MLST and antibiotic resistance determination of Swiss Campylobacter using a multiplex scheme and online database

Transcription

1 MLST and antibiotic resistance determination of Swiss Campylobacter using a multiplex scheme and online database Peter Kuhnert Institute of Veterinary Bacteriology

2 Overview > 1. Introduction Situation in Switzerland Characterization of Campylobacter > 2. Optimized and extended genotyping multiplex protocol online genotyping tool > 3. Application of optimized genotyping Swiss genotypes from different sources - overview Swiss genotypes determined from baseline study 2008 > 4. Conclusions and outlook 2

3 1. Introduction Situation in Switzerland > Reported campylobacteriosis cases > 2008: incidence of >100 per inhabitants 3

4 1. Introduction Characterization of Campylobacter > Identification can be problematic in not well trained laboratories > Standard typing tool for international comparison is missing > Increasing antibiotic resistance observed A universal genotyping tool could cover all aspects 4

5 1. Introduction Three level genetic characterization I Identification II Typing III Antibiotic resistance rpob gene MLST 7 house-keeping genes extended typing flaa (flagellin A) flab (flagellin B) macrolide 23S rrna quinolone gyra 5

6 1. Introduction Three level genetic characterization I Identification II Typing III Antibiotic resistance rpob gene MLST 7 house-keeping genes extended typing flaa (flagellin A) flab (flagellin B) macrolide 23S rrna quinolone gyra 6

7 1. Introduction MLST: Allel number Sequence type clonal complex 1 Compare sequence to database aspa allele number 15 Etc. for all 7 genes 2 Compare sequence to database aspa allele number 33 Etc. for all 7 genes aspa atpa glmm glna glta glya tkt Sequence type Strain Strain Clonal complex: formed by sequence types that share at least 5 allels 7

8 1. Introduction MLST as the method of choice advantages > Highly reproducible and standardisable method > Isolates easily and directly comparable between laboratories world-wide > Unambiguous sequence data > Protocols and primers easily distributed > Houskeeping genes evolve slowly: investigation of weakly clonal population limitations > Species/strain specific primers > Time- and cost-consuming > No species identification > Short-term investigation might be limited > No international collaborative and cumulative database > PubMLST: the reference but limited (e.g. data privacy, trace files, defined length, copy/paste) 8

9 1. Introduction Three level genetic characterization I Identification II Typing III Antibiotic resistance rpob gene MLST 7 house-keeping genes extended typing flaa (flagellin A) flab (flagellin B) macrolide 23S rrna quinolone gyra 9

10 1. Introduction Basis of antibiotic resistance > Macrolide resistance: 23S rrna gene Macrolides bind to the 23S rrna of the ribosome and inhibit protein synthesis 2 individual point mutations in the 23S rrna can neutralize the antibiotic (A2074G/C and A2075G) > Quinolone resistance: gyra gene Quinolones bind and inactivate gyrase involved in DNA replication 2 individual point mutations in the gyra gene can prevent binding of quinolones to gyrase (C257T and A256G) 10

11 2. Optimized and extended genotyping Identification, typing, antibiotic resistance > Universal primer set for C. jejuni and C. coli applicable for PCR and sequencing > Handling of PCR and sequencing > Easy data analysis end entry 11

12 2. Optimized and extended genotyping Universal primers compatible with PubMLST Target gene UniBern sequence size (bp) PubMLST sequence size (bp) Difference (bp) glmm aspa glna tkt glya atpa glta > Reduction from 65 primers to 14! > Fragments containing additional sequence information 12

13 2. Optimized and extended genotyping PCR > Universal primer sets for C. jejuni and C. coli rpob 7 MLST loci / flaa / flab 23S rrna / gyra > Problem: labour-intensive, costly and susceptible to mix-up 12 PCR reactions / strain! 12 PCR column-purifications / strain! 24 sequencing reactions / strain! Improper for large-scale investigation! 13

14 2. Optimized and extended genotyping Workflow 1500 bp bp bp bp bp bp bp bp bp bp - M AG1 AG2 AG3 1. PCR OF TWELVE TARGETS IN THREE AMPLIFICATION GROUPS AG AGAROSE GEL ELECTROPHORESIS 3. ENZYMATIC PURIFICATION Pooling of PCR products in a single tube, adding of ExoI/rAPid Alkaline Phosphatase mix and incubation in thermocycler. 4. PREPARATION OF SEQUENCING MIX Adding of BigDye and sequencing buffer to purified PCR products. 5. SEQUENCING Transfer of sequencing mix into wells containing dried primers and sequencing in thermocycler. AG3 AG2 glta atpa glya rpob L R L R L R L R flab tkt glna gyra L R L R L R L R flaa aspa glmm 23S rrna 6. ONE STEP ETHANOL PURIFICATION Adding of 60% EtOH / 0.5 mm MgCl 2 and precipitation by centrifugation. AG1 L R L R L R L R 7. RUN ON AUTOMATED SEQUENCER A B C D E F G H 14

