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Critical Reviews in Microbiology
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Beta-lactams and Beta-lactamase-inhibitors in current- or potential-clinical
practice: A comprehensive update
M. Shahid a; F. Sobia a; A. Singh a; A. Malik a; H. M. Khan a; D. Jonas b; P. M. Hawkey cd
a Section of Antimicrobial Resistance Researches and Molecular Biology, Department of Microbiology,
Jawaharlal Nehru Medical College & Hospital, Aligarh Muslim University, Aligarh, Uttar Pradesh, India b
Department of Environmental Health Sciences, Freiburg University Medical Centre, Breisacher Straße,
Freiburg, Germany c Antimicrobial Agents Research Group, Institute of Biomedical Research, University of
Birmingham, Edgbaston, Birmingham, UK d West Midlands Health Protection Agency, Heart of England NHS
Foundation Trust, Bordesley Green East, Birmingham, UK
Online Publication Date: 01 May 2009
To cite this Article Shahid, M., Sobia, F., Singh, A., Malik, A., Khan, H. M., Jonas, D. and Hawkey, P. M.(2009)'Beta-lactams and Beta-
lactamase-inhibitors in current- or potential-clinical practice: A comprehensive update',Critical Reviews in Microbiology,35:2,81 — 108
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Critical Reviews in Microbiology, 2009; 35(2): 81–108
R E VI EW A RTICLE
Beta-lactams and Beta-lactamase-inhibitors
in current- or potential- clinical practice:
A comprehensive update
M. Shahid1, F. Sobia1, A. Singh1, A. Malik1, H. M. Khan1, D. Jonas2, and P. M. Hawkey3,4
1Section of Antimicrobial Resistance Researches and Molecular Biology, Department of Microbiology, Jawaharlal
Nehru Medical College & Hospital, Aligarh Muslim University, Aligarh-202002, Uttar Pradesh, India, 2Department
of Environmental Health Sciences, Freiburg University Medical Centre, Breisacher Straße 115 BD-79106, Freiburg,
Germany, 3Antimicrobial Agents Research Group, Institute of Biomedical Research, University of Birmingham, Vincent
Drive, Edgbaston, Birmingham B15 2TT, UK, and 4West Midlands Health Protection Agency, Heart of England NHS
Foundation Trust, Bordesley Green East, Birmingham, B9 5SS, UK
Address for Correspondence: shahidsahar@yahoo.co.in; drmohdshahid123@yahoo.com
(Received 29 July 2008; revised 11 November 2008; accepted 08 January 2009)
Introduction
e history of B-lactam antibiotics began with Alexander
Fleming and his observation in 1928 that a strain of the
mould Penicillium produced a diusible antibacte-
rial agent, which he named penicillin (Fleming 1929).
However, the modern era of antimicrobial chemo-
therapy dates back to 1936, with the introduction of
sulfanilamide into clinical practice. Penicillin became
available in quantities sucient for clinical use in 1941,
and streptomycin, chloramphenicol, and tetracycline
were identied toward the end of or soon after World
ISSN 1040-841X print/ISSN 1549-7828 online © 2009 Informa UK Ltd
DOI: 10.1080/10408410902733979
Abstract
The use of successive generations of β-lactams has selected successive generations of β-lactamases includ-
ing CTX-M ESBLs, AmpC β-lactamases, and KPC carbapenamases in Enterobacteriaceae. Moreover, this
cephalosporin resistance, along with rising resistance to uoroquinolones, is now driving the use of carbap-
enems and unfortunately the carbapenem resistance has emerged markedly, especially in Acinetobacter
spp. due to OXA- and metallo-carbapenemases. The industry responded to the challenge of rising resist-
ance and recently developed some novel β-lactams such as ceftobiprole, ceftaroline etc. and many
β-lactam compounds, including β-lactamase-inhibitors, such as BMS-247243, S-3578, RWJ-54428, CS-023,
SMP-601, NXL 104, BAL 30376, LK 157, and so on are under trials. This review provides the comprehensive
accounts of the developments in penicillins, cephalosporins, carbapenems, and β-lactamase-inhibitors,
and the insight about medicinal chemistry, mechanism(s) of action and resistance, potential strategies to
overcome resistance due to β-lactamases, and also the recent advancements in the development of newer
β-lactam compounds; some of which are still under trials and yet to be classied. This review will ll the gap
since previously published reviews and will serve as a comprehensive update on the current topic.
