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Molecular detection of antibiotic resistance Katie L Hopkins PhD Laboratory of Gastrointestinal Pathogens HPA Microbiology Services.

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Presentation on theme: "Molecular detection of antibiotic resistance Katie L Hopkins PhD Laboratory of Gastrointestinal Pathogens HPA Microbiology Services."— Presentation transcript:

1 Molecular detection of antibiotic resistance Katie L Hopkins PhD katie.hopkins@hpa.org.uk Laboratory of Gastrointestinal Pathogens HPA Microbiology Services Colindale 20 th May 2011

2 Overview  Methods used for molecular detection of antibiotic resistance: In reference labs Commercially available systems  Considerations when choosing a molecular assay: What are the advantages over phenotypic susceptibility testing? What are the limitations?

3 Antimicrobial susceptibility testing  Antimicrobial susceptibility testing a core function of diagnostic labs.  Interpretation of R-patterns can suggest the underlying mechanisms.  Limitations: Time delay due to requirement for pure culture. May be affected by experimental conditions. No international consensus on methodology or interpretive criteria. Low-level resistance can be difficult to detect.  Rapid and reliable tests even more important with emergence of MDR organisms.

4 Resistance at the molecular level  Genetic basis for antimicrobial resistance includes: 1. Acquisition and expression of new DNA by horizontal transfer. 2. mutations in genes that alter targets or affect gene expression.  Informed development of methods: PCR. Hybridisation. Sequencing. Sundsfjord et al. 2004

5 Services at Colindale Services currently offered by ARMRL include detection of:  mecA in S. aureus with borderline methicillin/oxacillin resistance.  mupA in mupirocin-resistant S.aureus.  23S rRNA mutations responsible for linezolid resistance in enterococci, staphylococci or streptococci.  Genes conferring quinupristin/dalfopristin resistance in enterococci or staphylococci.  Genes encoding carbapenemases in Acinetobacter, Enterobacteriaceae* or Pseudomonas spp. (*Send Salmonellae, Shigellae to Laboratory of Gastrointestinal Pathogens).  Genes encoding acquired (plasmid-mediated) AmpC β-lactamases in E. coli and Klebsiella spp. resistant to cephalosporins, but with no synergy with clavulanic acid. Services offered by LGP:  PCR detection of CLA and TET resistance in H. pylori from culture-negative gastric biopsies.  Investigation of the genetic basis of antibiotic resistance in enteric bacteria. Typically acquired AmpC or ESBL confirmation.

6 “Conventional” PCR Acquired OXA carbapenemases in Acinetobacter (Woodford et al. 2006; Higgins et al. 2010) } Intrinsic to A. baumannii  Most commonly applied technique.  Amplification targets conserved or variable regions within gene of interest.  Separate post-PCR detection – usually agarose gel electrophoresis. Metallo-ß-lactamases Ellington et al. (2007) Acquired (plasmid-mediated) AmpCs) (Pérez-Pérez & Hanson, 2002)

7 PCR + restriction fragment length polymorphism (RFLP) E. faecium / E. faecalis (Woodford et al. 2002) 633-bp 591-bp R S S R R S. aureus (Tsiodras et al. 2001) S R R 526-bp 430-bp 168-bp 96-bp  wild-type GCGAGC vs. mutant GCTAGC leads to linezolid R.  creates a NheI cutting site in 23S rRNA.  Heterozygosity due to multiple copies of 23S rRNA.

8 Real-time PCR (RT-PCR) Detection of linezolid R E. faecalis/E. faecium (Woodford et al. 2002) Melting temperature is dependent on the degree of complementarity between the probe and target sequence. Metallo-ß-lactamase detection (Mendes et al. 2007) The temperature at which DNA dissociates (melting temperature) is dependent on amplicon length and GC content. GIM IMP SIM SPM VIM

9 Commercially available RT-PCR kits  Roche Molecular Systems Inc. LightCycler® MRSA Advanced Test: identify MRSA direct from nasal swabs. LightCycler® SeptiFast MecA Test: identify MRSA direct from blood samples. LightCycler® VRE Detection Kit (RUO): identify vanA, vanB, vanB2/3 in VRE (req. DNA extraction).  Becton, Dickinson U.K. Ltd./Cepheid SmartCycler® BD GeneOhm™ VanR: ID of VRE direct from perianal and/or rectal swabs. BD GeneOhm™ StaphSR: detection and differentiation of MRSA/SA from blood culture, wound and nasal swabs. BD GeneOhm™ MRSA: direct detection of MRSA from nasal swab.

