+ Clonal expansion of ciprofloxacin-resistant Campylobacter jejuni Jasna Kova č, Alison J. Cody, James E. Bray, Kate E. Dingle, Sonja Smole Mo ž ina, Martin C.J. Maiden

+ Oxford, United Kingdom Ljubljana, Slovenia

+ Campylobacter !

+ Importance of Campylobacter Food safety aspect 23.6% broiler meat on the market contaminated, in some EU countries up to 80% ! Public health aspect #1 bacterial cause of gatroenteritis in western world

+ Antibiotic resistance in Campylobacter Most of the human campylobacteriosis cases are caused by Campylobacter jejuni Disease is usually self-limiting, but severe cases need clinical treatment with antibiotics Quinolones (ciprofloxacin) and macrolides (erythromycin) are the first drugs of choice for campylobacteriosis treatment High prevalence of antibiotic resistance is becoming a serious problem

+ Prevalence of antibiotic resistance in Campylobacter EFSA, % !

+ Antibiotic resistance mechanisms Multidrug resistance (cmeABC) porA, omp50 Tetracycline (tetO) Quinolones (gyrA) Aminoglycosides (aphA-3 …) β -lactams (Cj0299) porA, omp50 Iovine, 2013 Plasmid encoded

+ Prevalence of antibiotic resistance - Studied population Campylobacter strains isolated from patients in Oxfordshire The hospital covers population of 600,000, which is about 1% of UK population  the results are representative for UK Time frame: 2 years, 1714 isolates Year 1 (June June 2012): 839 isolates Year 2 (June June 2013) : 875 isolates TotalYear 1Year 2 STs CCs3331 rSTs

+ Whole genome sequencing Illumina HiSeq bp paired-end reads generated Assembled with Velvet version Established automatized genome assembly and annotation pipeline

+ PubMLST database Consists of two separate databases that interact: Sequence and profile definitions (allele seq. definitions) PubMLST Isolate Database (metadata)

+ BIGS Bacterial Isolate Genome Sequence Database (BIGSdb) Pubmlst.org/campylobacter database runs on BIGS 1,667 loci defined in the database, including: MLST loci Ribosomal (rMLST) loci porA antigen-encoding genes etc. BIGSdb autotagger automatically: Identifies loci Assigns alleles Tags the sequences for future reference Automatic report of the MLST alleles, sequence types (STs), clonal complexes and porA alleles, genome comparison…

+ Genome comparator module in BIGS Calculates the distance matrix based on the differences in allelic types (number of loci that differ between each pair of isolates) The distance matrix is generated in NEXUS format NEXUS format is used as an input for SplitsTree phylogenetic analysis SplitsTree generates the phylogenetic network based on the split decomposition neighbor-net method Advantage of networks over trees is that they account for the recombination events

+ Antibiotic resistance prevalence Gene% Resistant Year 1Year 2 QuinolonesgyrA TetracyclinestetO β -lactamsCj AminoglicosidesaphA-3, aadE, sat40.7 MDRgyrA, tetO, Cj Unique variants gyrA243 tetO222 Cj aphA-34 aadE_B1 aadE_Ef2 sat44 cmeB267 porA442 MOMP227 omp50222 The phenotypic resistance to QN was determined in a subset of 685 isolates from year two  over 97% congruence between phenotypic and genotypic methods of resistance determination

+ Association of quinolone resistance with MLST genotypes Green colour (*) represents statistically significant association of genotype with with quinolone resistance.

+ Increased prevalence of ST-464 over the years

+ How does the quinolone resistance spread? By many individual events of resistance mutation or with increased survival fitness of specific resistant clones?

core loci Genome comparator (distance matrix) SplitsTree (wgs phylogenies) Blue = susceptible genotype Red = resistant genotype

+ Clonal expansion of quinolone resistance 1515 core loci 1525 core loci Blue = susceptible genotype Red = resistant genotype

+ Co-resistance to quinolones and tetracycline – chicken associated clonal complexes Co-resistance to CIP and TET Year 1: 221/839 (26.3%) Year 2: 217/875 (24.8%) Co-resistance to CIP, TET and BL Year 1: 179/839 (21.3%) Year 2: 193/875 (22.1%) CIP … ciprofloxacin TET … tetracyclin BL … β -lactams Isolates from year 1 and 2 TET R is chromosomally encoded in ST-464 and ST-354 Alignment, NJ tree produced in MEGA

+ Why are these two genotypes so successful? Random forest analysis in Orange data mining tool to identify genes that are associated with this genotype.

+ Conclusions High prevalence of quinolone resistance in Campylobacter in UK (38%) MLST clonal complexes ST-464 and ST-354 are highly associated with quinolone resistance Quinolone resistance is expanding clonally in ST-464 and ST-354 High level of co-resistance to quinolones, tetracyclines and β - lactams (21-22% MULTIDRUG RESISTANCE) New approaches to managing antibiotic resistance and preventing the proliferation of resistance along the food production chain are needed

Thank you for your attention! Now… Thanks to ICGEB, BIOS, and Genetic Society of Slovenia for travel grant.