Masters DissertationMasters Dissertation

Clostridium sordelliiClostridium sordellii and knee and knee infectionsinfections

Trefor MorrisTrefor Morris

BackgroundBackground

A public health dispatch from the A public health dispatch from the CDC in Atlanta, USA 19th November CDC in Atlanta, USA 19th November 2001 alerted clinicians to the 2001 alerted clinicians to the deaths of 3 patients following deaths of 3 patients following “uncomplicated knee surgery”“uncomplicated knee surgery”

All died 36-100hours after surgery All died 36-100hours after surgery from a septic shock like illnessfrom a septic shock like illness

Two of the patients had received Two of the patients had received total knee replacements whilst total knee replacements whilst the other had received a the other had received a cartilage graft implant taken cartilage graft implant taken from a cadaverfrom a cadaver

All elective knee surgery in All elective knee surgery in Minnesota was subsequently Minnesota was subsequently suspendedsuspended

c. 50 000 Anterior Cruciate knee c. 50 000 Anterior Cruciate knee ligament (ACL) operations ligament (ACL) operations performed in USA per annum and performed in USA per annum and infections are rare- with only 26 infections are rare- with only 26 reported in the previous 4 monthsreported in the previous 4 months

Cadaver knee tissue is often Cadaver knee tissue is often used to repair damaged kneesused to repair damaged knees

Blood cultures taken from one Blood cultures taken from one patient grew patient grew Clostridium sordelliiClostridium sordellii after 5 days incubation (obtained after 5 days incubation (obtained pre mortem)pre mortem)

This patient had received an This patient had received an osteochondral (bone and osteochondral (bone and cartilage) allograft and thus the cartilage) allograft and thus the source of the allograft came source of the allograft came under suspicionunder suspicion

C. sordelliiC. sordellii was isolated was isolated from non implanted knee from non implanted knee tissue from the same tissue from the same cadaveric donor cadaveric donor

Thus the allograft came Thus the allograft came under suspicion as the under suspicion as the source of the source of the C. sordelliiC. sordellii

No typing method was No typing method was available for available for C. sordelliiC. sordellii

A typing method would A typing method would become vital if other cases become vital if other cases

were to occurwere to occur

The isolates were sent to the The isolates were sent to the ARL and to FSML London in the ARL and to FSML London in the hope that they would be hope that they would be distinguishabledistinguishable

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

Clostridium sordelliiClostridium sordellii

Gas gangrene caused by clostridia can have Gas gangrene caused by clostridia can have devastating effectsdevastating effects

C. sordelliiC. sordellii has previously been isolated from has previously been isolated from many different sites and from the many different sites and from the environment. For example the ARL has isolates environment. For example the ARL has isolates from “theatre wall” and human appendixfrom “theatre wall” and human appendix

Non haemolytic (often spreading) growth - Non haemolytic (often spreading) growth - characteristic of characteristic of Clostridium sordelliiClostridium sordellii

Phenotypic characteristics of Phenotypic characteristics of C. C. sordelliisordellii

The organismThe organism produces many toxins produces many toxins including a lecithinase (but no lipase). including a lecithinase (but no lipase). This is easily demonstrable using egg This is easily demonstrable using egg yolk agaryolk agar

Spore stained smear showing spores (green) and Spore stained smear showing spores (green) and vegetative cells (pink)vegetative cells (pink)

C. sordelliiC. sordellii sporulates readily. Spores can sporulates readily. Spores can remain viable for yearsremain viable for years

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

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

Patellar tendon has been re Patellar tendon has been re attached previously, not very attached previously, not very successfullysuccessfully

Cadaveric achilles Cadaveric achilles tendon being “sized tendon being “sized up” to replace the up” to replace the patellar tendonpatellar tendon

The SurgeryThe Surgery

Cadaveric tendon being Cadaveric tendon being re-attached- using a re-attached- using a wedge made from heel wedge made from heel bonebone

The final product- a The final product- a fully attached fully attached cadaveric tendoncadaveric tendon

