1. Lisa Drummond University of Edinburgh Antibiotics and Clostridium difficile

2. Introduction Gram positive spore- former obligately anaerobic first described in asymptomatic neonates increased use of antibiotics led to an increase in C. difficile disease

3. Introduction cont. infection ranges from asymptomatic, mild diarrhoea, colitis to pseudomembranous colitis risk factors - antibiotics, age, environment and virulence of infecting strain third generation cephalosporins, clindamycin and amoxycillin associated with the greatest risk disease occurs after depletion of patient’s normal protective flora

4. Disease process ANTIBIOTIC THERAPY  ALTERATION OF COLONIC MICROFLORA  C.difficile EXPOSURE & COLONISATION  RELEASE OF TOXIN A & TOXIN B  COLONIC MUCOSAL INJURY AND INFLAMMATION Adapted from Kelly CP & LaMont JT (1998). Clostridium difficile infection. Annual Review of Medicine 49, 375-390.

5. Incidence of C.difficile in the population Subject populationC. difficile positive Pseudomembranous colitis 95-100% Antibiotic-associated diarrhoea 10-30% Hospital in-patients 20% Healthy adults 0-3% Healthy neonates and infants 25-80% Adapted from Kelly CP & LaMont JT (1998). Clostridium difficile infection. Annual Review of Medicine 49, 375-390.

7. Pathogenicity Locus (PaLoc) 19.6kb element replaced by 115bp in non- toxigenic strains tcdD alternative sigma factor tcdC putative negative regulator toxins transcribed on entry to stationary phase tcdD 552bp toxB 7098bp tcdE 501bp toxA 8133bp tcdC 695bp

8. PaLoc cont. toxin production affected by glucose, sub-inhibitory concs. of antibiotics, amino acids, temperature, oxidative stress, biotin insufficiency, biocarbonate concentration...

9. AIMS to analyse MIC data, patient antibiotic regimes, S-types, resistance to look at effects of sub-MICs on growth and toxin production investigate toxin transcripts using RT-PCR investigate total cell protein between controls and sub-MIC antibiotics using 2D gel electrophoresis and MALDI-TOF

10. MICs 186 strains and 6 antibiotics (NCCLS) the two treatment agents - vancomycin and metronidazole 4 precipitating agents - amoxycillin, clindamycin, cefoxitin and ceftriaxone database utilised for any connections

11. Clindamycin resistance 12 isolates tested had clindamycin MIC of  128  g/ml all contained ermB gene 2 different sizes noted smaller band lack leader peptide (Farrow et al., 2002)

12. Recurrences and reinfections some patients produced up to 12 samples over the 18 months allowed comparisons of their isolates over that time some patients had changing S-types over this time some patients also had different isolates in the same faecal sample

13. MIC conclusions no strains resistant to vancomycin or metronidazole no significant difference of resistance profiles between S-types no correlation between antibiotics given and resistance profiles evidence of mixed infections or recurrences

14. Sub-MIC antibiotics antibiotics have previously been shown to affect toxin production in C.difficile vast amounts of literature showing effects on other bacteria though there’s very little data for C. difficile

15. Sub-MIC experimental set-up used same 6 antibiotics as MIC work used reference strain NCTC 11223, locally endemic strain 338a and sequenced strain 630 1/2, 1/4 and 1/8 sub-MIC concs. used sampled 3X a day for 104 hours OD 600 measured each time and 1ml of supernate frozen for ELISA analysis

16. Controls from sub-MIC experiments each strain grown 6 times in total growth varied little between strains toxin elaborated at slightly different times in the growth curve toxin production by 338a and 630 exceeds assay by ca. 48h

19. Sub-MIC conclusions there’s often a lag in the growth of the bacteria compared to the control main effect on toxin is that it’s elaborated quicker under sub-MIC conditions heterogeneity common between strains for toxin production and growth in response to antibiotics

20. RT-PCR wanted to look for toxin transcripts to see if they correlate to sub-MIC work RNA concentrations low (ca. 5  g/ml) 16S transcripts easily seen but only with Sensiscript enzyme low concentrations of RNA probably made toxin transcripts difficult to see

21. Sensiscript Sensiscript vastly improves ability to pick up 16S RNA still no transcripts from toxins decide to cut losses as time extremely short

22. RT-PCR outcome Was unsuccessful in seeing transcripts for toxins, tcdC, tcdD and groEL use of Sensiscript led to clear signal from 16S RNA if had more time would have tried another technique e.g. Trizol, Tri reagent etc.

23. Proteomics use 2D gel electrophoresis and MALDI-TOF analysis of proteins protein profile still largely uncharacterised in C. difficile wanted to compare control vs. sub-MIC sample preparation reproducibility new MASCOT database being set-up

24. Control vs sub-MIC gels very reproducible - good for future manipulations no obvious difference between two sets of conditions (with and without ceftriaxone) 40 spots from 6 gels were taken for MALDI-TOF data still being analysed and new MASCOT database in the pipeline

25. Typical 2D gel

26. Conclusions - MICs no strains resistant to either of the treatment agents no significant difference of resistance profiles between S-types no correlation between antibiotics given and resistance profiles evidence of mixed infections or recurrences

27. Conclusions - sub-MICs sub-MIC antibiotics often cause a growth lag and shift forward the production of toxin there is heterogeneity between strains and their response to sub-MIC antibiotics the effect on toxin could not be seen mirrored in the toxin transcripts due to the sensitivity of the RT-PCR

28. Conclusions - proteomics reproducibility - good sample preparation the combination of strain 630 and ceftriaxone produced a protein profile unchanged to that of the control once new database available should get more high-scoring hits next stage - other antibiotics and strains

29. Acknowledgements Ian Poxton David Smith Bob Brown Jodie McCoubrey Microbial Pathogenicity Research Laboratory John Starr Becky Graham Functional Genomics Unit at MRI Pilar Alberdi MRC