Keywords: Newer- β-lactams; penicillins; cephalosporins; carbapenems; β-lactamase-inhibitors; resistance
Abbreviations: 3GCs: Third generation cephalosporins; 6-APA: 6-amino penicillanic acid; ABC:
Adenosine triphosphate binding cassette; CA-MRSA: Community-acquired methicillin-resistant S. aureus;
CFU: colony forming unit; ESBLs: Extended spectrum beta-lactamases; ISCRs: Insertion sequence common
regions; MATE: Multidrug and toxic compound extrusion; MBL: Metallo-beta-lactamases; MDR: Multi-drug
resistance; MFP: Membrane fusion protein; MFS: Major facilitator superfamily; MIC: Minimum inhibitory
concentration; MRCoNS: Methicillin-resistance coagulase negative Staphylococci; MRSA: Methicillin
resistant S. aureus; MRSE: Methicillin resistant S. epidermidis; MSSA: Methicillin susceptible S. aureus;
OMP: Outer membrane protein; PBPs:Penicillin binding proteins; PRSP: Penicillin-resistant S. pneumoniae;
RND: Resistance nodulation cell division; RTI: Reproductive tract infection; SMR: Small multidrug
resistance; UDP: Uridine diphosphate; UTI: Urinary tract infection; VRE: Vancomycin resistant Enterococci
http://www.informapharmascience.com/mcb
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82 Shahid et al.
War II (Mandell and Petri 1996). Since then, numerous
classes of antimicrobial agents have been discovered
and even continue to be discovered in recent years with
some potent antimicrobials including newer B-lactams
and carbapenems. However, the pace of antimicrobial
drug development has drastically slowed during the last
decade, with only few newer agents, some of which are
really novel.
Since the introduction of antimicrobials into clinical
practice a little over 6 decades ago, the resistant bacterial
strains have arisen due to selective pressure of their use.
During this period, numerous classes of antibiotics were
developed and, in response, antibiotics resistance genes
continued to emerge to prove the Darwinian dictum of
the survival of the ttest. Taking as an example, in 1950s,
the problem due to resistant bacterial isolates, espe-
cially due to Staphylococcus aureus, was tried to solve by
the development of penicillinase-stable penicillins such
as methicillin and the more clinically useful derivatives
cloxacillin and ucloxacillin. However, it was disappoint-
ing that following a year of the introduction of methicillin,
the rst methicillin-resistant isolate and clinical failure
was discovered (Jevons 1961). Similarly, the rapid rise of
resistance to ampicillin in the early 1960s turned out to
be due to a plasmid mediated B-lactamase, one of the
rst described in Gram-negative bacteria, known as TEM
(Hawkey 2008). In turn, industry responded by develop-
ing more sophisticated B-lactam compounds resistant
to hydrolysis by TEM and SHV B-lactamases that were
popularly known as third-generation cephalosporins
(3GCs). e further selection of resistant mutants and
acquisition of novel antibiotic resistance genes led to the
appearance of extended-spectrum B-lactamases (ESBLs)
that now compromise the use of 3GCs for treatment of
serious infections caused by Gram-negative bacteria.
e industry further responded with the introduction
through the 1990s of carbapenems, which are extremely
stable to degradation by B-lactamases and which was
claimed to be the answer (Hawkey 2008). However, a
variety of B-lactamases capable of hydrolyzing carbap-
enems, including IMP, VIM, GIM, SPM, KPC, and OXA,
are seen increasingly in clinical Gram-negative bacterial
isolates (Walsh et al. 2005; Hawkey 2008). To combat this
threatening problem of drug resistances, researchers
are still trying to develop newer compounds including
newer cephalosporins and carbapenems. e purpose
of this review is, therefore, to provide comprehensive
accounts of the developments in penicillins, cepha-
losporins, carbapenems, and B-lactamase-inhibitors,
and also the recent advancements in the development
of newer B-lactams; some of which are still under trials
and yet to be classied.