Aims and Objectives of Aims and Objectives of StudyStudy

To determine whether the 16S-23S To determine whether the 16S-23S intergenic spacer region would be a intergenic spacer region would be a useful target site for typing useful target site for typing C. C. sordelliisordellii

To investigate the similarity of the To investigate the similarity of the Minnesota isolatesMinnesota isolates

The Anaerobe Reference Laboratory already The Anaerobe Reference Laboratory already has a nationally recognised typing scheme has a nationally recognised typing scheme for for Clostridium difficileClostridium difficile based on differences based on differences inin the the 16S-23S intergenic spacer region16S-23S intergenic spacer region

This currently has 160 types and This currently has 160 types and employs the GelCompar software employs the GelCompar software analysis programmeanalysis programme

Here a consistent relationship is Here a consistent relationship is found between Ribotype and toxin found between Ribotype and toxin production- would this be the case production- would this be the case for for C. sordellii C. sordellii ??

As the organism is closely related As the organism is closely related to to C. difficile, C. difficile, the typing method the typing method used for this organism may be used for this organism may be useful for useful for C. sordelliiC. sordellii

Also as the toxins produced are Also as the toxins produced are thought to be the major thought to be the major virulence factor, the toxigenic virulence factor, the toxigenic capacity of the capacity of the C. sordelliiC. sordellii isolates warrants further isolates warrants further investigationinvestigation

PCR BASED FINGERPRINTING METHOD

Cells Infected tissueIsolated DNA PCR reactions

Electrophoresisof DNA

Image generated Pattern Analysis

Samples - Amplificationin ThermalCycler

GelCompar

Applied Maths

Cluster Analysis

LibraryComparison

Diagrammatic representation of the Diagrammatic representation of the 16S-23S spacer region of 16S-23S spacer region of C. sordelliiC. sordellii

spacer regionspacer region

1 40 1391 1543

474 2905

16S rRNA tRNA tRNA 23S rRNA

Deceased Patient

Operation Blood culture result

1 Total knee replacement No growth 2 Total knee replacement No growth 3 Cartilage graft

implantation C. sordellii @ 5 days

Minnesota Minnesota PatientsPatients

Cadaveric donorCadaveric donor

Non implanted tissue 1 Growth of C. sordellii Non implanted tissue 2 Growth of C. sordellii

Isolates of Isolates of C. sordelliiC. sordellii referred for typing by CDC referred for typing by CDC AtlantaAtlanta

Referred Blind CodedReferred Blind CodedReference Reference Number Number Actual Actual

SourceSourceP31P31

P32P32

P33P33

P34P34

P35P35

P36P36

Laboratory Strain Laboratory Strain 1980s1980s

B/C B/C Minnesota Minnesota

patientpatientLaboratory Laboratory

strainstrainCadaveric kneeCadaveric knee

Cadaveric Cadaveric kneeknee

ATCC 9714ATCC 9714

11

22

33

44

55

66

Investigated using the same primers to type C. sordellii. The results were not very clear cut. Some bands were difficult to distinguishFound C. sordellii specific primers (Sasaki et al 2000) and applied these to the typing of the CDC Atlanta isolates(x6)

Also adjusted the gel running time and added ethidium bromide to the gel

Results of typing the Minnesota Results of typing the Minnesota isolates(isolates(P32, 34 & 35P32, 34 & 35) and blind coded ) and blind coded isolates from CDC Atlanta (isolates from CDC Atlanta (P31, 33 and P31, 33 and 3636))

P31P31 P32P32 P33P33 P34P34 P35P35 P36P36

Investigated 120 isolates in the Investigated 120 isolates in the ARL collection to find out if ARL collection to find out if other different ribotypes other different ribotypes existed.existed.

