Mechanisms of colistin resistance in Gram negative bacteria

Transcription

1 Mechanisms of colistin resistance in Gram negative bacteria BARON Sophie Interne en biologie médicale 2 st year PhD student Supervisor: ROLAIN Jean-Marc

2 I. Increase of antibiotic resistance. Introduction Increase of carbapenemase producing Gram negative bacteria : Diene et al. Expert Rev Anti Infect Ther Colistin: An update on the antibiotic of the 21 st century. Biswas et al., Expert Rev Anti Infect Ther Colistin : therapeutic alternative? Fig 1. Number of citation found in pubmed between 1951 and 2011 using key words «colistin» and «colistin resistance

3 Introduction II. Mechanisms of colistin resistance : knowns and unknowns MgrB PhoPQ? PmrD CrrAB? PmrAB TupA/glycosyltransfer ase? ArnBCADTEF PmrC Mcr-1 4-amino-4-deoxy-Larabinose phosphoethanolamine Modification of lipid A portion of the lipopolysaccharide causing colistin resistance Various genes involved in activation of lipopolysaccharide-modifying genes : aminoarabinose, phosphoethanolamine (Baron et al, IJAA, 2016) 2015 : Emergence of a plasmid-mediated colistin resistance mcr-1 (Liu et al. Lancet Infect Dis., 2016)

4 Molecular mechanisms of polymyxin resistance: knowns and unknowns E. coli +++ but also in Klebsiella pneumoniae. Baron S. et al, Int J Antimicrob Agents, 2016

5 Review Authors: Sophie Baron, Linda Hadjadj, Jean-Marc Rolain* and Abiola Olumuyiwa Olaitan* PLAN : INTRODUCTION OVERVIEW OF COLISTIN RESISTANCE UPDATES ON WHAT IS KNOWN IN COLISTIN RESISTANCE Modification of the lipid A or Kdo with L-ara4N Modification of the lipid A or Kdo with PEtN. Others LPS modification Loss of complete LPS Efflux pump mechanism Capsule formation WHAT IS NEW Modification of lipid A CrrAB two component system. Co-association of beta-lactam and colistin resistance via unusual mgrb truncation Mcr-1 plasmid mediated resistant gene. WHAT IS STILL UNKNOWN To be submitted in IJAA, IF=4.296.

6 III. Colistin animal consumption and human resistance Emergence of colistin resistance in human without colistin usage : involvment of colistin wild usage in animal? Olaitan AO et al. IJAA, 2016 Evidence of clonal transmission of a Escherichia coli from pig to human. Olaitan AO et al., JAC First detection of plasmid mediated mcr-1 gene in a pig in China. Liu et al, Lancet Infect Dis, Detection in Algeria of mcr-1 gene in chicken. Olaitan AO et al., Lancet Infect Dis, 2016

7 Objectives Objective: To decipher mechanisms of colistin resistance in Gram negative bacteria by different approaches: Genomic comparative analysis Phenotypic tests Part I: Identify the molecular support of colistin resistance in intrinsically resistant bacteria: Project 1 : Comparison of a Proteus vulgaris susceptible to colistin strain with a resistant one. Project 2 : Comparison of a Serratia marcescens susceptible to colistin with a resistant to colistin, and by in vitro tests Part II: Identify the molecular support of colistin resistance in Enterobacteriaceae. Project 3 : Identify the molecular support of colistin resistance Enterobacteriaceae isolated in clinical samples at University Hospitals of Marseille

8 Objectives In Algeria : Isolation from a clinical sample (pus) of a Proteus vulgaris colistin-susceptible isolate along with a colistin-resistant P. mirabilis Isolation from a clinical sample (urine) of a Serratia marcescens colistin resistant isolate. MS : Proteus vulgaris MS : Proteus mirabilis MS: Serratia marcescens

9 Materiel & Methods BACTERIAL STRAINS PHENOTYPIC STUDY 2 P. vulgaris strains isolated from a clinical sample (pus): CSUR P1868_S to colistin CSUR P1867_R to colistin 2 S. marcescens strains isolated from a clinical sample (urine): S. marcescens G429_S to colistin S. marcescens oxa-48_r to colistn ANTIBIOTIC SUSCEPTIBILITY ELECTRONIC MICROSCOPY LIPOPOLYSACCHARI DE EXTRACTION & MS ANALYSIS PHENOTYPE MICROARRAY FOR MICROBIAL CELLS AMX SXT IPM CIP ATM AMC CTX OFX FOX CRO TCC TIC GM TOB AK CT

