Transcript Grumbling problems, etc ,etc

Molecular detection of antibiotic
resistance
Katie L Hopkins PhD
[email protected]
Laboratory of Gastrointestinal Pathogens
HPA Microbiology Services Colindale
20th May 2011
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?
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.
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
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.
“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.
Acquired (plasmid-mediated)
AmpCs)
(Pérez-Pérez & Hanson, 2002)
Separate post-PCR detection – usually
agarose gel electrophoresis.
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.
R
S S
R R
S R R
633-bp
526-bp
430-bp
591-bp
168-bp
96-bp
E. faecium / E. faecalis
(Woodford et al. 2002)
S. aureus
(Tsiodras et al. 2001)
Real-time PCR (RT-PCR)
GIM
IMP SIM SPM VIM
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 linezolidR E. faecalis/E. faecium
(Woodford et al. 2002)
Melting temperature is dependent on
the degree of complementarity between
the probe and target sequence.
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.
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/
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.
Strip assays
PCR-based reverse hybridisation DNA strip assays (Hain Lifescience).
GenoType
GenoQuick
results within
2.5 hrs.
MRSA.
results within 4 hrs.
MDR + XDR-TB.
VRE, MRSA.
Helicobacter pylori
http://www.hain-lifescience.de/en/
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
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 ßlactamases.
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
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)
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
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.
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?
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.