e penicillins
e history of B-lactams began with Alexander Fleming
and his observation in 1928 that a strain of the mould
Penicillium produced a diusible antibacterial agent,
which he named penicillin (Fleming 1929). Attempts
to isolate and purify penicillin in the 1930s were largely
unsuccessful and by the end of the 1930s interest in pen-
icillin had almost disappeared. A decade later, penicil-
lin was developed as a semisynthetic therapeutic agent
(Chain et al. 1940). Subsequently in the 1940s and 1950s,
the deep fermentation procedure for the biosynthesis of
penicillin marked a crucial advance in the large-scale
production of this antibiotic (readers are advised to see
Rolinson 1998 for detailed description of the fermen-
tative strategies, and development of semisynthetic
penicillins). During this period, B-lactams comprised
only two compounds, namely penicillin G and peni-
cillin V, and the major extension of the B-lactam eld
only occurred in the early 1960s with the development
of the semisynthetic penicillin, quickly followed by the
development of semisynthetic cephalosporins and
other B-lactam antibiotics. Until the middle of the 1970s
virtually all the signicant developments in B-lactam
N
SCH3
CH3
O
O
OH
R
O
HH
BA
H
N
N
SCH3
CH3
O
O
OH
H2NH
CH
O
R
Penicillins
Amidase
Penicillinase
O
HC
S
C
CH3
HC COOHN
H
CH3
NH CH
C
OH
CHR
O
Penicilloic Acids
R6- Aminopenicillanic Acid
Figure 1. Penicillins and product of their enzymatic hydrolysis.
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b-lactams in clinical practice 83
antibiotics had been achieved by adding dierent side
chains to the classical penam and cephem nucleus.
Chemistry of penicillins
e basic structure of penicillins consists of a thiazo-
lidine ring (B) connected to a B -lactam ring (A) {the
penicillin nucleus; 6-amino penicillanic acid (6-APA)}
and to which is attached a side chain (R) (see Fig. 1). e
penicillin nucleus (6-APA) is the chief structural require-
ment for biological activity and requires the presence of
an acid residue on the thiazolidine ring for binding to
the penicillin binding proteins. Chemical alterations of
this portion of the molecule cause loss of all signicant
antibacterial activity. e side chain determines many of
the antibacterial and pharmacological characteristics of
a particular type of penicillin (Mendell and Petri 1996).
Production of 6-APA by enzymatic hydrolysis
e penicillin nucleus, 6-APA (Fig. 2), was rst obtained
as a naturally occurring fermentation product (Batchelor
et al. 1959). However, the yields were always lower than
those of penicillin G or penicillin V obtained in fermen-
tations in which the appropriate precursor was used.
Subsequently, an alternative route to obtain 6-APA was
discovered by enzymatic removal of the side-chain of
the penicillin molecule (Rolinson et al. 1960). A process
for the manufacture of the 6-APA by deacylation was
rst developed in Beecham Research laboratories using
a deacylase obtained from Streptomyces lavendulae with
penicillin V as substrate. Subsequently the deacylase of
bacterial origin was discovered independently in a num-
ber of laboratories (Rolinson et al. 1960; Claridge et al.
1960; Huang et al. 1960; Kaufmann and Bauer 1960) and
6-APA was obtained by using this enzyme with peni-
cillin G as substrate. 6-APA is now produced in large
quantities with the aid of an amidase from Penicillium
chrysogenum (Rolinson et al. 1960).
Semisynthetic penicillins
e discovery of 6-APA, depleted of side chain pre-
cursors, led to the development of the semisynthetic
penicillins. Side chains can be added that alter the
susceptibility of the resultant compounds to inactivat-
ing enzymes, like B-lactamases, and that change the
antibacterial spectrum and pharmacological properties
of the drugs (Mandell and Petri 1996). e rst semi-
synthetic penicillin introduced in the clinical practice
was phenethicillin (a close analogue of Penicillin V).