10 Materiel & Methods GENOMIC STUDY SEQUENCING, ASSEMBLY, MAPPING & ANNOTATION Illumina/ionTorrent Mauve COMPARATIVE GENOMIC ANALYSIS Genes comparison Multiple alignment Phylogenetic analysis Genes of interest : Genes absents in susceptible strain Genes involved in polymyxin resistance : arnbcdatef operon Resistome with ARG-ANNOT RAST CLUSTALW MEGA5 PCR/SEQUENCING TRANSCRIPTOMIC STUDY GENES EXPRESSION Quantitative RT-PCR

11 a. AMX ATM 1. ANTIMICROBIAL SUSCEPTIBILITY AMC FOX IMP CTX CRO TCC TIC SXT CIP OFX GN TOB AK CT P1868_S b. c. P1868_S P1867_ P1867_R P1868_S R Fig 1. a. Antimicrobial susceptibility of P1868_S and P1867_R. b. Colistin E-test of P1868_S and P1867_R. c. Culture on cepacia medium AMX SXT IPM ATM AMC CTX FOX CRO TCC Project 1: Results CIP OFX TIC GM TOB AK CT P1867_R b. Genomes characteristics of P. vulgaris strains and reference strain ATCC Sequencing technology Number of contigs G427 (P1867_R) ATCC 49132_R G428 (P1868_S) Illumina Illumina Ion torrent Lenght (bp) %G+C 38.1% 37.9% 37.8% Plasmid - - Yes 140 kb Proteins Number of RNAs 10 rrna and 73 trna 12 rrna and 77 trna Resistome of P. vulgaris P1868_S 9 rrna and 74 trna Antibiotic class Gene % aa identity Beta-Lactams Oxa Per Aminoglycosides AadB AadA1 100 Aph(3 ) 100 -Ia AacA Fluoroquinolones Aac(6 ) Ib-cr Chloramphenicol catb2 100 Cm1A5 100 cata1 100 Sulfonamides Sul1 100 Trimethoprim DfrA1 100 DfrA5 100 Macrolides MphE 99 MsrE 100 EreA 100 lnuf 99.75

12 Project 1: Results 2. CELL WALL AND LIPID A ANALYSIS P1867_R P1868_S Lipid A Heptaacylated RESISTANT +131 Da m=13.8 nm MM=1960 Da m=2,10 nm Loss of aminoarabinose Decoration of lipid A Lipid A SUSCEPTIBLE Hepta-acylated a. b. c. Significant decrease of cell wall thickness (red ruthenium staining) Fig 1. Lipid A MALDI-TOF spectra of P1867_R and P1868_S

13 Project 1: Results 3. Genomic/transcriptomic : ArnBCADTEF operon in P. vulgaris In review for JAC. ArnF ArnE ArnT ArnD ArnA ArnC ArnB Inactivation of the arn operon by a mutation in ArnD SNP in P1867_R nssnp in P1868_S nssnp In Proteus sp. PROVEAN Score in P1868_S R29 K29 22/27 N (0.846) ArnD G119 A119 1/27 Deleterious (-4.489) To do: complementation of the colistin susceptible strain with ArnD Fig 1. ArnA alignment of proteins found in Proteus, Morganella, Providencia and Escherichia coli. Ct RplS Ct ArnB Ct ArnT1 Fold change of ArnB P1867_R P1868_S Fold change of ArnT Table 1. Results of quantitative RT-PCR of ArnB and ArnT in P. vulgaris P1867_R and P1868_S.

14 Project 2: Serratia marcescens Smar CS Smar oxa48 PHENOTYPIC STUDY Smar CS Smar oxa48 GENOMIC STUDY G429_S SM39_R Size 5,169 Gb 5,23 Contigs GC content 59,9 59,8 Number of proteins Number of RNAs 6 rrna, 83 trna, 1 other 22 rrna, 87 trna, 1 other plasmid Yes (73 kb, 52.5%) yes Resistant genes Bla CTX-M-15 +Bla TEM-169 Aac3-Iid + Aph3''Ia Table. General characteristics of the genome of S. marcescens - Antibiotic susceptibility of Serratia marcescens G429_S (left) SUSCEPTIBLE and oxa48_r (right) RESISTANT SXT AMX IPM OFX SXT AMX IPM OFX FOX AMC CRO CIP FOX AMC CRO CIP ATM CTX TIM TIC ATM CTX TIM TIC GM TOB AK CT GM TOB AK CT

