Article

Diagnostic multiplex PCR assay for the identification of the Liverpool Midlands 1 and Manchester CF epidemic strains of

Division of Medical Microbiology, University of Liverpool, Daulby Street, Liverpool L69 3GA, United Kingdom.
Journal of Cystic Fibrosis (Impact Factor: 3.48). 06/2008; 7(3):258-61. DOI: 10.1016/j.jcf.2007.09.002
Source: PubMed

ABSTRACT

Individual PCR amplification tests have been developed for three UK CF epidemic strains, the Liverpool epidemic strain (LES), Midlands 1 and the Manchester epidemic strain (MES). We report a simple diagnostic multiplex PCR test that can be used to screen for all three of these strains. To evaluate the test, we screened collections of LES, MES and Midlands 1 isolates, along with various CF and non-CF non-epidemic Pseudomonas aeruginosa strains. The test was 100% sensitive and 100% specific in the identification of these UK CF epidemic strains.

Download full-text

Full-text

Available from: Craig Winstanley
  • Source
    • "The presence of OXA-type carbapenemases in A. baumannii was determined using the assay described by Woodford et al. [11] with the addition of bla OXA-143 specific primers [12], whilst other ␤-lactamase genes were sought using the series of multiplex PCR assays described by Dallenne et al. [13]. Clinical isolates of E. coli and P. aeruginosa were assigned to epidemic lineages using the PCR-based assays described by Clermont et al. [14] and Fothergill et al. [15] "
    [Show abstract] [Hide abstract]
    ABSTRACT: The problem of antimicrobial resistance is exemplified by multidrug-resistant (MDR) isolates of Gram-negative species. Of particular concern are expanded-spectrum cephalosporin-resistant isolates of Enterobacteriaceae, epidemic lineages of Acinetobacter baumannii producing OXA-type carbapenemases, and MDR Pseudomonas aeruginosa. In this study, the in vitro activity of the novel monosulfactam BAL30072 was investigated both alone and in combination with meropenem against a diverse collection of commonly encountered Gram-negative pathogens. Thirty-one isolates were studied, including type strains and clinical isolates with defined mechanisms conferring resistance to various antimicrobial agents including to carbapenems, colistin and tigecycline. BAL30072 minimum inhibitory concentrations (MICs) were determined in the presence and absence of meropenem (1:1, w/w) by agar dilution. Potential synergy or antagonism between BAL30072 and meropenem was investigated using standard chequerboard assays. Versus MDR A. baumannii strains producing class D oxacillinases, BAL30072 MICs were all ≤4mg/L with the exception of the isolate belonging to the UK 'Burn' lineage. BAL30072 exhibited MIC values of 0.5mg/L to >64mg/L towards the five P. aeruginosa strains. Against three meropenem-susceptible Escherichia coli, including the CTX-M-15 extended-spectrum β-lactamase-producer, BAL30072 exhibited MICs of 0.25-2mg/L; higher MICs were recorded against some of the Enterobacteriaceae isolates tested. The in vitro data suggest that BAL30072 has a potential role in the treatment of infections due to Gram-negative pathogens, including those with important resistances to other agents. In addition, BAL30072 shows powerful synergistic activity in combination with meropenem, potentially expanding its coverage for the treatment of infections caused by problematic species.
    Full-text · Article · Jul 2013 · International journal of antimicrobial agents
  • Source
    • "Accessory genome profiling. PS21 and LESF9 (both markers for the Liverpool strain) were sought using previously published primers (Fothergill et al., 2008). Some targets used in the ArrayTube (AT) microarray (Wiehlmann et al., 2007) (PA0636, PA0722, PA0728, PA2185, PA2221, PA3835 and orfl) were sought using primers described inTable 1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Variable Number Tandem Repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals and from over 2,000 patients between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. MLST and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70% by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of 'cluster A' of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x' (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.
    Full-text · Article · Apr 2013 · Journal of Medical Microbiology
  • Source
    • "2008–2009: In March 2008, by combining the specific primers and modifying the product mix we identified LES, Manchester and Midlands1 strains from one test (combined multiplex PCR).26 Primer F9 added to the PCR mix increased the specificity of the test to identify LES. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To assess if cohort segregation policies are effective in preventing cross-infection in cystic fibrosis (CF) clinics. A prospective cohort study. A large adult CF centre in Northwest England. All CF patients cared for at the Liverpool adult CF centre 2003-2009. Regular sputum sampling with genotyping of pseudomonas aeruginosa (Psa) isolates led to a policy of inpatient and outpatient segregation by microbiological group. Prevalence and cross-infection/super-infection rates of a transmissible Psa strain, i.e. the Liverpool epidemic strain (LES) in adult CF patients at the Liverpool adult CF centre from 2003 to 2009. There was a decline in the proportion of patients with LES (71-53%) and an increase in those with unique strains (23-31%) and without Psa infection (6-17%) from 2003 to 2009. There were two cases of LES super-infection and one case of new chronic Psa infection (with a unique strain). There were no cases of transmissible strain infection in patients previously uninfected by Psa. Our segregation policy has halted the spread of the commonest highly transmissible strain in the UK (LES) in our clinic, without endangering patients who were not previously infected with Psa. It confirms that if genotypic surveillance is used, it is unnecessary to segregate patients infected with unique strains from those without Psa infection.
    Full-text · Article · Jan 2013
Show more