Qnr gene; a member of Plasmid Mediate Quinolones Resistance

Qnr gene; a member of Plasmid Mediate Quinolones Resistance

1. 1. Introduction: T he discovery of plasmid-mediated quinolone resistance 1994 in Birmingham, Alabama, USA Transconjugants from a porin-deficient K. pneumoniae containing a plasmid ( pMG252 , coding for FOX-5) from a clinical isolate A moderate increase in the MIC of ciprofloxacin and other quinolones compared with the recipient strain

2. 1. Introduction: T he discovery of plasmid-mediated quinolone resistance Further studies on pMG252 allowed the gene responsible for quinolone resistance qnr ( q ui n olone r esistance) 2002 qnrA was identified 2008 Multiple families of Qnr proteins were subsequently discovered Qnr proteins were shown to be members of the pentapeptide repeat protein (PRP) family

3. 1. Introduction: T he discovery of plasmid-mediated quinolone resistance Two other mechanisms unrelated to Qnr , but also encoded by plasmid genes, have been discovered Acetyltransferase [ AAC(6’)- Ib-cr ] Aminoglycoside modification(and resistance) QepA and OqxAB Active efflux pumps

4. 2. Qnr proteins 2.1. Nomenclature of qnr determinants Concept Number priority Allele number assignment Family assignment Function qnr genes on chromosome A common nomenclature, a database was set up in 2008 Efsqnr from Enterococcus faecalis

5. 2. Qnr proteins 2.1. Nomenclature of qnr determinants 2016 2016 7 QnrA 80 QnrB 1 QnrC 2 QnrD 9 QnrS 7 QnrVC A common nomenclature, a database was set up in 2008

6. 2. Qnr proteins 2.1. Nomenclature of qnr determinants Concept Criteria Number priority Published manuscripts Accepted manuscripts Submitted manuscripts GenBank submission date Allele number assignment Full-length sequence available Natural alleles Nucleotide changes resulting in amino acid change Changes in promoter are not considered Family assignment 1. ≥30% difference in nucleotides or amino acids from existing families A common nomenclature, a database was set up in 2008

7. 2. Qnr proteins 2.1. Nomenclature of qnr determinants Concept Criteria Function Demonstration of reduced susceptibility to quinolones: Required for defining a new family Desirable (but not required) for defining a new allele qnr genes on chromosome qnr from a given organism or use, distinguishing initials for the consideredspecies qnr letter designation only used when the chromosomal gene is ≥70% identical to one in an already recognized family

8. 2. Qnr proteins 2.2. The origin of qnr genes Most data suggest that qnr genes originated from water-dwelling or other environmental microorganisms QnrA1 QnrA -like determinants Shewanella algae a few amino acid substitutions QnrB5, QnrB19 qnr -like gene in the chromosome ( Smaqnr ) S.marcescens 80% amino acid identity with QnrB -like and is able to reduce fluoroquinolones susceptibility when expressed in E.coli QnrS QnrS -like genes Vibrio splendidus 84% and 88% amino acid identity with QnrS1 and QnrS2

9. 2. Qnr proteins 2.2. The origin of qnr genes Dendrogram obtained for representative Qnr proteins ( plasmid- and chromosomally-located ) by neighbor -joining analysis, and tertiary mfpA is present in Mycobacterium and several Actinobacteria species, although the native function of these genes remains unknown

10. 2. Qnr proteins 2.2. The origin of qnr genes It has been suggested that Qnr proteins may be antitoxins protecting type II topoisomerases or that they could be involved in the SOS response . QnrA could contribute to the adaptation of Shewanella to low temperatures QnrB proteins may have a natural role in protecting the cell from naturally occurring DNA-damaging agents

11. 2. Qnr proteins 2.3. structure and activity of mutant forms QnrB1 folds as a right-handed quadrilateral -helix with a highly asymmetric dimeric structure

12. 2. Qnr proteins 2.3. Mechanism of action: pentapeptide proteins (structure and activity of mutant forms) Qnr proteins Function QnrA to reverse the ability of fluoroquinolones to inhibit DNA gyrase QnrB1 The tertiary (dimeric) and secondary (monomeric) structures of QnrB1 are shown.

