Development of strategies for management of infections with carbapenem resistant bacteria

Myths and Facts

Dr. Bhoj R SinghAct. Head of Division of Epidemiology

Indian Veterinary Research Institute, Izatnagar-243122, India

Ph. No. +91-8449033222; Email: brs1762@ivri.res.in; brs1762@gmail.com

Objectives

• To understand epidemiology and emergenceof carbapenemase resistant infections inanimals.

• To look for strategies for management ofinfections with carbapenemase resistantbacteria.

Present scenario

• Antimicrobial drug resistance in common andconsistently emerging problem all over the globeincluding Indian sub-continent.

• Antimicrobial drug resistance is either flowvertically or horizontally.

• Genes for drug resistance may be either onchromosome or on mobile genetic elements (Rfactors, plasmids, transposons, Insertionelements, integrons, bacteriophages).

• Emergence of antimicrobial drug resistance isnatural.

Common Mechanisms of Antimicrobial drug resistance (Levy et al., 2004)

Target site of Antibiotics Inhibition of cell wall synthesisPenicillins, Cephalosporins, Carbapenems, Monobactams, Daptomycin, GlycopeptidesInhibition of protein synthesisTetracyclines, Chloramphenicol, Macrolides, Aminoglycosides, Lincosamides, Oxazolidinones, StreptograminsInterference of nucleic acid synthesisQuinolones, Nitroimidazoles, RifampicinDisruption of bacterial membranePolymixins, ColistinInhibition of folic acid pathwaySulphonamides, Trimethoprim

Antimicrobial drug resistance mechanisms

• Reduced permeability and active efflux: Gram-negative pathogens likeKlebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacterbaumannii show resistance to β-lactams by altering the porins or by loss ofporins. Another strategy is expelling the antibiotics out of the bacterial cell byactive efflux through membrane bound efflux pumps to counter action ofvariety of antimicrobials, P. aeruginosa harbour several efflux pumps likeMexAB-OprM, MexCDOprJ, and MexXY-OprM.

• Target alteration: Modification of penicillin binding proteins (PBPs, maintargets for β-lactams). MRSA is achieved by the acquisition of an altered PBP(PBP2a or PBP2’) by the mecA gene. Also in Gram-negative bacteria such as A.baumannii and P. aeruginosa altered PBPs have been implicated in resistancetowards β-lactams.

• Enzymatic inactivation or modification: Most of the antibiotics arecharacterized by ester or amide bonds, which are hydrolytically susceptible,targeted by certain bacterial enzymes, and render them inactive. β-lactamases are the major resistance mechanisms both in Gram positive andGram negative bacteria. Modification of the antibiotic molecule is a majorresistance mechanism in Gram-negatives to aminoglycosides conferred byaminoglycoside modifying enzymes

β-lactam antibiotics

•The β-lactam antibiotics comprise six different structural

subtypes, including penams, cephems, monobactams, cla-

vams, penems, and carbapenems.

•Penams: benzylpenicillin and ampicillin.

•Cephems: These Cephaloridine, nitrocefm, cefotaxime,

cephamycins (7-α-methoxy-cephalosporins) the classical

cephalosporins,

•Monobactams are monocyclic β-lactams, aztreonam.

•Penems have a 2, 3-double bond in the fused thiazolidine

ring (dihydrothiazole) and Carbapenems (Imipenem,

biapenem)

Common structure of β-Lactam antibiotics

β-Lactamases

More than 400 β-lactamases have been reported and new β-lactamases continue to

emerge worldwide (Jacoby, 2009). http://www.laced.uni-stuttgart.de/; http://www.lahey.org/Studies/

Now as on December 2014 about 800 β-lactamases~190 SHV ESBLs; ~239 OXA BLS of which >37 are carbapenemases; ~193 TEM ESBLs~100 MBLs (VIM-48 variants; IMP-44 variants; NDM-12 variants)

From Saradhi, 2012.

Molecular classification of B-lactamases

Molecular class (Ambler

classes) and common

names

Bush Jacoby groups Inhibited by

Clavulanic

acid,

subactam

Inhibited

by

EDTA

Mechanism of

Action

A (>500) ESBLs

TEM ~193; SHV ~190;

CTX-M ~90; GES~15;

PER~5; VEB~7;

KPC~9, SME~3)

2a, 2b, 2be, 2br, 2ber, 2c, 2e

2f (carbapenems and B-

lactams are ineffective)

Yes No Serine B-

lactamase

B (>100) MBLs

B1: IMP(1-44), VIM (1-

40), NDM (1-12), IND

(1-8), GIM-1, BcII,

CcrA

B2: CphA, Sfh-1

B3-FEZ; L1

3a (IMPs, VIMs, NDMs,

GIM-1, BcII, CcrA, L1,

AIM-1, FEZ-1).