is was followed by methicillin (Rolinson et al. 1960),
ampicillin (Rolinson and Stevens 1961), amoxycillin,
the isoxazolyl penicillins (Geraci et al. 1962) like oxa-
cillin, cloxacillin, dicloxacillin and ucloxacillin, the
B lactam active against Pseudomonas aeruginosa, like
carbenicillin (Rolinson and Sutherland 1967) and ticar-
cillin (Rolinson and Sutherland 1967) (for more detailed
description of historical aspects of B -lactam research,
the readers are encouraged to read Rolinson 1998).
e cephalosporins
e rst source of cephalosporins was Cephalosporium
acremonium and the culture ltrate in which the fungus
was grown was found to contain three distinct antibiotics,
namely, cephalosporin P, N, and C. e discovery of the
nucleus, 7-aminocephalosporanic acid (Fig. 3), made it
possible to introduce semisynthetic compounds, with anti-
bacterial activity greater than that of the parent substance,
by addition of side chains (for detailed historical review and
discussion of biochemistry, Abraham 1962; Flynn 1972).
Chemistry of cephalosporins
Cephalosporin C contains a side chain derived from
D-A-aminoadipic acid, which is condensed with a
dihydrothiazine B-lactam ring system (7-aminocepha-
losporanic acid). Cephalosporin C can be hydrolyzed
by acid to 7-aminocephalosporanic acid. Subsequent
modications had been achieved by addition of dier-
ent side chains to create a whole family of cephalosporin
antibiotics (Mandell and Petri 1996). e cephamycins
are similar to the cephalosporins, but have a methoxy
group at position 7 of the B-lactam ring of the 7-aminoc-
ephalosporanic acid nucleus (Fig. 2).
Classication and spectrum of activity of
cephalosporins
e classication by “generations”, as shown in Table 1 is
very useful, although it is somewhat arbitrary. is clas-
sication is based on several features of antimicrobial
N
SCH3
CH3
H
O
R1
O
OOH
H
N
Figure 2.6-APA Nucleus.
H
N
N
O
R1
O
S
R2
COOH
Figure 3. 7-ACA Nucleus.
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84 Shahid et al.
activity (Karchmer 2000). e rst generation cepha-
losporins have good activity against Gram-positive
bacteria and relatively modest activity against Gram-
negative microorganisms. Most Gram-positive cocci
are susceptible with exception of enterococci, methi-
cillin-resistant S. aureus (MRSA) and S. epidermidis.
Most anaerobes of gastrointestinal tract, including oral
cavity, except Bacteroides fragilis are susceptible. e
activity against Moraxella catarrhalis,Escherichia coli,
Klebsiella pneumoniae, and Proteus mirabilis is good.
e second-generation cephalosporins have somewhat
increased activity against Gram-negative organisms, but
are less active than the third-generation agents. A sub-
set of second-generation agents (cefoxitin, cefotetan,
and cefmetazole) is also active against B. fragilis
group. ird-generation cephalosporins are generally
less active than rst-generation agents against Gram-
positive cocci, but they are much more active against the
Enterobacteriaceae, including B-lactamase–producing
strains. A subset of third generation agents (ceftazidime
and cefoperazone) is also active against Pseudomonas
aeruginosa but less active than other third-generation
agents against Gram-positive cocci (Donowitz and
Mandell 1988). Fourth-generation cephalosporins
have an extended spectrum of activity compared to
the third-generation and have increased stability from
hydrolysis by plasmid and chromosomally mediated B-lactamases (Mandell and Petri 1996). It must be noted
that none of the cephalosporins on the market has reli-
able activity against penicillin-resistant Streptococcus
pneumoniae, MRSA, methicillin-resistant S. epidermidis
(MRSE) and other coagulase-negative staphylococci,
enterococci, Listeria monocytogenes,Legionella pneu-
mophila,Legionella micdadei,Clostridium dicile,
Stenotrophomonas maltophilia, Campylobacter jejuni,
and Acinetobacter species.
