being sensitive to certain antibiotics in vitro, some isolates were still able to cause serious bacteremia. We have also reported for the first time the susceptibilities of non-fermenting Gram-negative bacteria to common biocides used in hospital infection control, and have shown that some strains are able to persist at the "in-use" concentration of particular biocides. It is therefore important to study further this particular group of organisms, and, in particular, to examine whether there exists a link between resistance to antibiotics and resistance to biocides.

Anti-Bacterial Agents↗
PubMed2001

A modified population analysis profile (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital.

One hundred methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated between 1983 and 1999, were tested alongside the vancomycin hetero-resistant S. aureus (hVRSA) strain Mu 3, and vancomycin-resistant S. aureus (VRSA) strain Mu 50, for their resistance to vancomycin. This was achieved using the screening method described by Hiramatsu, gradient plates, agar incorporation, standard Etest, macrodilution Etest and a modified population analysis. Using Hiramatsu's screening method, 5% of the 100 MRSA were identified as VRSA and 5% identified as hVRSA, the gradient plates identified 7% hVRSA, and the standard and macrodilution Etests identified no hVRSA. Mu 3 appeared to be vancomycin-susceptible using both the agar incorporation and standard Etest methods, but was classified as hVRSA using the macrodilution Etest. The modified population analysis reliably detected vancomycin hetero-resistance in Mu 3 and identified no hVRSAs within the 100 MRSA sample.

Hospitals↗
PubMed2001

The penetration of ceftriaxone and cefamandole into bone, fat and haematoma and relevance of serum protein binding to their penetration into bone.

Thirteen patients undergoing total hip replacement were given ceftriaxone 1 g and cefamandole 1 g simultaneously, either immediately or 8 h before surgery. For both agents the concentrations seen in the bone and fat during the operation, and for haematoma fluid <or=24 h after the operation, exceeded the MIC for susceptible staphylococci and would be expected to provide adequate prophylaxis. Although ceftriaxone concentrations in bone and fat were significantly higher than those of cefamandole, after correction for the simultaneous blood concentrations, we were unable to detect any differences in the bone or fat penetration of the two agents. We conclude that there is no evidence that cephalosporins with a lower serum protein binding penetrate bone better than ones with higher serum protein binding.

Adipose Tissue↗
PubMed2001

Carriage of antibiotic-resistant bacteria by healthy children.

The frequency of carriage of antibiotic-resistant bacteria in healthy 7- and 8-year-old children in Bristol was studied. Children born in Avon between 1 April 1991 and 31 December 1992, attending the Avon Longitudinal Study of Pregnancy and Childhood (ALSPAC) 7 year follow-up clinic, formed the study population. Carriage was estimated using mouth and stool samples. None of 105 children on whom information was available had received tetracycline, chloramphenicol, ciprofloxacin or an extended-spectrum cephalosporin in the previous year. Staphylococcus aureus was isolated from mouthwashes from 200 (37.1%) of 539 children sampled. Six (3%) of the isolates were resistant to chloramphenicol or tetracycline and four (2%) were methicillin resistant. Haemophilus spp. were isolated from 369 (72%) of 513 samples and 63 (17%) were ampicillin resistant, 49 (13.3%) were erythromycin resistant and seven (1.9%) were tetracycline resistant. Branhamella catarrhalis was isolated from 333 (74%) of 450 samples. Twenty-eight (8.4%) were erythromycin resistant and 14 (4.2%) strains were tetracycline resistant. Group A beta-haemolytic streptococci were isolated from 17 of 507 children sampled. One (5.9%) was tetracycline resistant. Stool samples were returned from 335 (62%) of 539 children from whom they were requested. Eleven per cent of samples yielded Gram-negative bacilli with high-level resistance to chloramphenicol, which was frequently linked to resistance to ampicillin, spectinomycin and streptomycin. Isolates demonstrating resistance to the third-generation cephalosporin ceftazidime were recovered from 17 subjects (3.2%). Six (35%) of 17 isolates possessed extended-spectrum beta-lactamases. Healthy children carry bacteria resistant to antibiotics to which children are not usually exposed. Resistance to ceftazidime, chloramphenicol and tetracycline may be co-selected by exposure to other antibiotics used in children or may be acquired from family members, pets, other children or food. These results suggest that antibiotic-resistant bacteria are widely disseminated and may be acquired by children before exposure to specific selection pressure.

Bacteria↗
PubMed2001

Characterization, cloning and sequence analysis of the inducible Ochrobactrum anthropi AmpC beta-lactamase.

Ochrobactrum anthropi is resistant to most cephalosporins and penicillins due, at least in part, to the inducible expression of a single beta-lactamase. The beta-lactamase gene has been cloned and sequenced. It encodes an AmpC-type class 1 serine active-site enzyme that hydrolyses mainly cephalosporins and is resistant to inhibition by clavulanic acid. Expression of the ampC gene is inducible via a typical AmpR regulator, which is encoded upstream of ampC. Inducible expression is retained following cloning of O. anthropi ampR-ampC into Escherichia coli, confirming that the signal for AmpR activation in O. anthropi is the same as that used in the Enterobacteriaceae. This is the first reported example of an AmpC beta-lactamase outside of the gamma-subdivision of the bacterial kingdom. Genomic searches of other non-gamma-subdivision bacteria revealed a homologous ampR-ampC cluster in the plant symbiont, Sinorhizobium meliloti.

Amino Acid Sequence↗
PubMed2001

The activity of vancomycin against heterogeneous vancomycin-intermediate methicillin-resistant Staphylococcus aureus explored using an in vitro pharmacokinetic model.

Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) may account for treatment failure with vancomycin and act as a precursor of vancomycin-intermediate or -resistant S. aureus. The activity of vancomycin was assessed against vancomycinsusceptible, hVISA and VISA strains in a dilutional pharmacokinetic model. Over a 48 h period, total bacteria and cells with a vancomycin-intermediate phenotype were quantified. Total counts of hVISA were reduced by vancomycin in a similar way to a vancomycin-susceptible control. The vancomycin-intermediate sub-population was eradicated from the model within one dose interval. Exposure to low vancomycin concentrations did not result in an increase in the proportion of cells which were vancomycin intermediate. Short-term exposure of hVISA to vancomycin at gradient concentrations did not increase the proportion of cells with vancomycin-intermediate phenotype.

Anti-Bacterial Agents↗
PubMed2001

Plasmid location and molecular heterogeneity of the L1 and L2 beta-lactamase genes of Stenotrophomonas maltophilia.

An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 beta-lactamase genes. The location of L1 and L2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes, L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1 (L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lower k(cat)/K(m) ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, and L2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275 L2 (L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.

Alleles↗