3b (CphA, Sfh-1)

Carbapenems are

ineffective but aztreonam

may be effective.

No Yes Zinc metallo-

B-lactamase

C (CMY1-CMY50) ~50 1, 1e (most of the B-lactams

and Aztreonam ineffective)

No No Serine B-

lactamase

D (OXA-1 to OXA-

58)~239

2d, 2de, 2df (carbapenems

are also ineffective)

Partially

inhibited

No Serine B-

lactamase

Up to 2007: GIM- German imipenemase; GES - Guiana extended spectrum β-lactamase; BES- Brazil extended spectrum β-

lactamase; SPM-1-Sao Paulo metallo-β-lactamases; SIM-1 -Seoul imipenemase; CTX-M- cefotaxime Munich; MIR-1, Miriam

Hospital in Providence extended spectrum β-lactamase; DHA- the Dhahran Hospital in Saudi Arabia extended spectrum β-

lactamase.; VEB -Vietnam extended-spectrum β-lactamase ; TLA- Tlahuicas Indians ESBL; TEM-1- Temoneira ESBL

(1965); BIL1 was named after the patient Bilal in 2002.

In 2008: NDM-New Delhi Metallo-β-lactamase.

Genetics of β-lactamses

• Chromosomal: AmpC ESBLs of many Gram-negative bacteria, including Citrobacter, Serratia and Enterobacter. blaSHV of K. pneumoniae, blaCTX-M ofKluyvera,

• Mobile genetic elements (MGEs), such as insertion sequences (ISs), integrons, transposons, plasmids and phage-related elements.– Plasmids: AmpC of E. coli and Klebsiella and many other ESBLs viz., DHA-1, MIR-

1, 2, BIL-1, CMY, FOX, LAT; blaCMY-13 on an IncN plasmid from Escherichia coli, blaCTX-M genes on IncI1 and IncFII and other plasmids.

– Transposons- Most of the blaTEM variants are associated with Tn1, Tn2 and Tn3 transposition, blaCTX-M-15

– Integrons and IS Elements: IBC (integron-borne cephalosporinase), IS-5 mediated bleomycin resistance; blaSHV of K. pneumoniae on IS26; ISEcp1 andISCR1 responsible for transposition of blaCTX-M ; blaGES-1 and blaVEB-1 gene cassettes are on class 1 integrons, blaGES-1 gene cassette on class 3 integron, insertion sequence ISEcp1 has been identified in association with many blaCMY

genes, Citrobacter blaCMY-13 gene is bound to IS26 elements; blaCTX-M-15

associated with ISEcp1 in Enterobacteriaceae.– Phage related/ mediated: blaCTX-M , blaTEM , and mecA , genes

(qnrA, qnrB and qnrS) conferring reduced susceptibility to fluoroquinolones,

Carbapenemases

• Class A (serine based)

– KPC, GES, SME, NMC, IMI

• Class B (metallo-enzymes)

– NDM (NDM-1 in 2008 at New Delhi K. pneumoniae andE. coli), IMP (IMP-1 in 1988 in Japan P. aeruginosa), VIM(VIM-1, in 1999 in Italy P. aeruginosa, VIM-2 in France1996 isolate), GIM, SIM, SMP, L1, BCII, Ccra

• Class D (serine)– OXA (37 of 239) Mostly from A. baumannii isolate. First from

Scotland in 1985. OXA-48 was isolated from a clinical isolate of K.

pneumoniae from Turkey.

Genetic regulation of carbapenemases• Chromosomal

– Class A• SME (1982), NMC (1984), IMI (1990)

– Class BCphA & SPM-1-Aeromonas spp., BCI,

BCII- Bacillus cereus, L1-Stenotrophomonas maltophilia, CcrA-Bacteroides fragilis; GOB1, FEZ1, Mbl1b, CAU1, BJP1

– Class D• OXA

• Plasmid– Class A

• KPC (1996), GES (2000)

– Class BBla-IMP, bla-VIM, bla-GIM, bla-SIM, blaKMH, NDM (2008), IMP, L1, AIM1, SMB1

– Class D• OXA

Integrons- on Class I integrons IPM and VIM (Verona integron-encoded MBL )

IS elementsIn A. baumannii, the insertion sequences of ISAba1 type carrying strong promoters are present upstream of chromosomal OXA genes.

