1. Development of strategies for management of infections with carbapenem resistant bacteria Myths and Facts
Dr. Bhoj R Singh
Act. Head of Division of Epidemiology
Indian Veterinary Research Institute, Izatnagar , India
Ph. No ;

2. Objectives
To understand epidemiology and emergence of carbapenemase resistant infections in animals.
To look for strategies for management of infections with carbapenemase resistant bacteria.

3. Present scenario
Antimicrobial drug resistance in common and consistently emerging problem all over the globe including Indian sub-continent.
Antimicrobial drug resistance is either flow vertically or horizontally.
Genes for drug resistance may be either on chromosome or on mobile genetic elements (R factors, plasmids, transposons, Insertion elements, integrons, bacteriophages).
Emergence of antimicrobial drug resistance is natural.

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

5. Target site of Antibiotics
Inhibition of cell wall synthesis
Penicillins, Cephalosporins, Carbapenems, Monobactams, Daptomycin, Glycopeptides
Inhibition of protein synthesis
Tetracyclines, Chloramphenicol, Macrolides, Aminoglycosides, Lincosamides, Oxazolidinones, Streptogramins
Interference of nucleic acid synthesis
Quinolones, Nitroimidazoles, Rifampicin
Disruption of bacterial membrane
Polymixins, Colistin
Inhibition of folic acid pathway
Sulphonamides, Trimethoprim

6. Antimicrobial drug resistance mechanisms
Reduced permeability and active efflux: Gram-negative pathogens like Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii show resistance to β-lactams by altering the porins or by loss of porins. Another strategy is expelling the antibiotics out of the bacterial cell by active efflux through membrane bound efflux pumps to counter action of variety of antimicrobials, P. aeruginosa harbour several efflux pumps like MexAB-OprM, MexCDOprJ, and MexXY-OprM.
Target alteration: Modification of penicillin binding proteins (PBPs, main targets 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 resistance towards β-lactams.
Enzymatic inactivation or modification: Most of the antibiotics are characterized 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 and Gram negative bacteria. Modification of the antibiotic molecule is a major resistance mechanism in Gram-negatives to aminoglycosides conferred by aminoglycoside modifying enzymes

7. 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)

8. Common structure of β-Lactam antibiotics

10. β-Lactamases More than 400 β-lactamases have been reported and new β-lactamases continue to emerge worldwide (Jacoby, 2009).
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)

11. From Saradhi, 2012.

12. 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.
Zinc metallo-B-lactamase
C (CMY1-CMY50) ~50
1, 1e (most of the B-lactams and Aztreonam ineffective)
D (OXA-1 to OXA-58)~239
2d, 2de, 2df (carbapenems are also ineffective)
Partially inhibited
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.

13. Genetics of β-lactamses
Chromosomal: AmpC ESBLs of many Gram-negative bacteria, including Citrobacter, Serratia and Enterobacter. blaSHV of K. pneumoniae, blaCTX-M of Kluyvera,
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 and ISCR1 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,

14. Carbapenemases Class A (serine based) Class B (metallo-enzymes)
KPC, GES, SME, NMC, IMI
Class B (metallo-enzymes)
NDM (NDM-1 in 2008 at New Delhi K. pneumoniae and E. coli), IMP (IMP-1 in 1988 in Japan P. aeruginosa), VIM (VIM-1, in 1999 in Italy P. aeruginosa, VIM-2 in France 1996 isolate), GIM, SIM, SMP, L1, BCII, Ccra
Class D (serine)
OXA (37 of 239) Mostly from A. baumannii isolate. First from Scotland in OXA-48 was isolated from a clinical isolate of K. pneumoniae from Turkey.

15. Genetic regulation of carbapenemases
Chromosomal
Class A
SME (1982), NMC (1984), IMI (1990)
Class B
CphA & 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 B
Bla-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 elements
In A. baumannii, the insertion sequences of ISAba1 type carrying strong promoters are present upstream of chromosomal OXA genes.

16. Mettalo-B-lactamases Susceptible to inhibition by aztreonam and metal ion chelators (EDTA)
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
Zn 1 site present
Zn 1 site absent
Zn 2 ligand in Cys22
Zn 2 ligand in His121
Only Zn 2 site is active

17. 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.

18. 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 inhibitor combinations.
Imipenem, panipenem, and doripenem are potent antibiotics against gram-positive bacteria whereas
Meropenem, biapenem, ertapenem (and doripenem) are slightly more effective against Gram-negative bacteria.

19. Our observations on Veterinary Clinical isolates of bacteria
In Vitro Drug Resistance in Veterinary Clinical Isolates of Bacteria ( ) 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?

20. Changing Resistance pattern of Drug resistance in last three years period ( ) Figures are shown as % of total isolates
All clinical isolates of Bacteria (1317)
Gram Negative isolates of Bacteria (901)
Resistance is increasing in bacteria due to ESBL and Carbapenemase production ability at alarming rate in veterinary clinical isolates.
Drug resistance is emerging faster in Gram Negative bacteria than Gram positive bacteria.
Gram Positive isolates of Bacteria (416)

21. Probability of Drug resistance and its type changes with types of bacteria (Based on clinical isolates of bacteria (1317) identified in Epidemiology Laboratory of IVRI, Izatnagar ).

22. 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 the strain carrying the plasmid
MIC
Preparedness to think laterally

23. MIC MIC ≤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
Mero
1-64

24. 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?

25. Herbal drugs MIC 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?

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

27. What should we do? Review of 298 published cases (244 BSI) Treatment
Tzouvelekis et al, CMR 2011
Treatment
Failure rate
2 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.

28. 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 not useful 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 Indian context more important.
Aminoglycoside if possible (Strains with KPC, VIM, IMP and OXA-48 enzyme are variably resistant to aminoglycosides). Our data indicates their potential as one of the best option.
Fosfomycin and Colistin: never alone, the last resort antibiotics for multidrug-resistant P. aeruginosa, and A. baumanni. Colistin resistance is quite 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.

29. Herbal drugs can modulated action of some of the potential drugs which can be used for treatment of infections with ESBL, MBL and MDR strains
Colistin antibacterial activity enhanced by Cinnamon oil
E. coli 26
Bacillus 7
Enterobacter 3
Pasteurella
Staphylococcus 5
Streptococcus 1
Colistin antibacterial activity enhanced by Carvacrol
Micrococcus
Flavobacter
Imipenem antibacterial activity on Carbapenemase positive strains enhanced by Carvacrol
Imipenem antibacterial activity on Carbapenemase positive strains enhanced by Cinnamon oil 10

30. Newer areas of Resaerch
Search for clinically usable modulators of carbapenem drugs: Till date no clinically usable inhibitor of MBLs is known. ESBLs can be managed due to availability of clinically usable inhibitors- Sulbactam, Tazobactam, Clavulanic acid).
Expoloitation of herbal drugs for their role as antimicrobial drug modulators. There are indications that some herbs can modulate the effect of antimicrobials including carbapenems and drugs of last resort as colistin and polymyxin B. If we can reduce the effective dose of these potentially toxic drugs it can be miracle.
Finding the ways for clinical use of herbal oils to treat infections: Herbal oils can inhibit growth of ESBL/ carbapenemase/ MBL producer strains in vitro.

31. Your Ideas! Welcome Anticipated Valued May be ice-breaking
May change the life- Visionary
May help to sustain the life
May open new era of science and scientific thinking.