Molecular typing insight on diversity and antimicrobial resistance of Campylobacter jejuni from Belgian chicken meat

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1 Molecular typing insight on diversity and antimicrobial resistance of Campylobacter jejuni from Belgian chicken meat Ihab Habib Ghent University Department of Public Health and Food Safety. Contents: Molecular typing, what and why? Multi-locus sequence typing!! Diversity and clonality of C. jejuni genotypes in chicken meat Antimicrobial resistance and clonality of C. jejuni genotypes in chicken meat Correlation between C. jejuni genotypes from chicken meat and human cases with diarrhoea

2 Molecular Typing: A methodology used to characterise certain genetic markers of microbial isolates at the subspecies level Epidemiological value!! Tracing sources (e.g. outbreak) and route of transmission Identifying and monitoring temporal and geographical patterns Monitoring critical control points within the food chain Identification of virulent subtypes: virulotyping. First generation: -RAPD -Ripotyping Trend in Campylobacter typing methods Later generation: -PFGE -AFLP -PCR RFLP (Fla) Going from fragment-based methods to sequence-based methods Next generation: -SVR sequencing -MLST (Multi-locus sequence typing) -Combination of these

3 MLST (Multi-locus sequence typing) Chromosomal DNA PCR Amplify 450-bp internal fragments of seven housekeeping genes Sequencing Sequence the seven gene fragments Each different sequence at a locus is given a different allele number The allele numbers at the seven MLST loci gives the allelic profile of the isolate Compare the allelic profile of isolate to those of all isolates within a central database on the web via the internet ( Housekeeping loci (genes) should be conserved with only minimal nucleotide changes due to conserved protein function Sequence variation allows for the assignment of alleles: Isolate A ATT CGG CAT allele 1 Isolate B ATT CGC CAT allele 2 A combination of alleles for all (7) loci provides an allele profile which can then be assigned to a sequence type (ST) Isolate A (1, 5, 6, 3, 4, 3, 1) ST-5 Isolate B (1, 5, 6, 3, 3, 8, 1) ST-51 Sequence types are grouped into clonal complexes based on similarity to a central allelic profile

4 Freely accessible Webbased data editing Free analysis tools e.g. eburst Motivation: Limited information on the molecular epidemiology of C. jejuni in Belgium No population-based surveys have been performed to investigate the molecular epidemiology of C. jejuni in chicken meat meant for human consumption. Aim of the work: Multi-locus sequence typing to study C. jejuni isolated from chicken meat preparations meant for human consumption. Characterize antimicrobial resistance in C. jejuni isolated from Belgian chicken meat for human consumption.

5 Methodology: 145 C. jejuni isolates from 135 samples of chicken meat preparation (Ready-to-cook) : Obtained from 5 companies (those with highest Campylobacter incidence), over 10 months. Isolated by direct plating Genotyping: MLST + PFGE Antibiotic susceptibility testing: Microbroth dilution method 6 antimicrobials: Erythromycin, Nalidixic acid, Ciprofloxacin, Tetracycline, Gentamicin, and Streptomycin. 20 st-50 [1] Diversity of MLST sequence types and clonal complexes: sequence types (STs) in 145 C. jejuni from 135 chicken meat samples -St-50 (19 isolates), ST-45 (10 isolates), ST-257 (10 isolates): Number of isolates st-19 st-21 st-2135 st-1728 st-290 st-3128 st-3550 st-44 CC-21 - These 3 sequence types together present almost quarter (26.8%) of the total collection studied by MLST st-45 st-583 st-418 st-11 st-2219 st-25 CC-45 st-257 st-990 CC-257 st-354 st-1073 CC-354 st-572 st-122 st-51 st-2034 st-2807 st-443 CC-206 CC-443 st-267 CC-283 st-3544 st-2274 st-2037 st-3546 st-2899 st-305 st-3548 st-3551 st-42 st-464 st-53 st-824 Not assigned

6 CC-21 CC-45 CC-257 CC-206 CC-354 CC-443 CC-42 CC-446 CC-575 CC-1034 CC-22 CC-48 CC-607 CC-702 NA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% B (n= 17) C (n= 23) D (n= 53) E (n= 19) I (n=33) Processing companies C. jejuni in clonal complex (CC)-21 and CC-45 were presented in chicken meat from all the five companies st-2324 st-1759 st-2883 st-2037 st-2899 st-3544 st-3546 st-2274 st-775 [2] Clonal population structure st-305 st-990 st-257 st-2807 st-824 st-2034 st-354 st-443 st-1073 G st-51 i H F st-3549 st-464 st-3551 st-3030 st-607 J st-2896 st st-1409 st-11 st-905 st-45 E st-704 st-2219 A st-25 st-3548 st-418 st-3547 D st-1045 st-267 B C st-572 st-583 st-22 st-122 st-48 st-42 st-1721 st-19 st-21 st-50 st-53 st-3128 st-1728 st-3550 st-883 st-290 st-44 st-2135 st-2258 C. jejuni population in Belgian chicken meat has some degree of clonality: 10 Clonal groups (eburst analysis) Group A: 24 isolates All in CC-45, limited antibiotic resistance Group B: the biggest, 32 isolates All in CC-21, high incidence of ciprofloxacin and tetracycline resistance Presented in all 5 companies, and in different product batches. Its genotypes are recurrent over 7 different months