13. 2. Qnr proteins 2.4. Regulation and information about the natural role of qnr genes LexA is the central regulator of the SOS response to DNA damage. Expression of qnrB / qnrD was augmented up to 9-fold by exposure to DNA-damaging agents C iprofloxacin , known to be a good inducer of the SOS system qnrB

14. 2. Qnr proteins 2.4. Regulation and information about the natural role of qnr genes No LexA binding site is found upstream from these qnr genes, although an upstream sequence is required for quinolone stimulation to occur qnrS

15. 2. Qnr proteins 2.4. Regulation and information about the natural role of qnr genes Expression of the qnrA gene is stimulated up to 8-fold by a cold shock qnrA Other conditions such as DNA damage, Oxidative Osmotic stress Starvation Heat shock

16. 2. Qnr proteins 2.4. Regulation and information about the natural role of qnr genes Someroles have been related to the following aspects: ( i ) the presence of chromosomally located qnr genes in species like S. algae, S. mal- tophilia and the Vibrionaceae family suggests that qnr genes can be acquired by pathogenic bacteria from water-dwelling or other environmental microorganisms; (ii) it has been suggested that Qnr may have been an antitoxin protecting DNA gyrase and topoisomeraseIV from some naturally occurring toxins (Ellington and Woodford,2006), although the theoretical toxin that Qnr protects them from has not been found. Qnr may work in a similar way to GyrI , a DNAgyrase regulator that is also capable of some antiquinolone effect( Chatterji et al., 2003); (iii) plasmid-borne qnrB alleles bear LexA -binding sites involved in the SOS response, suggesting that QnrB may play a natural role in protecting the cell from naturally occur-ring DNA-damaging agents (Da Re et al., 2009); (iv) cold shock induces qnrA expression in S. algae during growth arrest, suggesting that it may contribute to the adaptation of Shewanella to low temperatures (Kim et al., 2011)

17. 3. Other PMQR genes 3.1. Acetylation of quinolones [ AAC(6’)- Ib-cr ] AAC(6 ’ )- Ib-cr , a variant of an aminoglycoside acetyltransferase two amino acid changes 1. Trp102Arg 2. Asp179Tyr classic AAC(6’)- Ib causing resistance to tobramycin, kanamycin and amikacin

18. 3. Other PMQR genes 3.1. Acetylation of quinolones [ AAC(6’)- Ib-cr ] Able to acetylate ciprofloxacin , norfloxacin and other quinolones at the secondary amino nitrogen N4 on its piperazinyl substituent Quinolones that lack this substitution (i.e. levofloxacin) are not affected by this acetyltransferase o OH Ciprofloxacin

19. 3. Other PMQR genes 3.2. QepA and OqxAB efflux pumps It is able to eliminate hydrophilic fluoroquinolones from the cell (ciprofloxacin and norfloxacin especially) by an active efflux mechanism OqxAB

20. 4. Genetic background of PMQR genes Quinolone resistance genes have been found on plasmids of Varying sizes Incompatibility groups Associated resistance and almost all of them carry multiple resistance determinants

21. 4. Genetic background of PMQR genes qnrA found as part of the complex sul1-type integron containing the presumed recombinase IS CR 1  (Insertion sequence Common Region)

22. 4. Genetic background of PMQR genes qnrB2 , qnrB4 , qnrB6 , and qnrB10 alleles are associated with ISCR1 qnrB19 has been found in large plasmids associated with ISEcp1C-based transposons, large plasmids bracketed by IS26, and in smallColE1-type plasmids

23. 4. Genetic background of PMQR genes The qnrS1 gene is linked to a Tn3-like transposon or to IS26, IS2 or ISEcl2. The qnrS2 gene identified was part of a novel genetic structure corresponding to a mobile insertion cassette element, called the MIC (for Mobile Insertion Cassette)

24. 4. Genetic background of PMQR genes qnrC gene is found downstream of an insertion sequence also belonging to the IS3 family The qnrS2 gene identified was part of a novel genetic structure corresponding to a mobile insertion cassette element, called the MIC (for Mobile Insertion Cassette)