Mechanism of action of penicillins and
cephalosporins
Penicillin acts by inhibiting the bacterial cell wall syn-
thesis i.e. the inhibition of the transpeptidation reaction
of the peptidoglycan synthesis. Numerous researchers
have provided information that allows understand-
ing of the basic phenomenon of mechanism (Ghuysen
1991; Du Bois et al. 1995; Bayles 2000). Peptidoglycan
is a heteropolymeric component of the cell wall that
provides rigid mechanical stability. e peptidoglycan is
composed of glycan chains, which are linear strands of
two alternating amino sugars (N-acetylglucosamine and
N-acetylmuramic acid) that are cross-linked by peptide
chains. e biosynthesis of the peptidoglycan involves
about 30 bacterial enzymes and may be considered in
Table 1. Classication of Cephalosporins.
First Generation Second Generation ird Generation Fourth Generation Yet to be classied
Cefacetrile* Cefonicid* Cefcapene Cefepime* Cefaclomezine
Cefadroxil* (Monocid) Cefdaloxime (Maxipime) Cefaloram
(Cefadroxyl; Duricef) Cefprozil*(Cefproxil; Cefdinir* (Omnicef) Cefclidine Cefaparole
Cefalexin*(Cephalexin; Cefzil) Cefditoren* Ceuprenam Cefcanel
Keex) Cefmandole* Cefetamet* Cefoselis Cefedroler
Cephaloglycin* Cefuroxime* (Zinnat, Cexime* (Suprax) Cefozopran Cefempidone
Cefalonium Zinacef, Ceftin, Cefmenoxime* Cefpirome* Cefetrizole
Cefaloridine* Biofuroksym) Cefodizime* Cefquinome Cevitril
Cefalotin* Ceforanide* Cefoperazone* (Cefobid) Cefmatilen
(Cephalothin; Cefuzonam Cefotaxime* (Claferan) Cefmepidium
Cefadryl) Cefotium* Cefpimizole Cefovecin
Cefapirin*(cephapirin; Cefacler* (cecler, Cefpodoxime* (Vantin) Cefoxazole
Cefadryl) Distaclor, Keor, Cefpiramide* Cefrotil
Cefatrizine* Raniclor) Cefteram Cefsumide
Cefazaur Ceftibuten* (Cedax) Ceftioxide
Cefazedone* With antianaerobic Ceftiofur Ceftobiprole
Cefazolin* activity Ceftiolene (Previously BAL 9141 &
Ro 63-9141)
(Cephazolin; Ancef, Cefoxitin Ceftizoxime* (Cezax) Cefuracetime
Kefzol) Cefmetazole* Cefsulodin* Ceftriaxone*
(Rocephin)
Cefradine* cefotetan Antipseudomonal
(Cephradine; Velosef) Ceftazidime* (Fortum,
Cefroxadine* fertaz)
Ceftezole* Cefoperazone
* Antibacterials for systemic use.
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b-lactams in clinical practice 85
three stages (Mandell and Petri 1996). In the rst stage,
which takes place in cytoplasm, the product Uridine
diphosphate (UDP)-acetylmuramyl-pentapeptide accu-
mulates in the cells when subsequent synthetic stages
are inhibited. e last reaction in the synthesis of this
compound is the addition of a dipeptide, D-analyl-
D-alanine. Synthesis of the dipeptide involves prior
racemization of L-alanine and condensation catalyzed
by D-alanyl-D-alanine synthetase. During the second
stage reactions, UDP-acetylmuramyl-pentapeptide and
UDP-acetylglucosamine are linked, with the release
of uridine nucleotides, to form a long polymer. In the
third stage, the transpeptidation reaction occurs outside
the cell membrane, though the transpeptidase itself is
membrane bound. e terminal glycine residue of the
pentaglycine bridge is linked to the fourth residue of the
pentapeptide (D-alanine), releasing the fth residue
(also D-alanine). It is this last step in the peptidoglycan
synthesis that is inhibited by the B-lactam antibiotics.
Stereo models reveal that the conformation of penicillin
is very similar to that of D-analyl-D-alanine (Waxman
et al. 1980; Kelly et al. 1982). e transpeptidase is
probably acylated by penicillin, i.e., penicilloyl enzyme
is formed with cleavage of the −CO-N- bond of the
B-lactam ring (Mandell and petri 1996).
e targets for the action of penicillins and cepha-
losporins are collectively termed as penicillin-binding
proteins (PBPs) (Spratt 1980; Ghuysen 1991). e
higher molecular weight PBPs of E. coli (PBP 1A and 1B)
includes the transpeptidases responsible for synthesis
of peptidoglycan. Enzymes inhibited by B-lactams are
PBPs (these are transpeptidases and are involved in nal
transpeptidation step in synthesis of peptidoglycan).