Mettalo-B-lactamasesSusceptible to inhibition by aztreonam and metal ion chelators (EDTA)

B1 B3 B2

Broad spectrum-hydrolyse penicillins, cephalosporins and carbapenems

Narrow spectrum- Hydrolyse carbapenems only

Require two Zn ions bound to active site Requires only one Zn ions bound to active site

Clinically more important (NDM,

VIM, IMP)

Clinically less important

Clinically less important

Zn 1 site present Zn 1 site present Zn 1 site absent

Zn 2 ligand in Cys22 Zn 2 ligand in His121 Only Zn 2 site is active

Carbapenems

Imipenem is susceptible to hydrolysis by dehydropeptidase found in renal brush

border. Hence, they have to be co-administered with the inhibitors such as

cilastatin (or betamipron). Subsequently, meropenem, biapenem, doripenem

and ertapenem were developed by addition of methyl group to 1-β position to be

protected from dehydropeptidase hydrolysis.

How Carbapenems act?

• Carbapenems enter Gram-negative bacteria through OMPs(porins) and reach periplasmic space.

• Carbapenems have ability to bind to multiple different PBPs.• Permanently acylate the PBPs.• Inhibit peptide cross linking and other peptidase reactions.• Weakening of cell wall leading to autolysis and death of the

bacterium.• Carbapenems have broader antimicrobial spectrum than

penicillins, cephalosporins or β-lactam/β-lactamase inhibitorcombinations.

• Imipenem, panipenem, and doripenem are potentantibiotics against gram-positive bacteria whereas

• Meropenem, biapenem, ertapenem (and doripenem) areslightly more effective against Gram-negative bacteria.

Our observations on VeterinaryClinical isolates of bacteria

5.5

11

.9

4.6

68

18

.3

38

.3

8.4

20 20

.7 25

.4

20

.6

16

.5 18

.9

49

.6

10

.7

79

.5

61

.3

34

.5

26

.2

10

7.6

2.5

57

.5

33

.9

19

.4

14

.2

13

38

.1

49

.6

24

.9

16

.5

14

.3

64

.9

6.5

40

.6

30

.8

59

.6

24

.6

0

10

20

30

40

50

60

70

80

G-ve Bacteria (901) G +ve Bacteria (416)

In Vitro Drug Resistance in Veterinary Clinical Isolates of Bacteria (2011-14) at IVRI,Izatnagar Bareilly (Figures are shown as % of total isolates resistant to the drug).Do herbal drugs may be an option for antibiotic resistant bacterial infection?

Changing Resistance pattern of Drug resistance in last three years period (2012-2014) Figures are shown as % of total isolates

20.9

27.9

63.7

13.9 12.3

47

5.4

30.5

0

10

20

30

40

50

60

70

2012 2013 2014

ESBL+Carbapenemase+ESBL+ Carbapenemase+

All clinical isolates of Bacteria (1317)

25.5

32.8

73.2

13.8 13.7

41.7

6.9

31.3

0

10

20

30

40

50

60

70

80

2012 2013 2014

ESBL+Carbapenemase+

Gram Negative isolates of Bacteria (901)

10.413.6

52.4

14.3

8

54.1

1

29.4

0

10

20

30

40

50

60

2012 2013 2014

ESBL+Carbapenemase+ESBL+ Carbapenemase+

Gram Positive isolates of Bacteria (416)

Resistance is increasing inbacteria due to ESBL andCarbapenemase productionability at alarming rate inveterinary clinical isolates.Drug resistance is emergingfaster in Gram Negativebacteria than Gram positivebacteria.

Probability of Drug resistance and its type changeswith types of bacteria (Based on clinical isolates of bacteria (1317)

identified in Epidemiology Laboratory of IVRI, Izatnagar 2011-2014).

29.9

26.9

18.119.5

50.0 50.0

20.0

32.3

21.7

12.6

19.4

13.8

21.119.4

37.5

5.0

15.4

8.76.7

9.0

5.63.3

8.1

12.5

2.5

7.76.5

0.0

10.0

20.0

30.0

40.0

50.0

60.0 % ESBL +ve % Carbapenemase +ve % ESBL & Carbapenemase +ve

Successful therapy of Infections needs

• Knowledge of local epidemiology• Local situation: Antimicrobial drug resistance

trends i.e.• clonal spread (all isolates have the same

antibiogram) or• polyclonal, transmission of plasmid

– sensitivities vary depending on the background of thestrain carrying the plasmid

– MIC

– Preparedness to think laterally

MICMIC ≤8 mcg mL-1 Mortality 29%, MIC>8 mcg mL-175%

Mortality Carmeli et al. CMI 2010; Daikos et al, AAC 2009

S I R

Erta ≤0.5 1 >1

0.5->64

Imi ≤2 4-8 >8

0.5->64

Mero ≤2 4-8 >8

1-64

Options

• Exhausted: B-lactam antibiotics (~80% or more bacteria are resistant)

• Still possible (Need Antibiogram studies to execute)– Quinolones (~75% isolates with ESBL and Carbapenemase production were sensitive)

– Aminoglycosides (~77% isolates with ESBL and Carbapenemase production were sensitive)

– Tigecycline (Only 65% isolates with ESBL and Carbapenemase production were sensitive)

– Colistin (Only 40-60% isolates with ESBL and Carbapenemase production were sensitive)

– Trimethoprim (Only 50% isolates were sensitive)

– Chloramphenicol (>85% isolates with ESBL and Carbapenemase production were sensitive)

– Fosfomycin

– Temocillin

– Combinations (which ones?)