Early investigations showed 10 Early investigations showed 10 different ribotypesdifferent ribotypes

The 16 different PCR ribotypes of The 16 different PCR ribotypes of C. C. sordelliisordellii

Each isolate was typed 3 times and gave Each isolate was typed 3 times and gave consistent results. Before being allocated a new consistent results. Before being allocated a new type- two or more differences had to be type- two or more differences had to be observedobserved

Toxin testing performed using Vero cells Toxin testing performed using Vero cells and McCoy cellsand McCoy cells

Toxin Toxin positivepositive

Toxin negativeToxin negative

Toxin production amongst Toxin production amongst C. sordelliiC. sordellii is is variable therefore I investigated the variable therefore I investigated the toxigenicity of the strains in the studytoxigenicity of the strains in the study

Initially 7 isolates were toxin positive by cell Initially 7 isolates were toxin positive by cell culture these were P23 (known toxin culture these were P23 (known toxin positive from Pasteur institute), P31 (CDC positive from Pasteur institute), P31 (CDC lab strain), P32 (B/C from deceased patient), lab strain), P32 (B/C from deceased patient),

P33 (CDC lab strain)P33 (CDC lab strain), , P36 (ATCC type P36 (ATCC type

strain)strain), , P38(foot abscess) and P47 (B/C)P38(foot abscess) and P47 (B/C)

However upon repeat subculture toxin However upon repeat subculture toxin production in these strains was lost. production in these strains was lost. Literature states that Literature states that C. sordellii C. sordellii is is notorious for “losing” its toxinnotorious for “losing” its toxin

Detection of the Detection of the C. sordelliiC. sordellii toxin gene toxin gene may be more advantageous as definite may be more advantageous as definite marker of toxigenic capabilitymarker of toxigenic capability

Specific primers for the Specific primers for the C. sordelliiC. sordellii toxin toxin gene are available (Hofmann gene are available (Hofmann et alet al 1998). 1998). Attempted to use these to detect the gene Attempted to use these to detect the gene in the 7 in the 7 C. sordelliiC. sordellii isolates that were isolates that were toxin positive by cell culturetoxin positive by cell culture

Toxin gene found in 6 of the 7 toxin Toxin gene found in 6 of the 7 toxin positive isolates. The PCR product was positive isolates. The PCR product was sequenced and found to be sequenced and found to be C. sordelliiC. sordellii toxin gene (99%)toxin gene (99%)

P53 P62 P97 P120 CD+ CD- P23 P31 P32 P33 P36 P38 P47 M

Key P= Project number CD= Clostridium difficileP23 = Positive control+= toxin positive, - = toxin negativeM = Molecular marker

Recently two cases involving Recently two cases involving C. sordelliiC. sordellii in postpartum in postpartum death have been typed and death have been typed and tested for toxintested for toxin

No toxin gene was detected in No toxin gene was detected in either of these isolates.either of these isolates.

Toxin gene should be stable even Toxin gene should be stable even where toxin expression is lost. where toxin expression is lost. Unknown whether gene is plasmid Unknown whether gene is plasmid or chromosomalor chromosomal

Marc Fischer (CDC Atlanta) stated that Marc Fischer (CDC Atlanta) stated that interest in the Minnesota cases had interest in the Minnesota cases had declined post September 11th and declined post September 11th and 29th anthrax cases29th anthrax cases

So could source have been the So could source have been the environment? No environmental swabs environment? No environmental swabs were mentioned. Or was the isolate that were mentioned. Or was the isolate that killed the patient just a different toxigenic killed the patient just a different toxigenic strain not isolated from the cadaveric strain not isolated from the cadaveric donor?donor?Cadaveric tissue in this case was found Cadaveric tissue in this case was found to have been harvested 23.5 hrs after to have been harvested 23.5 hrs after death- this may have allowed graft to death- this may have allowed graft to become seeded with bowel florabecome seeded with bowel flora

Conclusions and further Conclusions and further researchresearchThe The C. sordelliiC. sordellii isolates from Minnesota isolates from Minnesota were dissimilar.were dissimilar.

Possibility of using primers as an Possibility of using primers as an identification tool for clostridia? or for identification tool for clostridia? or for typing other important clostridia e.g typing other important clostridia e.g C. septicum, C. novyi C. septicum, C. novyi

ARL now has a valid ribotyping and ARL now has a valid ribotyping and toxin detection method for toxin detection method for C. sordelliiC. sordellii

The end.The end.

Many thanks to my colleagues at Many thanks to my colleagues at the ARL! the ARL!