Diagnostic microarrays for antimicrobial resistance bacterial gene (ABG) identification.

1. Diagnostic microarrays for antimicrobial resistance bacterial gene (ABG) identification. Philippe Garneau1, Donald Tremblay1, Olivia Labrecque1,3, Christine Maynard2, Serge Messier1, Luke Masson2, Marie Archambault1 and Josée Harel1 1Université de Montréal, Faculté de Médecine Vétérinaire, 2 Institut de Recherche en Biotechnologie, CNRC 3Ministère de l’Agriculture et des Pêcheries du Québec

2. Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Traditionally, the predominant techniques used to identify pathogens have relied upon culture-based morphological approaches. Various culture-independent molecular characterizations, especially those involving PCR amplification of pathogen-specific nucleic acid targets. Effective Detection of few pathogens Microarrays may enable highly parallel detection of diverse organisms and their characterization. Introduction

3. Comparison between various techniques of molecular diagnosis

4. A microarray is a solid substrate (ex:glass slide) on which DNA or oligonucleotides are attached. Spotted DNA microarrays Oligonucleotides arrays Samples of DNA are fluorescently labelled with fluorochromes Cy5. Hybridization with the labelled sample and immobilised probes. Scanning of the slide. The intensity of the color is proportional to the amount of Cy5-labelled DNA hybridized. Microarray technology

5. Micro-array Whole pathogen genome Design of oligonucleotides Labeling Synthesis Printing on slides Laser PMT Hybridization Scanning Advantages Cost-effective method Simultaneous gene analysis (max. 25 000 genes) Highly specific technique Detection of viable but non-cultivable organisms Uses Molecular diagnostic Molecular epidemiology Detection of specific genes Gene expression profiling

6. Diagnosis Organism identification Novel pathogens Epidemiology Outbreaks Historical stuides Evolution Antimicrobial resistance Bryant PA, et al. 2004. Lancet Infect Dis. 4. Microarray Diagnosis

7. Global project Main goal: To develop a powerful molecular diagnostic tool (micro-array) Escherichia coli virulence and antimicrobial resistance gene micro-array Virulence genes detection micro-array Bacterial identification micro-array Antimicrobial resistance genes detection micro-array Detection of transfer genes between bacteria Detection of present and emerging bacteria To develop a software to simplify the result interpretation Appropriate antibiotherapy Surveillance Prevention Control Efficient infection disease treatment Epidemiology of Escherichia coli

8. Design and synthesis of the oligonucleotides, printing on slides Oligonucleotides printed in triplicate Optimization of the hybridization conditions Validation of the prototype by  Hybridization of a collection of reference and well-characterized AMR strains  Reproducibility  Comparison of the results with those obtained by PCR (Source: www.nrc-cnrc.gc.ca) (Source: personal) Microarray Development Methodology

9. Antimicrobial resistance oligonucleotides Design of the oligonucleotides • Bibliographic searches: Resistant gene sequence characterized in gram-negative and gram-positive bacteria • Published PCR primers lengthened to 70 bases • Selectivity tested through BLAST searches using Oligopicker software Correspond to antimicrobial agents and related resistance genes • Corresponding to 63 antimicrobial acquired resistance genes and their variants found in various gram-positive strains (Maynard et al., 2003 and 2004; Bruant et al., 2006)

10. Labelling of bacterial DNA with Cy5-dCTP Lysed single colonies or small overnight cultures 38 Gram+ strains resistant to at least one antimicrobial Control strains for 63 Gram+ genes Hybridization Overnight, 50°C, with 1 mg of labeled DNA Microarray hybridization analysis Scanarray Express software Normalization & present/absent determination on MS-Excel . Biological and technical replicates Hybridization methodology Hybridization (50°C) Cy5-dCTP Analysis of microarray S. aureus DNA (lysate) Labeled DNA

11. Hybridization result Positive results Negative controls Positive controls

12. Validation Validation of our microarray results • Reference strains, and PCR Excellent reproducibility • Biological and technical replicates: Identical results for 2 hybridizations at 2 different times Significant reduction of effort and time Our oligonucleotide microarray is suitable for the detection of multiple antimicrobial resistance genes in gram positive and gram-negative bacteria.

13. Test STUDY Correlation between AMR phenotype and genotype in Gram+ ISOLATES

14. Tested resistance genes specific to Gram positive bacteria (n=63)

15. Resistance genotypes of 38 different staphylococcal isolatesa a The 418 S. aureus isolates were tested for phenotypic resistance to penicillin G and, oxacillin (beta-lactams), to tetracycline, gentamycin (aminoglycoside), lincomycine and pirlimycine (lincosamides), erythromycin (macrolide) and, enrofloxacin (quinolone). Thirty-eight isolates were resistant to at least one antimicrobial.

16. Several strains (22) were blaZ positive Two (2) strains were positive for tet genes (one tetM, the other tetK) Although many strains (14) were phenotypically resistant to lincomycin, one strain was linA and 2 were ermB positive Most strains were norA positive (36/37) No methicillin resistance genes were found Microarray results were confirmed by PCR. AMR genes in S. aureus Resistance genes Very few isolates carried more than one gene • Two strains carried both blaZ and tet genes • One strain was multigenic ant(9)-Ia(aadA9), ermA, spc

17. Relationship between resistance gene status determined by microarray and phenotypic resistance • Sensitivity was calculated as the number of resistance gene-positive strains with phenotypic resistance/the number of strains with phenotypic resistance (indicated in parentheses). • Specificity was calculated as the number of resistance gene-negative strains with phenotypic susceptibility/the number of strains with phenotypic susceptibility (indicated in parentheses). • Pen, penicillin; Ery, erythromycin; Lin, lincomycin; Tet, tetracycline. The status of the related gene(s) was determined by microarray hybridization.

18. The presence of more than one antibiotic resistance determinant appears to be a rather rare occurrence in the bovine S. aureus strains isolated from dairy cows in Québec. Among resistant isolates many isolates carried beta-lactamase gene (blaZ), some carried macrolide/clindamycin genes (ermA, ermC)or tetracycline resistance genes (tetK and tetM)and lincomycin (ermB, linA) gene. The microarray platform has the advantageof rapidly screening bacteria for the presence of known antibiotic resistance genes in bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance. Conclusion

19. Microarrays available at UdeM molecular diagnostic service and BRI • AMR microarray • 182 probes: • 14 antibiotic families from Gram positives • 15 antibiotic families from Gram negatives E. coli virulence & AMR microarray 289 virulence and AMR genes detected, covering several variants (~480 probes) 11 virotypes & >12 antibiotic families represented • MiniVir microarray • 208 probes (with controls): • 14 antibiotic families for Gram negatives • E. coli virulence genes

20. Acknowledgments This work was supported in part by a grant from the Fond Québécois de la Recherche sur la Nature et les Technologies (FQRNT)/Centre de Recherche en Infectiologie Porcine (CRIP) (#11946). We are indebted to many researchers for kindly providing us with control strains. We thank Jacinthe Lachance for the graphical work of the presentation. Martinez et al. Dev Biol (Basel) 2006 Bruant et al. AEM 2006 Maynard et al. AAC 2003 Perreten et al. JCM 2004 Zhu et al. JCM 2007 References