– Herbal drugs? Which? How? What do they do?

Herbal drugsMIC for the best effective Herbal oils as

antimicrobials Lemon Grass oil 5 mcg to >5000 mcg mL-1

Holy Basil oil 20 mcg to >2560 mcg mL-1

Cinnamon oil 10 mcg to > 1280 mcg mL-1

Carvacrol from Oregano oil 5mcg to >5000mcg mL-1

The Questions are: •How we can administer these effective herbal oil safely to achieve the required systemic concentrations?•What are the toxicities and safety limits for Herbal oils while treating infection?•How they interact with other drugs used simultaneously?

In Vitro Sensitivity of Veterinary Clinical Isolates of Bacteria Having Different Types of Drug-Resistance

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0ESBL+ve Carbpenemase +ve ESBL & Carbapenemase +ve

What should we do?• Review of 298 published cases (244 BSI)

Tzouvelekis et al, CMR 2011

Treatment Failure rate2 drugs, inc carbapenem (MIC<8) 8%

2 drugs, no carbapenem 29%

Aminoglycoside alone 24%

Carbapenem alone (MIC<8) 25%

Tigecycline alone 36%

Colistin alone 47%

Inappropriate Rx 54%

On the basis of our data on in vitro antimicrobial sensitivity of the isolates at Indian Veterinary Research Institute, Izatnagar, similar predictions could be made.

What should we do? Studies!• Understanding of Resistance mechanism: Chromosome or MGEs.• Individual patient approach.• Treatment: Usually based on sensitivities of previous screening or the

current clinical isolates.• Combination therapy: Aztreonam, ceftazidime and aminoglycoside

(amikacin/ genatmicin)• Some broad principles: 2 or more agents• Aztreonam: Aztreonam is stable to metallo-carbapenemases IMP, VIM and NDM

but ineffective in isolates that also co-produce AmpC or ESBL. It seems to be notuseful in Indian context with high percentage of Am,p C and ESBL producers.

• B-lactams (co production of AmpC or an ESBL make them useless) In Indiancontext more important.

• Aminoglycoside if possible (Strains with KPC, VIM, IMP and OXA-48enzyme are variably resistant to aminoglycosides). Our data indicates theirpotential as one of the best option.

• Fosfomycin and Colistin: never alone, the last resort antibiotics formultidrug-resistant P. aeruginosa, and A. baumanni. Colistin resistance isquite common in Indian isolates from aniamls.

• Tigecycline: effective for in Enterobacteriaceae and Acinetobacter spp.Seems to be one of the best option for veterinary cases in India.

Herbal drugs can modulated action of some of the potential drugs which can be used for treatment of

infections with ESBL, MBL and MDR strainsColistin antibacterial activity enhanced by Cinnamon oil

E. coli 26

Bacillus 7

Enterobacter 3

Pasteurella 3

Staphylococcus 5

Streptococcus 1

Colistin antibacterial activity enhanced by CarvacrolE. coli 1

Bacillus 5

Micrococcus 1

Flavobacter 1

Staphylococcus 3

Streptococcus 1

Imipenem antibacterial activity on Carbapenemase positive strains enhanced by Carvacrol

E. coli 5

Imipenem antibacterial activity on Carbapenemase positive strains enhanced by Cinnamon oil

E. coli 10

Newer areas of Resaerch

• Search for clinically usable modulators of carbapenemdrugs: Till date no clinically usable inhibitor of MBLs isknown. ESBLs can be managed due to availability ofclinically usable inhibitors- Sulbactam, Tazobactam,Clavulanic acid).

• Expoloitation of herbal drugs for their role asantimicrobial drug modulators. There are indications thatsome herbs can modulate the effect of antimicrobialsincluding carbapenems and drugs of last resort as colistinand polymyxin B. If we can reduce the effective dose ofthese potentially toxic drugs it can be miracle.

• Finding the ways for clinical use of herbal oils to treatinfections: Herbal oils can inhibit growth of ESBL/carbapenemase/ MBL producer strains in vitro.

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