Masters Dissertation

1. Masters Dissertation Clostridium sordellii and knee infections Trefor Morris

2. Background A public health dispatch from the CDC in Atlanta, USA 19th November 2001 alerted clinicians to the deaths of 3 patients following “uncomplicated knee surgery” All died 36-100hours after surgery from a septic shock like illness

3. Two of the patients had received total knee replacements whilst the other had received a cartilage graft implant taken from a cadaver All elective knee surgery in Minnesota was subsequently suspended

4. Cadaver knee tissue is often used to repair damaged knees c. 50 000 Anterior Cruciate knee ligament (ACL) operations performed in USA per annum and infections are rare- with only 26 reported in the previous 4 months

5. Blood cultures taken from one patient grew Clostridium sordellii after 5 days incubation (obtained pre mortem) This patient had received an osteochondral (bone and cartilage) allograft and thus the source of the allograft came under suspicion

6. C. sordellii was isolated from non implanted knee tissue from the same cadaveric donor Thus the allograft came under suspicion as the source of the C. sordellii

7. No typing method was available for C. sordellii The isolates were sent to the ARL and to FSML London in the hope that they would be distinguishable A typing method would become vital if other cases were to occur

8. Clostridium sordellii C. sordellii is a member of the Gas gangrene causing, spore forming clostridia. It therefore had the potential to be responsible for the death of these patients

9. Gas gangrene caused by clostridia can have devastating effects C. sordellii has previously been isolated from many different sites and from the environment. For example the ARL has isolates from “theatre wall” and human appendix

10. Phenotypic characteristics of C. sordellii Non haemolytic (often spreading) growth - characteristic of Clostridium sordellii

11. The organismproduces many toxins including a lecithinase (but no lipase). This is easily demonstrable using egg yolk agar

12. C. sordellii sporulates readily. Spores can remain viable for years Spore stained smear showing spores (green) and vegetative cells (pink)

13. Example of a preoperative patient about to undergo knee surgery in the USA- “dead Mike”

14. Dead mikes (Seriously) damaged knee- he is about to receive an allograft of cartilage from a cadaver

15. The Surgery Patellar tendon has been re attached previously, not very successfully Cadaveric achilles tendon being “sized up” to replace the patellar tendon

16. Cadaveric tendon being re-attached- using a wedge made from heel bone The final product- a fully attached cadaveric tendon

17. Aims and Objectives of Study • To determine whether the 16S-23S intergenic spacer region would be a useful target site for typing C. sordellii • To investigate the similarity of the Minnesota isolates

18. The Anaerobe Reference Laboratory already has a nationally recognised typing scheme for Clostridium difficilebased on differences in the 16S-23S intergenic spacer region This currently has 160 types and employs the GelCompar software analysis programme Here a consistent relationship is found between Ribotype and toxin production- would this be the case for C. sordellii ?

19. As the organism is closely related to C. difficile, the typing method used for this organism may be useful for C. sordellii Also as the toxins produced are thought to be the major virulence factor, the toxigenic capacity of the C. sordellii isolates warrants further investigation

20. PCR BASED FINGERPRINTING METHOD Samples - Amplification in Thermal Cycler Cells Infected tissue Isolated DNA PCR reactions Cluster Analysis Library Comparison GelCompar Applied Maths Electrophoresis of DNA Pattern Analysis Image generated

21. 1 40 1391 1543 474 2905 16S rRNA tRNA tRNA 23S rRNA Diagrammatic representation of the 16S-23S spacer region of C. sordellii spacer region

22. Minnesota Patients Cadaveric donor

23. Isolates of C. sordellii referred for typing by CDC Atlanta Reference Number Referred Blind Coded Actual Source Laboratory Strain 1980s 1 P31 P32 P33 P34 P35 P36 2 B/C Minnesota patient 3 Laboratory strain 4 Cadaveric knee 5 Cadaveric knee 6 ATCC 9714

24. Investigated using the same primers to type C. sordellii. The results were not very clear cut. Some bands were difficult to distinguish Found C. sordellii specific primers (Sasaki et al 2000) and applied these to the typing of the CDC Atlanta isolates(x6) Alsoadjustedthe gel running timeandaddedethidium bromide to thegel

25. Results of typing the Minnesota isolates(P32, 34 & 35) and blind coded isolates from CDC Atlanta (P31, 33 and 36) P31 P32 P33 P34 P35 P36

26. Investigated 120 isolates in the ARL collection to find out if other different ribotypes existed. Early investigations showed 10 different ribotypes

27. The 16 different PCR ribotypes of C. sordellii Each isolate was typed 3 times and gave consistent results. Before being allocated a new type- two or more differences had to be observed

28. Toxin production amongst C. sordellii is variable therefore I investigated the toxigenicity of the strains in the study Toxin testing performed using Vero cells and McCoy cells Toxin negative Toxin positive

29. Initially 7 isolates were toxin positive by cell culture these were P23 (known toxin positive from Pasteur institute), P31 (CDC lab strain), P32 (B/C from deceased patient), P33 (CDC lab strain), P36 (ATCC type strain), P38(foot abscess) and P47 (B/C) However upon repeat subculture toxin production in these strains was lost. Literature states that C. sordellii is notorious for “losing” its toxin

30. Detection of the C. sordellii toxin gene may be more advantageous as definite marker of toxigenic capability Specific primers for the C. sordellii toxin gene are available (Hofmann et al 1998). Attempted to use these to detect the gene in the 7 C. sordellii isolates that were toxin positive by cell culture

31. Toxin gene found in 6 of the 7 toxin positive isolates. The PCR product was sequenced and found to be C. sordellii toxin gene (99%) P53 P62 P97 P120 CD+ CD- P23 P31 P32 P33 P36 P38 P47 M Key P= Project number CD= Clostridium difficile P23 = Positive control += toxin positive, - = toxin negative M = Molecular marker

32. Recently two cases involving C. sordellii in postpartum death have been typed and tested for toxin No toxin gene was detected in either of these isolates. Toxin gene should be stable even where toxin expression is lost. Unknown whether gene is plasmid or chromosomal

33. Marc Fischer (CDC Atlanta) stated that interest in the Minnesota cases had declined post September 11th and 29th anthrax cases So could source have been the environment? No environmental swabs were mentioned. Or was the isolate that killed the patient just a different toxigenic strain not isolated from the cadaveric donor? Cadaveric tissue in this case was found to have been harvested 23.5 hrs after death- this may have allowed graft to become seeded with bowel flora

34. Conclusions and further research The C. sordellii isolates from Minnesota were dissimilar. ARL now has a valid ribotyping and toxin detection method for C. sordellii Possibility of using primers as an identification tool for clostridia? or for typing other important clostridia e.g C. septicum, C. novyi

35. The end.

36. Many thanks to my colleagues at the ARL!