15 2. Optimized and extended genotyping Data analysis: SmartGene IDNS technology > Web-based solution for the analysis, archiving and networking of complex data > Password protected multi-point access via an encrypted and secured connection > Archival database with epidemiological info > Integrated sequence analysis tool (Proofreader ) > Automated typing via link to PubMLST > Mutation finder for antibiotic resistance determination 15

16 2. Optimized and extended genotyping Database access 16

17 2. Optimized and extended genotyping SmartGene MLST workflow Storage Direct Upload Quality link of check with Proofreading Create of trace and Public Typing report, typing of files segmentation DB of compare result trace with files batch Auto-fill of upload sequences sample samples the corresponding of tool typing fields form Sequencer Upload of trace files with batch upload tool Direct link with Public DB Auto-fill of typing form PubMLST Typing of sample Proofreading of trace files Quality check and segmentation of sequences Create report, compare samples Storage of typing result in the corresponding fields IDNS TM Contains information from 17

18 2. Optimized and extended genotyping Data collector Data Collector Centralized data analysis (read only) read only Lab 1 shared data online encrypted Lab 2 UBERN Lab x Basis for tracing and monitoring Allows collaborative studies 18

19 3. Application of optimized genotyping > Swiss genotypes from various sources & years > Swiss genotypes found in broiler during baseline study

20 3. Application of optimized genotyping Swiss genotypes from various sources > Validation of the optimized approach > Total of >320 isolates (180 C. jejuni, 141 C. coli) > Sampled between 1993 and 2003 > Human, cattle, pets, poultry, pig, water 20

21 3. Application of optimized genotyping Multiplex results > rpob, MLST, gyra, and 23S rrna gene targets were determined for all isolates > flaa and flab were problematic with C. coli With 1/3 of strains no amplification with flaa flaa and flab sequences were ambiguous for a few strains Alternative primers or single PCR solved the problem 21

22 3. Application of optimized genotyping MLST results > 118 different STs, 34 of them new (~30%) > Predominant C. jejuni genotypes: CC21 (23%), CC45 (15%), CC48 (9%), CC206 (8%) > Predominant C. coli genotypes: 90% of isolates belonged to CC828 including 13 new STs > Differences in flanking sequences for a few ST 22

23 3. Application of optimized genotyping fla-typing: cluster analysis > flaa (DI=0.855) higher resolution than flab (DI=0.799) > High congruence between the two (98.5%) but <5% congruence with MLST > fla sequences can be combined with MLST to increase resolution power MLST, DI=0.788 MLST/flaA, DI=0.958 MLST/flaB, DI=

24 3. Application of optimized genotyping Antibiotic resistance > Quinolone: 31% C. jejuni 40% C. coli > Macrolide: 0% C. jejuni 21% C. coli > All genetically identified resistances were confirmed by MIC resistance testing 24

25 3. Application of optimized genotyping Summary > The optimized genotyping approach was successfully applied with >320 isolates > Many new STs were determined for Swiss isolates from various sources > flab might be the better choice than flaa (stability, PCR/sequencing) > High antibiotic resistance towards quinolones 25

26 3. Application of optimized genotyping Swiss genotypes from baseline study 2008 > 5 slaughterhouses that cover 80% of broiler production in Switzerland > Isolates were received from the Swiss NRL (ZOBA) > 340 isolates (114 caecum and 226 neck skin) > 95 paired samples 26

27 3. Application of optimized genotyping Summary > STs found in poultry in Switzerland are the ones found in other countries > Few regional differences > Types found on neck skin derive in >50% of cases from flock > Other ways of contamination > Antibiotic resistance situation is less pronounced that in other countries 27

28 4. Conclusions and outlook > Optimized multiplex approach suitable for routine investigations (ID, typing and antibiotic resistance) > SmartGene IDNS global typing database for networked data analysis and epidemiological comparison (EFSA) > Future studies: analysis of geographically and temporally associated isolates from human cases, (retail) poultry meat and other potential sources for Campylobacter infection > Source attribution can disclose possible intervention and prevention strategies 28

29 Acknowledgements Bożena Korczak Monika Zurfluh Jacqueline Kuhn Gudrun Overesch Simone Wirz Bern Lausanne Lausanne Stefan Emler (SmartGene) 29