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Harris K.A.,Hospital NHS Foundation Trust | Kenna D.T.D.,Center for Infections | Blauwendraat C.,Hospital NHS Foundation Trust | Hartley J.C.,Hospital NHS Foundation Trust | And 3 more authors.
Journal of Clinical Microbiology | Year: 2012

Forty-one Mycobacterium abscessus complex isolates from 17 pediatric cystic fibrosis (CF) patients were typed using a novel variable-number tandem repeat (VNTR) scheme and an automated repetitive-PCR (rep-PCR) system. Both VNTR and rep-PCR typing methods differentiate between members of the M. abscessus complex. The isolates from individual patients are indistinguishable, and the data strongly suggest that individual CF patients are persistently infected with one strain and also suggests that different CF patients can harbor the same strain. Copyright © 2012, American Society for Microbiology. All Rights Reserved.


Espinel-Ingroff A.,Virginia Commonwealth University | Diekema D.J.,University of Iowa | Fothergill A.,University of Texas Health Science Center at San Antonio | Johnson E.,Center for Infections | And 5 more authors.
Journal of Clinical Microbiology | Year: 2010

Clinical breakpoints have not been established for mold testing. Wild-type (WT) MIC distributions (organisms in a species/drug combination with no detectable acquired resistance mechanisms) were defined in order to establish epidemiologic cutoff values (ECVs) for five Aspergillus spp. and itraconazole, posaconazole, and voriconazole. Also, we have expanded prior ECV data for Aspergillus fumigatus. The number of available isolates varied according to the species/triazole combination as follows: 1,684 to 2,815 for A. fumigatus, 323 to 592 for A. flavus, 131 to 143 for A. nidulans, 366 to 520 for A. niger, 330 to 462 for A. terreus, and 45 to 84 for A. versicolor. CLSI broth microdilution MIC data gathered in five independent laboratories in Europe and the United States were aggregated for the analyses. ECVs expressed in μg/ml were as follows (percentages of isolates for which MICs were equal to or less than the ECV are in parentheses): A. fumigatus, itraconazole, 1 (98.8%); posaconazole, 0.5 (99.2%); voriconazole, 1 (97.7%); A. flavus, itraconazole, 1 (99.6%); posaconazole, 0.25 (95%); voriconazole, 1 (98.1%); A. nidulans, itraconazole, 1 (95%); posaconazole, 1 (97.7%); voriconazole, 2 (99.3%); A. niger, itraconazole, 2 (100%); posaconazole, 0.5 (96.9%); voriconazole, 2 (99.4%); A. terreus, itraconazole, 1 (100%); posaconazole, 0.5 (99.7%); voriconazole, 1 (99.1%); A. versicolor, itraconazole, 2 (100%); posaconazole, 1 (not applicable); voriconazole, 2 (97.5%). Although ECVs do not predict therapy outcome as clinical breakpoints do, they may aid in detection of azole resistance (non-WT MIC) due to cyp51A mutations, a resistance mechanism in some Aspergillus spp. These ECVs should be considered for inclusion in the future CLSI M38-A2 document revision. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Johnson A.P.,Center for Infections | Davies J.,Center for Infections | Guy R.,Center for Infections | Abernethy J.,Center for Infections | And 4 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2012

Since 2001 it has been mandatory for acute hospital Trusts (groups of hospitals under the same management) in England to report all cases of bacteraemia due to Staphylococcus aureus together with information on their susceptibility or resistance to methicillin. This allowed the incidence of methicillin-resistant S. aureus (MRSA) bacteraemia (expressed as the number of cases per 1000 occupied bed days) to be determined for each Trust. In late 2005, the scheme was enhanced to collect demographic, clinical and epidemiological information on each case using a web-based data collection system. Analysis of this mandatory dataset has provided important information on the trends in MRSA bacteraemia in England and has documented a year-on-year decrease in incidence since 2006, following a government initiative in which Trusts were tasked with halving their MRSA bacteraemia rates over a 3year period. In addition, the enhanced mandatory surveillance scheme has captured a wealth of data that have helped to further define the epidemiology of MRSA bacteraemia. It is to be hoped that based on the English experience of mandatory surveillance, other countries will consider the implementation of similar schemes, not only for MRSA but for other pathogens of public health importance. © The Author 2012. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Abubakar I.,Center for Infections
International Journal of Tuberculosis and Lung Disease | Year: 2010