7 [3] Antimicrobial resistance Cipro Tetra Strepto Nal Resistance if >1 >2 >2 > No. of isolates with MIC (mg/litre) > No. (%) 77 (53.1) 70 (48.2) 6 (4.1) 80 (55.2) - Erythromycin-resistant C. jejuni: 1 strain out of the Almost half of C. jejuni from chicken meat were resistant to Ciprofloxacin - High level of MIC for Ciprofloxacin and Tetracycline-resistant C. jejuni - 42 C. jejuni isolates were simultaneously resistant to Cipro and Tetra Example: [4] Antimicrobial resistant C. jejuni from Belgian chicken meat showed some level of clonality Dice (Opt:1.30%) (Tol 1.6%-1.6%) (H>0.0% S>0.0%) [0.0%-100.0%] PFGE smai PFGE smai CMP 13/3/2007 F cip+tet 78-2 CMP 13/3/2007 F cip+tet 78E1 CMP 13/3/2007 F cip+tet 79E1 CMP 13/3/2007 F cip+tet CMP 12/04/2007 M cip+tet+strep+gent CMP 18/04/2007 P cip+tet CMP 18/04/2007 P cip+tet CMP 21/08/2007 F cip+tet CMP 18/09/2007 M cip+tet 629 CMP 06/11/2007 C cip+tet CMP 21/08/2007 F cip+tet 11 isolates with simultaneous resistance to ciprofloxacin and tetracycline shared the same PFGE type. MLST revealed that all of the 11 isolates share same sequence type (ST), and that was ST-50 of the Clonal Complex 21. These 11 isolates From 5 different producers; Sampled over 5 different months; Obtained from 9 different samples those represent 6 distinguishable slaughter batches.

8 [5] C. jejuni genotypes in Human cases versus Chicken meat: In an on-going work: (primary results) Comparing C. jejuni genotypes from Belgian patients with diarrhoea in summer 2007 (seasonal peak), with genotypes profiles in chicken meat 40 C. jejuni isolates 39 human cases Brussels 17 out of the 40 (42.5%) C. jejuni strains from human diarrhoeal samples were found to share sequence types with strains from chicken meat preparations. 145 C. jejuni isolates 135 chicken samples Across-Belgium [6] New sequence types in Belgian chicken meat: - 8 sequence types representing 17 (11.7%) isolates were unreported previously (only identified in Belgian chicken meat, and not yet in other countries) one of the novel sequence type characterised in chicken meat was also characterised in a Belgian patient with diarrhoea (in Brussels) ST chicken meat isolates (different batches) -17/04/ /05/ /09/ Human case: 13/06/ All Ciprofloxacin resistant, share same PFGE type

9 Conclusion Now we know more? Successful C. jejuni genotypes!! ST-50, ST-45, and ST-257, CC-21, CC-45 and CC-257 -Are more prevalent -Recurrent over time (up to 7 months) High resistance to ciprofloxacin and tetracycline was detected in abundant number of C. jejuni isolates, and there is might be an evidence of dissemination of antimicrobial resistant clone C. jejuni from chicken meat share Faculty some of genotypes found in human cases with diarrhoea + a new ST (first to be reported in Belgium) was characterised in both Belgian chicken meat and a human case of But we still need to know more? Weather certain clonal groups are so common in the environment that is repeatedly infects Belgian broiler flocks or weather it has the potential to persist on farms or in slaughterhouses needs to be investigated.??? Does that reflect improper use of antimicrobials in broiler farms??!!??? Or could that be due to origin and transmission of resistance genes and/or propagation and spreading of resistant clones??!! More MLST to correlate between human/chicken isolates: Are certain genotypes more virulent than others (virulotyping!!!)? Acknowledgements: Supervisors: Prof. Lieven De Zutter (UGent- Fac Vet Med) Prof. Mieke Uyttendaele (UGent- Fac Bioscience Eng) Collaborators: Prof. Kurt Houf (UGent- Fac Vet Med) Dr. Olivier Vandenberg (National Reference Centre for Enteric Campylobacters- Brussels) Dr. William G. Miller (USDA-California)

10 Thank you