10 Cepheid GeneXpert system  Fully integrated and automated sample preparation, RT-PCR and detection.  Specimens don’t need to be batched.  <2 mins hands-on time.  Results in <1hr – 6 targets per sample.  MRSA/SA – orfX-SCCmec junction + mecA + spa.  VRE – vanA.  MTB/RIF – mutations in rpoB (RUO). http://www.cepheid.com/

11 DNA probe-based hybridisation assays  EVIGENE  (AdvanDX): mecA mupA vanA and vanB.  No expensive equipment required.  No risk of cross-contamination with amplicons.  10 min of hands-on time, with a 3.5-h turnaround time (not incl. DNA extraction). “…the EVIGENE kit was user friendly for the routine microbiology laboratory, with results available within 7 h of recognition of a blood culture positive for GPCC. Rapid and accurate testing of GPCC-positive blood culture samples should facilitate infection control measures, reduce empirical use of vancomycin, and improve the management of MRSA bacteremia…” Levi & Towner, 2003.

12 Strip assays  PCR-based reverse hybridisation DNA strip assays (Hain Lifescience). GenoType  GenoQuick  http://www.hain-lifescience.de/en/  results within 2.5 hrs.  MRSA.  results within 4 hrs.  MDR + XDR-TB.  VRE, MRSA.  Helicobacter pylori

13 PCR – ELISA: Hyplex  assays Avlami et al. 2010  kits for MßL, MRSA, VRE, ESBLs (TEM, SHV, CTX-M and OXA) and OXA carbapenemases (OXA-23, -40 and -58).  identifies genes in 2.5 – 4 hrs directly from clinical specimens.  Only one target per well – cost-effective?

14 Microarray: Check-Points assays  TEM, SHV and CTX-M ESBLs.  Plasmidic AmpC.  KPC, OXA-48, IMP, VIM, NDM.  Can detect SNPs that differentiate between narrow and broad-spectrum ß- lactamases.  Assay time 6hr.  Positive evaluations in: France (Naas et al. 2011). USA (Endimiani et al. 2010). Netherlands (Cohen Stuart et al. 2010).  Requires purified DNA. http://www.check-points.com

15 Liquid array: Luminex xTAG assay  Detects multiple targets (genes or SNPs) simultaneously.  Allele-specific primers adds tag sequence to amplicon – complementary to sequence on bead set.   susceptibility in Salmonella Typhi and SPA due to 11 SNPs in gyrA, gyrB and parE (Song et al. 2010).  Luminex technology also used in StaphPlex (Qiagen) and MVPlex MRSA (Geneco Biomedical Products).  Protocol labour-intensive. Song et al. (2010)

16 Pyrosequencing® technology  ‘sequencing by synthesis’ method.  Extremely rapid SNP detection – 15min.  Built in QC.  Can detect novel mutations.  Quantifies heterozygotes.  Also MTb, FQ-resistance.  No commercial assays. Homo-S Homo-R Hetero-R Sinclair et al. 2003 Detecting linezolid R enterococci

17 Phenotypic vs. genotypic: advantages  Can be performed direct from clinical specimens: Rapid. Good for difficult to culture organisms or slow-growers. May reduce biohazard risk.  Potential for automation.  Simple yes/no answer - not dependent on S/I/R categories.  Sort out ambiguous phenotypic results.  Good for resistance mechanisms that encode low-level resistance.  Inform epidemiological studies.

18  False –ves due to new mechanisms or mutations.  False +ves due to silent genes or partial sequence. Phenotypic vs. genotypic: limitations Correlation between resistance genotype and phenotype of staphylococci Martineau et al. 2000 Nearly perfect correlation (n = 394): 98% OXA, 100% GEN, 98.5% ERY  Low sensitivity when applied directly to clinical specimens. Specificity?  Still need culture for confirmation of ID + epidemiological typing.  One assay/platform unlikely to cover all resistance mechanisms - cost?

19 Summary  Molecular assays for detection of AMR have yielded a wealth of information.  Unlikely to replace, but instead augment, phenotypic susceptibility testing.  Commercial kits seem to be promising but thorough evaluation in multicentre studies required.  Several choices for MRSA, VRE, ESBLs.  For now characterisation of new resistance genes and mechanisms are best undertaken in reference laboratories.


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