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 Gram negative grows ID and sens ? ESBL – grow with and without clavulante to confirm 24 h ESBL + Control ESBL- 72 HOURS

5. Major ESBL Types Type (Class)OriginsNo. Variants TEM (A)Mutants of TEM penicillinase, origins unknown>100 SHV (A)Mutants of SHV penicillinase, originated as a chromosomal type in K. pneumoniae > 50 CTX-M (A)Chromosomal ESBL in Kluyvara spp.>40, 5 sub- classes VEB (A)Unknown3 PER (A)Unknown2 OXA-15 (D)*Mutant of OXA-2, a long-known penicillinase of uncertain origin 1 OXA-11, 14, 15, 16 17 (D)* Mutant of OXA-10, a long-known penicillinase of uncertain origin At least 5 * 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 ESBLs CTX-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: 1 st 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. 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 Number of cycles 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. Towards a Faster Diagnosis Extract DNA and set up PCR Run PCR and detect CTX-M DNA in real time Observe GNR in sample (eg Blood culture) 2 hours

10. 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 10 4 bacteria/ml (Positive blood cultures 10 7 -10 8 bacteria/ml) 1 x 10 4 cells /ml 1 x 10 9 cells /ml Fig 1: Detection of CTX-M-9 ESBLs in blood cultures containing control organism at 1 x 10 9 cells/ml to 1 x 10 2 cells/ml. The detection limit was 1 x 10 4 cells/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