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1 Please note: The presenters consented to share only some of the slides from this session.

2 Polymyxin resistance A major breach for the last line of antimicrobial therapy Samir N. Patel, PhD, FCCM Roberto G. Melano, MSc, PhD Public Health Ontario Laboratories Toronto, ON, Canada

3 Outline Polymyxins: history, structure, mode of action Challenges with colistin PK / PD Why are we talking about colistin? Mechanisms of resistance in Enterobacteriaceae 2

4 History Polymyxin is an old class of cyclic polypeptide antibiotics that was discovered in 1947 from the soil bacteria Paenibacillus polymyxa (former Bacillus polymyxa var. colistinus) This bacteria produces polymyxin B and colistin (polymyxin E) as secondary metabolite nonribosomal peptides Colistin has been available since 1959 for the treatment of infectious diseases caused by Gram-negative bacteria Colistin use was restricted when the potentially less-toxic aminoglycosides and other antipseudomonal agents became available As a result, the use of colistin in the treatment of infections caused by Gramnegative pathogens has declined from the early 1970s to the early 2000s 3

5 Expert Rev. Anti Infect. Ther. 10(8): (2012) 4

6 Susceptible Resistant Variable Gram-negative bacilli Polymyxins: spectrum of action Pseudomonas aeruginosa Proteus spp. Stenotrophomonas maltophilia Acinetobacter spp. Providencia spp. Aeromonas spp. Escherichia coli Serratia spp. Vibrio spp. Klebsiella spp. Morganella morganii Enterobacter spp Edwardsiella tarda Salmonella spp. Chromobacterium spp. Shigella spp. Burkholderia spp. Pasteurella spp. Helicobacter pylori Legionella pneumophila Gram-negative cocci: Haemophilus influenzae Neisseria spp. Bordetella pertussis Moraxella catarrhalis Anaerobic GNB: Other anaerobic GNB Prevotella spp. All Gram-positive cocci Fusobacterium spp. Colistin is now often considered as the last option to treat MDR Gram-negatives, particularly CPE, A. baumannii and P. aeruginosa Expert Rev. Anti Infect. Ther. 10(8): (2012); Indian J Med Microbiol, 29(3): (2011) 5

7 Polymyxins: chemistry/structure Thr Cationic polypeptide ring R Leu Lipophilic fatty acyl side chain (hydrophobic tail) Polymyxins are cyclic amphipathic polycationic peptide with a short aliphatic side chain Thr R: D-Phe in polymyxin B, D-Leu in colistin 6

8 CM Periplasm OM Lipopolysaccharide (LPS), which makes up to 70% of the outer leaflet of the outer membrane, consists of the lipid A moiety attached to glycan chain of variable length and composition Polymyxins: mode of action LPS OMP LP CW IMP Cytoplasm 7 Cold Spring Harb Perspect Biol doi: /cshperspect.a000414;

9 CM Periplasm OM Ca 2+ Mg 2+ Polymyxin binds to LPS Electrostatic interaction displacement of divalent cations (which usually serve as a bridge between the adjacent LPS molecules to stabilize monolayer) Membrane lysis pathway Increased permeability of the cell envelope Membrane disruption Leakage of cell contents Polymyxin disrupts OM by inserting its hydrophobic N-terminal fatty acyl chain into OM Vesicle-vesicle contact pathway Induction of lipid exchange between IM and OM Loss of specificity of phospholipid composition Cell death Osmotic imbalance Biomed Res Int. 2015: doi: /2015/ (2015) 8

10 CM Hydroxyl Radical Death Pathway Polymyxin would induce formation of reactive oxygen species (ROS), including superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( OH) in GNB A A. O 2 will be induced when polymyxin enter into and cross OM and CM B. O 2 will be converted to H 2 O 2 by superoxide dismutases SOD O 2 - C. H 2 O 2 oxidizes ferrous iron (Fe 2+ ) to ferric iron (Fe 3+ ), along with the formation of OH H 2 O 2 Fe 2+ Fenton reaction D. When concentration of OH reaches high levels, it results in oxidative damage of DNA, lipids, and proteins Cell death B C. OH Fe 3+ DNA damage Lipid damage Protein damage Biomed Res Int. 2015: doi: /2015/ (2015) D Cell death 9

