This document discusses antibiotic resistance in the probiotic bacteria Bacillus clausii. It finds that B. clausii has intrinsic resistance to several classes of antibiotics due to chromosomal genes. The resistance genes are not located on mobile genetic elements and resistance was not transferred to other bacteria in vitro, suggesting a low risk of resistance gene spread. Sequence analysis found the aadD2 gene, which confers aminoglycoside resistance, is localized in the B. clausii chromosome between other housekeeping genes.

1. Antibiotic resistance and probiotics P. Courvalin

2. The two types of resistance . Intrinsic or natural (insensitivity): - present in all the strains of the genus/species - not transferable - defines the spectrum of activity of an antibiotic . Acquired: - exogenous gene - mutation

3. Probiotics: Antibiotic multiresistance and safety . Pros: - co-administration with antibiotics . Cons: - transfer of resistance to human bacterial pathogens . directly . indirectly via the commensal flora - acquisition of resistance from human commensals - shortage of antibiotics in case of infection

4. Resistance mechanisms

5. Bacterial genome

7. Plasmid transfer by conjugation

8. Intra- and Inter-cellular mobility of Tn 916 Tn 916 plasmid chromosome Integration (Int) Excision (Int + Xis) Integration (Int) Donor Recipient Transfer (Tra)

10. Combinatorial genetics of antibiotic resistance Vector Bacteria Plasmids Transposons Host Mammals Bacteria Replicons

11. Potential for spread of resistance genes . High: - gene carried by a mobile genetic element (self-transferable plasmid or transposon) . Low: - chromosomal gene(s) responsible for insensitivity - present in all the strains of the genus/species - absence of in vitro / in vivo transfer - absence of known acquired genes - genus/species specific gene - co-localisation with chromosomal genes - flanking sequences: . house-keeping genes . no mobile genetic element

12. Phenotype Susceptible Resistant Transferability Yes No Acquired genes Yes No Insensitivity or mutation No convincing evidence Convincing evidence OK Not OK Algorithm for decision making

13. Antibiotic resistance in Bacillus clausii Resistance to No of resistant strains Penicillins Cephalosporins Aminoglycosides (kanamycin, tobramycin, amikacin) Macrolides Tetracyclines Chloramphenicol Rifampicin 4 4 SIN 4 T 4 NR Susceptible to carbapenems, co-trimoxazole, fluoroquinolones, gentamicin, glycopeptides, oxazolidinones, streptogramins

14. Risk assessment of resistance dissemination . Identification of the resistance genes - cloning and deduced sequence comparison . Characterisation of the biochemical mechanisms . Elucidation of the genetic basis - co-localisation with a chromosomal gene - sequence analysis of the flanking DNA . Attempts of in vitro resistance transfer to: - Enterococcus faecalis and Enterococcus faecium - Bacillus subtilis

15. Sequence comparison of aminoglycoside 4’- O -nucleotidyltransferases

16. Localisation of aadD2 in Bacillus clausii Driver NR SIN T Ref. OC NR SIN T Ref. OC Probe rrs aadD2

17. Sequence comparison of aadD2 promoters in B. clausii S I N A A C AG C T A TG A CA T G A TT A C G AA T T C AA G C G TG C G A AA T C G TC A C T AC C T G AA A C A AA T G D S M 8 71 6 A A C AG C T A TG A CA T G A TT A C G AA T T C AA G C G TG C G A AA T C G TC A C T AC C T G AA A C A AA T G * * * ** * * * ** * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * - 3 5 - 1 0 -3 5 S I N G C A AA C A A TT A TA T C A TT T T C C T T G A C A A T G A AA T G C CT T T T T G G T A A C C TT T T C TT A A T - 3 5 - 1 0 D S M 8 71 6 G C A A AC A A T TA T G T CA T T T TC C T T G C C A A T G A AA T G C CT T T T A G G T A A C C TT T T C TT A A T * * * ** * * * ** * * * * * ** * * * ** * * ** * * * ** * * * ** * * * * * * * ** * * * ** * * * ** * * - 1 0 S I N C A A A C A TA T A G G GG T G G TT C A A GT G C A CC G A A AA A AA G T C CC C C G TT A A T CT C G A GG T C C D S M 8 71 6 C A A AC A T A TA G GG G T G GT T C A AG T G C AC C G A AA A A A AG T C C CC C G T TA A T C TC G A G GT C C * * * ** * * * ** * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * * ** * * R B S S I N T C C AT T C A TC G TT C A T GT C A C AC A C T CA A A T TG A A A C A A A AG G A G A T G G A AG T G A A T G D S M 8 71 6 T C C AT T C A TC G TT C A T GT C A C AC A C T CA A A T TG A A A C A A A AG G A G A T G G A AG T G A A T G

18. The aadD2 genomic environment aadD2 blaI blaR blaZ ytrA gntR ABC Transporter 1232 3073 4322 3552 3477 3097 1176 181

19. Antibiotic resistance in Bacillus clausii Resistance to Mechanism Known gene Genetic basis Presence in ref. strains Penicillins Inactivation No Chromosome + Cephalosporins Inactivation No Chromosome + Aminoglycosides Inactivation No Chromosome + (kanamycin, tobramycin, amikacin) Macrolides Target modification No Chromosome + Tetracyclines ? ? ? - Chloramphenicol Inactivation No Chromosome - Rifampicin Target modification Mutation Chromosome - R genes - not located on a plasmid (Tc?) - not transferable in vitro

20. Acknowledgements . Service de Microbiologie, CHU Côte de Nacre, Caen Roland Leclercq, Bülent Bozdogan, Sébastien Galopin . Unité des Agents Antibactériens, Institut Pasteur, Paris Guy Gerbaud