The UK has issued IGRA Guidelines by the National Institute for Health and Clinical Excellence (NICE), but the science is changing rapidly. The Health Protection Agency (HPA) has released a position statement to address some of the changes. The crux of the UK guidelines is a two-step testing approach, the basis of which is a cost-effectiveness analysis. All household contacts are investigated, irrespective of the disease site in the index case. The NICE guidelines call for an initial TST followed by an IGRA if the TST is positive (≥15 mm if the person had BCG or ≥5 mm if the person had not received BCG). The IGRA (if available) is then given if the TST is positive. Occasionally, wider screening beyond household contacts is required. Three examples were cited where the IGRA was successfully used for expanded investigations. Regarding TB immigrant screening, NICE recommends screening for latent infection only for those from countries in sub-Saharan Africa or a country with TB incidence >500/100 000. For those aged <16 or 16-35, if they have a chest X-ray that is normal, they are given a TST. A TST is also given to pregnant women. To address the issue of boosting, the HPA position statement now recommends IGRA at time of reading of the TST. There are other IGRA implementation issues, such as when to do the IGRA test following exposure and what should be the role for the test in children. While IGRAs are here to stay, it would be useful if we could develop studies on IGRA cost-effectiveness based on predictive value to help judge the test. The impact of this test is likely to be modest in England because of the low overall incidence. © 2010 The Union.


Ciplys E.,Vilnius University | Samuel D.,Center for Infections | Juozapaitis M.,Vilnius University | Sasnauskas K.,Vilnius University | Slibinskas R.,Vilnius University
Microbial Cell Factories | Year: 2011

Background: The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach.Results: Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response.Conclusions: Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway. © 2011 Čiplys et al; licensee BioMed Central Ltd.


Livermore D.M.,Center for Infections | Mushtaq S.,Center for Infections | Warner M.,Center for Infections | Zhang J.,Center for Infections | And 3 more authors.
Antimicrobial Agents and Chemotherapy | Year: 2011

Combinations of NXL104 with ceftazidime and aztreonam were tested against carbapenem-resistant members of the Enterobacteriaceae. Ceftazidime-NXL104 was active against strains with the OXA-48 enzyme or with combinations of impermeability and an extended-spectrum β-lactamase (ESBL) or AmpC enzyme and also against most Klebsiella spp. with the KPC enzyme, but metallo-β-lactamase producers were resistant. Aztreonam-NXL104 was active against all carbapenemase producers at 4 and 4 μg/ml, including those with metallo-β-lactamases. Copyright © 2011, American Society for Microbiology. All Rights Reserved.


Mushtaq S.,Center for Infections | Warner M.,Center for Infections | Livermore D.M.,Center for Infections
Journal of Antimicrobial Chemotherapy | Year: 2010

Background: NXL104 potentiates ceftazidime and ceftaroline against Enterobacteriaceae with extended-spectrum, AmpC, KPC and OXA β-lactamases. We examined whether similar potentiation was obtained against non-fermenters, which are less permeable than Enterobacteriaceae and have more potent efflux. Methods: MICs of ceftazidime+NXL104 (with NXL104 at 4 mg/L) and comparators were determined by CLSI agar dilution for: (i) Pseudomonas aeruginosa AmpC mutants and extended-spectrum β-lactamase (ESBL)-producing transconjugants; (ii) clinical P. aeruginosa isolates with AmpC enzymes, ESBLs or up-regulated efflux; (iii) P. aeruginosa and Burkholderia cepacia complex isolates from cystic fibrosis patients; and (iv) Acinetobacter baumannii with OXA carbapenemases, which also compromise ceftazidime. Results: NXL104 reversed AmpC-mediated ceftazidime resistance in P. aeruginosa, reducing MICs for fully derepressed mutants and isolates to ≤8 mg/L. NXL104 also reversed ceftazidime resistance caused by the ESBL PER-1, but not that due to OXA ESBLs or VEB-1 enzyme. Efflux-mediated resistance was unaffected. Resistance to ceftazidime in isolates of P. aeruginosa and the B. cepacia complex from patients with cystic fibrosis was variably overcome, generally to greater effect for B. cepacia. NXL104 had little effect on MICs of ceftazidime for A. baumannii isolates with OXA carbapenemases. Conclusions: The potentiation of ceftazidime against many β-lactamase-producing P. aeruginosa and B. cepacia complex strains confirms that NXL104 penetrates these organisms. The utility of the combination against these pathogens will depend on the local prevalence of strains with β-lactamase-versus efflux-mediated resistance. The lack of potentiation against A. baumannii may reflect failure of NXL104 to penetrate these bacteria to inhibit relevant (OXA-23, -40, -51 and -58) carbapenemases. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Livermore D.M.,Center for Infections | Mushtaq S.,Center for Infections | Warner M.,Center for Infections
Journal of Antimicrobial Chemotherapy | Year: 2010

