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1、Lisa Drummond University of Edinburgh,Antibiotics and Clostridium difficile,Introduction,Gram positive spore- former obligately anaerobic first described in asymptomatic neonates increased use of antibiotics led to an increase in C. difficile disease,Introduction cont.,infection ranges from asymptom
2、atic, 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 patients normal protective flora,D
3、isease 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.,Incidence of C
4、 .difficile in the population,Adapted from Kelly CP & LaMont JT (1998). Clostridium difficile infection. Annual Review of Medicine 49, 375-390.,Pathogenicity Locus (PaLoc),19.6kb element replaced by 115bp in non-toxigenic strains tcdD alternative sigma factor tcdC putative negative regulator toxins
5、transcribed on entry to stationary phase,PaLoc cont.,toxin production affected by glucose, sub-inhibitory concs. of antibiotics, amino acids, temperature, oxidative stress, biotin insufficiency, biocarbonate concentration.,AIMS,to analyse MIC data, patient antibiotic regimes, S-types, resistance to
6、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,MICs,186 strains and 6 antibiotics (NCCLS) the two treatment agents - vancomycin an
7、d metronidazole 4 precipitating agents - amoxycillin, clindamycin, cefoxitin and ceftriaxone database utilised for any connections,Clindamycin resistance,12 isolates tested had clindamycin MIC of 128g/ml all contained ermB gene 2 different sizes noted smaller band lack leader peptide (Farrow et al.,
8、 2002),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,MIC conclusions,no strains resista
9、nt 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,Sub-MIC antibiotics,antibiotics have previously been shown to affect toxin production in C.d
10、ifficile vast amounts of literature showing effects on other bacteria though theres very little data for C. difficile,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.
11、 used sampled 3X a day for 104 hours OD600 measured each time and 1ml of supernate frozen for ELISA analysis,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
12、338a and 630 exceeds assay by ca. 48h,Sub-MIC conclusions,theres often a lag in the growth of the bacteria compared to the control main effect on toxin is that its elaborated quicker under sub-MIC conditions heterogeneity common between strains for toxin production and growth in response to antibiot
13、ics,RT-PCR,wanted to look for toxin transcripts to see if they correlate to sub-MIC work RNA concentrations low (ca. 5g/ml) 16S transcripts easily seen but only with Sensiscript enzyme low concentrations of RNA probably made toxin transcripts difficult to see,Sensiscript,Sensiscript vastly improves
14、ability to pick up 16S RNA still no transcripts from toxins decide to cut losses as time extremely short,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.
15、 Trizol, Tri reagent etc.,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,Control vs sub-MIC,gels very repr
16、oducible - 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,Typical 2D gel,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,