11 Untreated E. coli A cell treated with 25 µg/ml of polymyxin B for 30 min The CM appears to be damaged, and part of the cytoplasmic material is released in fibrous forms through the cracks J Bacteriol 97: (1969) Blebs are composed of the OM 10

12 Why are we talking about colistin? 11

13 Clin Microbiol Infect 20: (2014) 12

14 Susceptibility profile of pandrug-resistant Klebsiella pneumoniae Sci Rep. 5:15082 (2015) 13

15 Death risk ratios for patients infected with CRE vs. CSE Emerg Infect Dis. 20: (2014) 14

16 Mortality caused by KPC-producers Author Study design Underlying disease No. of patients Mortality Atb Combination n (% mortality) Monotherapy n (% mortality) Qureshi et al 2012 Tumbarello 2012 Zarkotou 2011 Retrospective Cohort Single-centre Retrospective multicentre Prospective Single centre Antimicrob Agents Chemother. 58: (2014) 53% ICU, renal dialysis, cancer 13% shock, DM, heart failure, CRF, cancer 34 At 28 days Coli-Carba 5(20) Coli-Tige 1(0) Coli-FQ 1(0) Tige-Carba 3(0) Carba-FQ 1(100) Azt-FQ 1(0) Cfpm-Gen 1(0) 125 At 30 days Tige-Coli 23(30) Tige-Gen 12(50) Coli-Gen 7(57) Tige-Coli-Carba 16(13) Tige-Gen-Carba 6(17) Coli-Gen-Carba 1(100) 71% ICU 35 Infected related Tige-Coli, 9(0) Tige-Gen 3(0) Tige-Coli-Carba 1(0) Tige-Coli-gen 1(0) Tige-Carba 1(0) Coli-Gen 2(0) Carba-Gen 1(0) Coli 7(57) Tige 5(80) Carba 4 (50) Gen 1(0) Tige 19(53) Coli 22(50) Gen 5(80) Coli 7(57) Tige 5(40) Gen 2(0) Carba 1(100) 15

17 Challenges with treatment recommendations Delay in appropriate antibiotics Site of infection Co-morbidity Lack of studies with sufficient sample size Different combination of antibiotics Heterogeneous organisms and their susceptibility profiles Emergence of R to last line of useful antibiotics (e.g. colistin R ) 16

18 Polymyxins Mechanisms of resistance 17

19 CM ph Mg 2+ Polymyxin Chromosomally mediated colistin R - PhoQ PmrB MgrB Kpn PhoP P PhoP Response regulator PmrA Mutations in mgrb or phop/phoq typically lead to the constitutive induction of the PhoP/PhoQ two-component system P PhoP + + PmrD Activation of LPSmodifying enzymes involved in polymyxin R Biomed Res Int. 2015: doi: /2015/ (2015); Front Microbiol. 5:643. doi: /fmicb (2014); Nat Rev Microbiol. 11(7): doi: /nrmicro3047 (2013) 18

20 CM L-Ara4N: 4-amino-4-deoxy-l-arabinose (aminoarabinose) petn: phosphoethanolamine ph Fe 3+ PmrE ArnT CptA PmrC L-Ara4N PmrB petn PmrA P PmrA LpxT PhoP P + + PmrD P PmrA ArnT PmrE PmrC CptA PmrR LpxT Activation of LPSmodifying enzymes involved in polymyxin R Biomed Res Int. 2015: doi: /2015/ (2015); Front Microbiol. 5:643. doi: /fmicb (2014); Nat Rev Microbiol. 11(7): doi: /nrmicro3047 (2013) - 19