Background: BAL30376 combines the siderophore monobactam BAL19764 (Syn/PTX 2416) with the bridged monobactam BAL29880 to inhibit AmpC enzymes and with clavulanate to inhibit extended-spectrum β-lactamases (ESBLs). We tested BAL30376 and its components versus isolates and laboratory strains of Enterobacteriaceae and non-fermenters. Methods: MICs were determined on Mueller-Hinton agar supplemented with 2,2′-bipyridyl to chelate Fe 3 + and induce TonB-mediated uptake. Results: Unprotected BAL19764 had MICs≤1 mg/L for most cephalosporin-susceptible Enterobacteriaceae, but values for a few isolates ranged up to 8 mg/L; its MICs were substantially raised for isolates with AmpC β-lactamases and ESBLs. Those of BAL30376 were ≤1 mg/L for 84% of ESBL producers and ≤4 mg/L for 85% of AmpC producers, excluding isolates with exceptional impermeability. Laboratory transformants with metalloor OXA-48 carbapenemases were susceptible to unprotected BAL19764, but many clinical isolates with these enzymes were resistant, probably having additional mechanisms; BAL30376, by contrast, was active at 4 mg/L versus 31/35 metallo-β-lactamase producers and 14/19 with OXA-48, although those with KPC carbapenemases were resistant. AmpC-mediated resistance to BAL19764 in Pseudomonas aeruginosa was overcome by BAL30376, as was that due to PER-1 enzyme; but MICs >16 mg/L were frequent for cystic fibrosis isolates. Many Burkholderia cepacia and carbapenemase-producing Acinetobacter baumannii were susceptible to BAL19764 and BAL30376 at ≤4 mg/L, but others were highly resistant, with MICs≥128 mg/L. Conclusions: BAL30376 overcomes most AmpC-, ESBL- and carbapenemase-mediated resistance in Enterobacteriaceae, though strains with KPC carbapenemases are resistant. It was active against many problem nonfermenters, though resistance was common in P. aeruginosa from cystic fibrosis. Raised MICs for some isolates were independent of β-lactamase. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Johnson A.P.,Center for Infections
Journal of Antimicrobial Chemotherapy | Year: 2011

Pan-European surveillance of bacteraemia caused by methicillin-resistant Staphylococcus aureus (MRSA) shows it to be a problem affecting all European countries, although there is marked geographical variation in prevalence. Although the proportion of S. aureus bacteraemia due to MRSA is declining in many countries, data from the European Antimicrobial Resistance Surveillance System (EARSS) for 2008 showed that in more than onethird of countries the proportion remained .25%. In contrast to bacteraemia, community-associated MRSA infection in Europe remains relatively uncommon. However, there appears to be an increasing problem involving transmission of MRSA (particularly sequence type 398) from colonized livestock, particularly pigs, to farm workers, abattoir workers and veterinarians who are in contact with such animals. Molecular analysis of isolates of MRSA has shown that there has been spread of only a limited number of MRSA clones in Europe and that many of these clones show geographical clustering due to dissemination through regional healthcare networks. Despite our increasing understanding of the epidemiology of MRSA in Europe, MRSA infections continue to pose a significant public health challenge. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Livermore D.M.,Center for Infections | Mushtaq S.,Center for Infections | Ge Y.,Calixa Therapeutics
Journal of Antimicrobial Chemotherapy | Year: 2010

Background: The developmental oxyimino-cephalosporin CXA-101 (FR264205) is notable for having greater antipseudomonal activity than ceftazidime. It is active against Enterobacteriaceae too, but is compromised by extended-spectrum, AmpC and carbapenem-hydrolysing β-lactamases. We investigated the tazobactam concentrations needed to potentiate this cephalosporin against strains with these mechanisms. Methods: MIC chequerboards were prepared between CXA-101 and tazobactam (1-32 mg/L) using CLSI agar dilution methodology and a challenge panel of 'difficult' Enterobacteriaceae isolates. Results: Only 20% of 59 extended-spectrum β-lactamase (ESBL) producers were susceptible to unprotected CXA-101 at 8 mg/L (5% at 2 mg/L), but 76% were susceptible to CXA-101+tazobactam at 8+4 mg/L and 93% at 8+8 mg/L. Among 20 AmpC-derepressed organisms, three of four Serratia spp. were susceptible to CXA-101 at 1-2 mg/L, but other species with the mechanism were more resistant; nevertheless, 70% were susceptible to CXA-101+tazobactam at 8+4 mg/L and 95% at 8+8 mg/L. The six least-susceptible AmpC-derepressed isolates were all Enterobacter spp. The MICs of CXA-101 for Klebsiella oxytoca isolates hyperproducing K1 enzyme were 4 mg/L and were not significantly reduced by tazobactam: those for Klebsiella pneumoniae with KPC enzymes were ≥128 mg/L and, in four out of five cases, were not significantly reduced by tazobactam. Conclusions: Tazobactam achieved concentration-dependent potentiation of CXA-101 versus ESBL producers and AmpC hyperproducers. If a breakpoint of 8+8 mg/L can be justified pharmacokinetically, CXA-101+ tazobactam should be active versus >90% of ESBL producers, AmpC hyperproducers and K1 hyperproducers. Most isolates with KPC or other carbapenemases will remain resistant. © The Author 2010. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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