15 Project 2: Serratia marcescens PHENOTYPE MICROARRAY Colistin resistant 2701 Colistin susceptible Differences PM1 A2 : L-arabinose D3: D-glucosaminic acid PM2A C7: B-methyl-D-galactoside G4: L-arginine G5: Glycine

16 Project 2: Serratia marcescens Efflux pump mechanism: SoxRS is activator of acrab-tolc efflux pump and induces colistin hetero-resistance in Enterobacter asburiae and Enterobacter cloacae, Telke et al., in review in JAC. E. cloacae NH52 E. asburiae E. cloacae NH52 E. asburiae RESISTANT Oxa-48 Test in S. marcescens RESISTANT 2701 RESISTANT Oxa-48 RESISTANT 2701 Temoin PAβN Temoin PAβN 42 strains of S. marcescens tested: 1 active (oxa-48) Others Efflux pump inhibitors: Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) Verapamil

17 Efflux pump inhibitor assays on Serratia marcescens Smar 1237 Smar 101 Smar 1237 Smar 101 Smar 1237 Smar 101 MH MH + verapamil MH + CCCP 21 strains of S. marcescens tested Strains CS Oxa X MH 0,25 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 MH + verapamil 0,25 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 ND ND ND ND ND ND >256 >256 >256 >256 MH + CCCP 0,25 0,094 0,19 0,125 0,125 0,125 0,064 0,125 0,19 0,125 0,094 0,125 0,19 0,,19 0,125 0,125 0,094 0,125 0,19 0,125 0,125 Table. CMI of colistin determined by E-test method in mg/l on Mueller-Hinton, supplemented or not with efflux pump inhibitor.

18 Efflux pump inhibitor assays on Serratia marcescens Temoin MH Strain Mechanism of resistance to colistin Number MH Mean MIC colistin in mg/l (range) MH+Verapamil Mean MIC colistin in mg/l (range) MH + CCCP Mean MIC colistin in mg/l (range) E. coli mcr (14-18) 17.2 (16-18) 24 (23-NG) Unknown (15-17) 16.2 (15-17) 24.5 (22-NG) mcr (16-16) 17 (17-17) 23 (23-23) K. pneumoniae Unknown (8-18) (6-20) 21.9 (19-NG) Susceptible K. oxytoca Unknown E. aerogenes Efflux 1 6 (HR) 6 (HR) 23 E. asburiae Unknown 2 6 (HR) 6 (HR) 24.5 (22-27) E. cloacae Unknown 4 18 (17-20) (17-20) (22-25) Susceptible S. enterica Unknown 2 17 (17-17) 17 (17-17) 35.5 (34-37) P. aeruginosa Unknwon 3 27 (23-33) 25 (20-30) 28.3 (23-32) A. xylosoxidans Unknwon NG S. maltophilia Unknwon 3 24 (17-30) 25.6 (20-33) NG Chryseobacterium sp. Unknown 2 6 (6-6) 6 (6-6) NG P. mirabilis 2 6 (6-6) 6 (6-6) NG P. vulgaris 1 6 (6-6) 6 (6-6) NG S. marcescens 1 6 (6-6) 6 (6-6) NG M. morganii Intrinsic 3 6 (6-6) 6 (6-6) NG P. alcalifaciens resistance 2 6 (6-6) 6 (6-6) NG P. rettgeri 2 6 (6-6) 6 (6-6) NG Burkholderia sp. 3 6 (6-6) 6 (6-6) NG B. diminuta 1 6 (6-6) 6 (6-6) NG P. pulmonicola 1 6 (6-6) 6 (6-6) NG NG: No growth HR: Heteroresistance MH+Verapamil MH+CCCP

19 Project 3: Colistin resistance in Enterobacteriaceae in Hospital MATERIEL & METHODS: Colistin resistant Enterobacteria Global Strategy for the management of resistant Enterobacteriaceae colistin RT-PCR Oxa-48 NDM-1 KPC YES Carbapenem resistance associated? Colistin and Imipenem E-test RT-PCR Strains: Enterobacteriaceae colistin resistant isolated since Nov 2015 in the laboratory of Microbiology of La Timone in Marseille. mcr-1 - Functional genomic Transposon mutagenesis Efflux pump inhibitor test - PCR and sequencing PmrA-PmrB PhoP-PhoQ mgrb (K. pneumoniae only) crrab-crrc (K. pneumoniae only)

