Molecular detection of antibiotic resistance

1. Molecular detection of antibiotic resistance Katie L Hopkins PhD katie.hopkins@hpa.org.uk Laboratory of Gastrointestinal Pathogens HPA Microbiology Services Colindale 20th 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: • Acquisition and expression of new DNA by horizontal transfer. • 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 Metallo-ß-lactamases Ellington et al. (2007) } Intrinsic to A. baumannii Acquired OXA carbapenemases in Acinetobacter (Woodford et al. 2006; Higgins et al. 2010) • Most commonly applied technique. • Amplification targets conserved or variable regions within gene of interest. • Separate post-PCR detection – usually agarose gel electrophoresis. Acquired (plasmid-mediated) AmpCs) (Pérez-Pérez & Hanson, 2002)

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

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

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

13. PCR – ELISA: Hyplex assays • 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? Avlami et al. 2010

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 Hetero-R Homo-R Detecting linezolidR enterococci Sinclair et al. 2003

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. Phenotypic vs. genotypic: limitations • False –ves due to new mechanisms or mutations. • False +ves due to silent genes or partial sequence. 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.