21 L-Ara4N: 4-amino-4-deoxy-l-arabinose (aminoarabinose) petn: phosphoethanolamine L-Ara4N petn The addition of petn to lipid A by petntransferases is a major mechanism for resistance to polymyxin in Neisseria spp. and Acinetobacter since these species do not synthesize L-Ara4N The addition of L-Ara4N to lipid A is commonly seen in Enterobacteriaceae 20

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23 phnshp45 (64,105 bp) from E. coli strain SHP45 GC content 43 0% (E. coli GC content ~50 0%) 60 predicted ORFs Typical IncI2-type backbone P. vortex P. dendritiformis mcr-1 1,626-bp, 49% GC content Located downstream of an IS GenBank KP The deduced aminoacid sequence MCR-1 aligned closely with petntransferases (EptA) found in Paenibacillus sophorae (63% identity) Lancet Infect Dis 16:161-8 (2016) 22

24 Year Positive /number of isolates Pigs at slaughter 2012/ /804 (20 6%) Retail meat 2011/14 78 /523 (14 9%) Inpatient 2014 E.coli : 13 /902 (1 4%) K. pneumoniae 3 /420 (0 7%) Prevalence of colistin resistance gene mcr-1 by origin Origin MIC (µg/ml) Colistin Polymyxin B Escherichia coli SHP45 (mcr-1 ) Pig 8 4 E coli C600 Lab strain E coli C600+ phnshp45 mcr-1 ) Transconjugant 8 4 E coli E11 (ST131, KPC-2-producer) Human E coli E11 (ST131, KPC-2-producer) + phnshp45 (mcr-1 ) Transformant 4 2 Klebsiella pneumoniae MPC11 Human K pneumoniae MPC11 + phnshp45 (mcr-1 ) Transformant 8 4 K pneumoniae 1202 (ST11, KPC-2-producer) Human K pneumoniae 1202 (ST11, KPC-2-producer) + phnshp45 (mcr-1 ) Transformant 4 4 Pseudomonas aeruginosa HE26 Human P aeruginosa HE26 + phnshp45(mcr-1 ) Transformant 8 4 E coli W3110+ puc18 Lab strain E coli W puc18-mcr-1 Transformant 2 2 Table 1. Minimum inhibitory concentration for parental strain, transformants, and transconjugant Lancet Infect Dis 16:161-8 (2016) 23

25 OM Peptidoglycan DAG PtdEtN Catalytic site IM Mcr-1? PmrC Proc Natl Acad Sci 107(11): (2010); and Nat Rev Microbiol 7(9): (2009) petn: phosphoethanolamine PtdEtN: Phosphatidylethanolamine DAG: Diacylglycerol 24

26 Food animals (chickens, veal calves, pigs, cattle, turkeys) Geographic distribution of the mcr-1 gene (as of March 1 st, 2016) Environment (river water) Switzerland, Malaysia Food (ground beef, retail meat -chicken, pork-, vegetables) 12 Humans (fecal carriage, salmonellosis, blood infect, wound infect) 8 Euro Surveill. 21(9):pii=

27 Jan March 2016 Status in Ontario Colistin MIC (µg/ml) Grand Total Enterobacter spp Escherichia coli Klebsiella spp Grand Total EUCAST R 4µg/ml 2.9% resistant One E. coli clinical isolate, recovered in 2011, had Col MIC=4 µg/ml mcr-1 positive Diagn Microbiol Infect Dis. 76(3): (2013); Lancet Infect Dis. 16(3): (2016) 26

28 Summary Colistin is the last choice for treatment of MDR Gram-negative bacteria High toxicity, dosage is not optimal Emergence of Mcr-1 in Enterobacteriaceae is concerning (horizontal dissemination of the resistance!) Preventing spread of colistin R Enterobacteriaceae: Minimize use of colistin in humans and food Minimize spread of CPE through screening and aggressive infection control practises 27

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