20 Project 3: Colistin resistance in Enterobacteriaceae in Hospital Strain name Sexe Date of isolation Unit Nature Germe MIC Colistin (mg/l) Carbapenemase associated a (CMI imipenem) mcr-1/2 gene mgrb insertion ECL1CR F 04/03/2016 Palliative care unit-timone Drain fluid swab E. cloacae ND ND EC1CR M 02/08/2016 Intensive care unit-nord Rectal swab. E. coli ND EC EC2CR M 04/08/2016 Intensive care unit-nord Pharynx E. coli ND EC KP1CR M 11/01/2016 Intensive care unit-timone Rectal swab K. pneumoniae 8 Oxa-48 - ABS EC (1,5 mg/l) KP2CR M 21/01/2016 Palliative care unit Timone Blood K. pneumoniae No EC KP3CR M 02/03/2016 Intensive care unit-timone Rectal swab K. pneumoniae 6 Oxa-48 - No EC (32 mg/l) KP4CR M 05/03/2016 Infectious disease Rectal swab K. pneumoniae 3 Oxa-48 - No EC department (0,38 mg/l) KP5CR F 22/06/2016 Intensive care unit-nord Rectal swab K. pneumoniae No EC KP6CR M 22/08/2016 Intensive care unit-nord Rectal swab K. pneumoniae No EC KP7CR M 28/08/2016 Cardiology intensive care unit KP8CR M 04/11/2015 Cardiology intensive care unit KP9CR M 24/10/2016 Infectious disease department Sequencing others Urine K. pneumoniae Oxa-48 - No EC Drain fluid K.pneumoniae No EC Stool K. pneumoniae 16 Oxa-48 (1,5 mg/l) a. Quantitative real-time PCR for oxa-48, NDM and KPC genes. ABS: mgrb absent ABS EC

21 Letter for JAC Project 3: Colistin resistance in Enterobacteriaceae in Hospital Report: First detection of a mcr-1 plasmid mediated colistin resistant gene in an Enterobacter cloacae clinical isolate in France. Author: Sophie BARON, Lucie BARDET, Gregory DUBOURG and Jean-Marc ROLAIN Enterobacter cloacae isolated from a flow of an abdominal drain in a patient with Algerian origin hospitalized in oncology unit. sample PE1 (Ct) PE2 (Ct) T Mcr-1 RT-PCR patient patient ATB AMX AMC TCC TZP FOX OXA CRO CTX FEP IPM ERM MEM ATM AK TOB GN SXT NA CIP FT FOS RA DOX TIG MIN CT S SD SPT Diameter (mm) interpretation R R R R R R R R R S R R R S R R R R R S R R R S R R R R R

22 Articles Published: Baron S, Hadjadj L, Rolain JM, Olaitan AO. Molecular mechanisms of polymyxin resistance: knowns and unknowns. IJAA 2016 Submitted: Baron S., Leulmi Z., Villard C., Olaitan AO., Telke AA., Labas N., Nguyen TT., Rolain JM. Inactivation of arn operon and loss of aminoarabinose on lipopolysaccharide as the cause of susceptibility to colistin in an atypical clinical isolate of Proteus vulgaris. For JAC. Baron S., Bardet L., Dubourg G., Fichaux M., Rolain JM. mcr-1 plasmid-mediated colistin-resistant gene detection in an Enterobacter cloacae clinical isolate in France. For JAC Orfanos S., Gomez C., Baron S., Rolain JM., Reynaud-Gaubert JM. Impact of Gram negative bacteria recolonization and de-novo colonization of lung allograft on chronic lung allograft dysfunction in a cystic fibrosis population. For Heart and Lung transplantation In progress: Baron S., Dubourg G, Couderc C, Fournier PE., Raoult D. Actinomyces ihumii, discovered by microbial culturomics studies, a common agent of actinomycosis Baron S., Dubourg G., Rolain JM., Raoult D. Decreasing level of carbapenem resistance in Marseille, France Baron S., Le Page S., Rolain JM. Microbiome and Multi-drug resistance bacteria Osteitis caused by Carbapenemase-producing Klebsiella pneumoniae harboring both NDM and OXA-48 genes Abou Abdallah R, Cimmino T, Baron S, Cadoret F, Michelle C, Raoult D, Fournier PE, Bittar F. Description of Chryseobacterium timonense sp. nov. Isolated from a Patient with Pneumonia.

23 Thank you for your attention