This document discusses strategies for managing infections caused by carbapenem-resistant bacteria. It begins by outlining the objectives of understanding the epidemiology of carbapenemase resistant infections in animals and identifying management strategies. It then provides background on the increasing issue of antimicrobial drug resistance globally and in India. The document discusses common resistance mechanisms like reduced permeability, target alteration, and enzymatic inactivation. It also summarizes different classes of beta-lactam antibiotics and carbapenemases, and the genetics of beta-lactamase resistance. Finally, it presents the author's observations on increasing drug resistance in veterinary clinical isolates in India.

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-243122,
India
Ph. No. +91-8449033222; Email: brs1762@ivri.res.in;
brs1762@gmail.com

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). 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)

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.
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.

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)
– 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 1985. 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)
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

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
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?

20. 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)
10.4
13.6
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 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.

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 2011-2014).
29.9
26.9
18.1
19.5
50.0 50.0
20.0
32.3
21.7
12.6
19.4
13.8
21.1
19.4
37.5
5.0
15.4
8.7
6.7
9.0
5.6
3.3
8.1
12.5
2.5
7.7
6.5
0.0
10.0
20.0
30.0
40.0
50.0
60.0 % ESBL +ve % Carbapenemase +ve % ESBL & Carbapenemase +ve

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
0.5->64
Mero ≤2 4-8 >8
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
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
ESBL+ve Carbpenemase +ve ESBL & Carbapenemase +ve

27. What should we do?
• Review of 298 published cases (244 BSI)
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 3
Staphylococcus 5
Streptococcus 1
Colistin antibacterial activity enhanced by Carvacrol
E. 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

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.