Rapid detection of resistant Gram- negatives directly from specimens

1. Rapid detection of resistant Gram- negatives directly from specimens Dr Gemma Vanstone Royal Free Hampstead NHS Trust

2. Introduction • Development of methods for the rapid detection of ESBL and AmpC producing organisms • Empirical therapy does not cover ESBL/AmpC producing organisms • Delayed treatment of infections caused by ESBL/AmpC producing organisms associated with increased mortality

3. Multi-resistance • Isolates that have an ESBL/AmpC are often multiresistant with few treatment options • Plasmids often carry genes for resistance to other classes of antibiotics • often no oral options available for minor infections • necessitates use of broad-spectrum carbapenems • Could lead to other resistance mechanisms developing

4. Laboratory Detection Sample received/flags positive ESBL + 24 h ControlESBL- 72 HOURS Gram negative grows 24 h ID and sens ? ESBL – grow with and without clavulante to confirm 24 h

5. Major ESBL Types * The definition of ESBLs is sometimes stretched to include Class D β-lactamase mutants that hydrolyse oxyimino-cephalosporins, but they are inhibitor resistant Taken from: Pocket Guide to ESBLs in Resistance. D. Livermore & D. Paterson

6. Epidemiology of ESBLsCTX-M types • Split into phylogenetic groups • eg CTX-M-1, -2, -8, -9 and -26. • First reported late 1980s (Japan and Germany) • Remained rare for a long time in Europe • 2000: 1st report in UK (CTX-M-9 Single K. oxytoca isolate) • 2001: CTX-M-26 and CTX-M-15 • NOW: THE MOST COMMON ESBL TYPE SEEN IN THE UK

7. Number of cycles Detection of ESBLs directly from blood cultures • A real-time multiplex PCR that can detect and type blaCTX-M genes has been described (Birkett et al, 2007) • One set of primers that bind to all CTX-M groups • Specific probes for the different groups • Performed on isolates Normalized fluorescence • Can this PCR be performed on blood cultures containing Gram negative rods?

8. Bacterial DNA Extraction direct from blood cultures • BD GeneOhm StaphSR assay extraction • Rapid • Simple • Automation?

9. 2 hours Observe GNR in sample (eg Blood culture) Extract DNA and set up PCR 2 hours Run PCR and detect CTX-M DNA in real time Towards a Faster Diagnosis

10. 1 x 109 cells /ml 1 x 104 cells /ml Fig 1: Detection of CTX-M-9 ESBLs in blood cultures containing control organism at 1 x 109 cells/ml to 1 x 102 cells/ml. The detection limit was 1 x 104 cells/ml ESBL PCR on spiked blood cultures • Control organisms representing the 5 CTX-M groups were spiked into negative blood cultures • Rapid DNA extraction and real-time PCR performed • The PCR was able to detect all groups at a limit of 104 bacteria/ml (Positive blood cultures 107-108 bacteria/ml)

11. Clinical samples, so far... • 230 positive blood cultures containing Gram negative rods tested by PCR • Phenotypic data: 12/230 shown to contain ESBL • PCR: 11/230 shown to contain ESBL • On further testing – discrepant isolate shown to contain OXA type ESBL • Results are available within 4 hours of the blood culture flagging positive

12. ESBL PCR – Future work... • Evaluation of other samples/fluids • Detection of other ESBL-types • Currently a negative result doesn’t mean ‘ESBL negative’ • On-going project for SHV/TEM/OXA type ESBLs

13. AmpC • Infection with AmpC producing organisms is associated with many of the risk factors associated with ESBL infection • Routine methods for detection of AmpC producing organisms are similar to those described for ESBLs

14. Detection of plasmidic AmpC genes • A conventional PCR that can detect the 6 plasmidic groups of AmpC has previously been described • This PCR had previously been performed on isolates • We wanted to convert this PCR into a real-time format that can be performed directly on samples Perez Perez & Hanson 2002

15. Real-time AmpC PCR: The story so far... • Probes to distinguish between the AmpC families not described • Initially investigating SybrGreen and HRM as a method to detect and distinguish between the AmpC families. • ACC, CIT and FOX can be easily distinguished • ENT and DHA are more similar in melting temperature

16. AmpC PCR: Work in progress... • Clinical isolates • Collected 5 CIT producing isolates • Spiked blood cultures • Clinical samples • Detected and AmpC in 2/92 blood cultures • Data so far agrees with phenotypic data.

17. Future Work • Continuation of ‘work in progress’ • Evaluation of PCR methods on other specimens • Detection of other resistance genes • Non CTX-M type ESBLs • Carbapenemase

18. Acknowledgements • Dr. Indran Balakrishnan • Dr. Bambos Charalambous • Medical Microbiology Laboratory, Royal Free Hampstead NHS Trust • Project students • Katie Mouskos • Laila Ramzi • Lois Wilkie • Ayse Yorgancioglu • Kit Ying Lam • Belinda Tse • Noam Roth