25. 4. Genetic background of PMQR genes qnrA and qnrB plasmids often harbor other antibiotic resistance genes that confer resistance to β -lactams Aminoglycosides Chloramphenicol Tetracycline Sulfonamides Trimethoprim Rifampin Contrast with qnrS plasmids. This difference could be related to the fact that qnrS -harboring plasmids are smaller in size or to their different origins

26. 5. Contribution of PMQR to quinolone resistance 5. Contribution of PMQR to quinolone resistance 5.1. Qnr (and other PMQR genes) expression and impact on MIC All PMQR determinants lead to a reduced susceptibility to fluoroquinolones Two or more unrelated plasmid-mediated quinolone resistance genes are present can be the cause higher levels of quinolone resistance

27. 5. Contribution of PMQR to quinolone resistance 5. Contribution of PMQR to quinolone resistance 5.2. Interaction with chromosomal mechanisms of quinolone resistance Clinical isolates of enterobacteria containing PMQR mechanisms generally show high levels of resistance to quinolones

28. 5. Contribution of PMQR to quinolone resistance The effect of PMQR on the MPC is similar to the effect of Ser83Leu substitution on the GyrA protein. The MIC values are higher if combined with additional mechanisms. These low-level resistance mechanisms facilitated the emergence of single step fluoroquinolone-resistant mutants in the population 5.3. Influence of PMQR on mutant prevention concentration(MPC)

29. 5. Contribution of PMQR to quinolone resistance 5.3. Influence of PMQR on mutant prevention concentration(MPC)

30. 6. Interplay between PMQR and bacterial fitness The acquisition of resistance plasmids by horizontal gene transfer Reduction in bacterial fitness due to the energy costs induced by the replication or expression of plasmids that can affect bacterial growth Fitness of bacteria is defined as the ability to adjust metabolism to suit environmental conditions, in order to survive and grow, and is a major physiological determinant

31. 6. Interplay between PMQR and bacterial fitness qnrS1 gene , alone or in combination with chromosomally-mediated mechanisms, seems to increase bacterial fitness qnrB1, qnrC or qnrD1 , alone or in combination with chromosomal mutations) is variable and seems to depend on the chromosomal mutations present in the bacteria.

32. ( i ) combinations causing no fitness cost; (ii) combinations causing a decrease in fitness cost; and (iii) combinations enhancing bacterial fitness. 6. Interplay between PMQR and bacterial fitness

33. ) Relationship between antibioticresistance and bacterial fitness (adapted from Machuca et al., 2014). Resistanceto fluoroquinolones in E. coli develops in multiple genetic steps. The progressiveaccumulation of plasmid- and/or chromosomally-mediated mechanisms leads tomodifications of the level of resistance to quinolones, as well as of bacterial fitness.S83L and S80R refer to specific amino acid modifications in GyrA and ParC proteins,respectively . marR means deletion of the marR gene. The discontinuous verticalred line indicates the critical ciprofloxacin concentration (1 mg/L) separating sus- ceptible from non-susceptible E. coli strains, according to CLSI guidelines

34. 7. Geographical distribution of PMQR determinants 1 66 Number of publications

35. 8. Detection of PMQR in the clinical laboratory Detection of PMQR is clinically important Increased risk of selecting higher levels of quinolone resistance conferred by additional mechanisms Failure of quinolone treatment.

36. 8. Detection of PMQR in the clinical laboratory The interpretation of susceptibility testing results has mostly been based on clinical breakpoints Defined taking into account data for strains harbouring mutations in the QRDR of the topoisomerase genes Presence of transferable resistance determinants Molecular methods

37. 9. Clinical relevance of PMQR The antibacterial activity of fluoroquinolones and the emergence of resistant mutants can be explained by pharmacokinetic/pharmacodynamic (PK/PD) parameters Concentration-Dependent Killing AUC/MIC

38. 39 Percent colonies recovered (100%) 100 1 Antibiotic concentration MIC The lowest antibiotic concentration that inhibits growth of 99% of a bacterial population 9. Clinical relevance of PMQR