Bacteria have multiple PBPs for eg. E.coli has seven,
each with a distinct role. PBP 1A and B are important in
cell elongation, PBP 2 maintain rod shape of cell wall,
PBP 3 forms septum between dividing cells. PBPs 4, 5,
and 6 are non-essential or redundant, since mutants
lacking any of them were viable. Individual PBPs have
dierent sensitivity with individual B-lactams, although
at clinical doses most drugs bind to more than one PBP
(Tomasz 1986). Inhibition of the transpeptidases causes
spheroplast formation and rapid lysis. However, inhibi-
tion of other PBPs may cause delayed lysis (PBP 2) or
the production of long, lamentous forms of bacterium
(PBP 3). Non-lytic killing by penicillin may involve
holin-like proteins in the bacterial membrane that col-
lapse the membrane potential (Bayles 2000).
Newer cephalosporins
RWJ-54428
RWJ-54428 is a parenteral cephalosporin (Fig. 4) with
a high level of activity against Gram-positive bacteria
including MRSA (Hecker et al. 2000; Chamberlandt et al.
2001). RWJ-54428 was also found to be the most active
agents against drug-resistant enterococci. e potency
of this new cephalosporin against MRSA is related to a
high anity for Penicillin binding protein 2a (PBP 2a)
(Dudley et al. 2003). RWJ-54428 also displayed excel-
lent anity for PBP 5 from Enterococcus hirae R40, with
a MIC50 of 0. 8mg/L. RWJ-54428 also showed stability to
hydrolysis by puried type A B-lactamase isolates from
S. aureus PC1. RWJ-54428 was 8-fold more active than
vancomycin and 32-fold more active than ampicillin
against vancomycin susceptible E. faecalis (Hecker et al.
2000). Vancomycin resistant E. faecalis remained sus-
ceptible to RWJ-54428, which was found to be 1000-fold
more active than vancomycin against these isolates. e
potent activities of RWJ-54428 against B-lactam resist-
ant Staphylococci and enterococci were correlated with
high anities for Staphylococcal PBP 2a and PBP 5 from
enterococci, both of them being major determinants of
resistance to B-lactam antibiotics. e enhanced bind-
ing of RWJ-54428 to MRSA PBP 2a is correlated to a
pharmacophore that is structurally distinct from that of
other reported anti- MRSA B-lactam (Hecker etal. 2003).
e pharmacophore of RWJ-54428 also provides bind-
ing anity to E.hirae PBP 5, which shares some com-
mon features with MRSA PBP 2a and E.faecium PBP 5
(Hartman and Tomasz. 1984; El Kharroubi et al. 1991).
Recent surveys of the susceptibilities of multidrug-resist-
ant Gram-positive bacteria to RWJ-54428 indicate that
this agent has the potential to be a useful agent against
Gram-positive bacteria. However, this compound cur-
rently does not appear to be in active development.
S-3578
S-3578 is a parental cephalosporin with a chemical for-
mula (6R,7R)-7-[(Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-
2-ethoxyiminoacetylamino]-3-{1-[3-(N-methylamino)
pr op yl]}-1H-imi dazo(4,5- b]pyrid ine-4 -i um-4- yl )
methyl-8-oxo-5-thia-1-azabicyclo[4.2.0] Oct-2-ene-2-
carboxylate monosulfate. It is an inner salt monosulfate
with an imidazopyridinium group in the side chain at
position 3 and displays broad and potent activities in vitro
against both MRSA and P. aeruginosa (Yoshizawaet al.
2002; Fujimura et al. 2003). is may be attributed to:
(1) S-3578 possesses better bactericidal activity than
H
N
N
S
S
S
NH2
Cl
S
N
H2N
N
OH
O
O
CO2H
N
CH3SO3H
Figure 4. RWJ-54428 (MC-02, 479).
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86 Shahid et al.
vancomycin, (2) Levels of binding of S-3578 to serum
protein was 22% and that of vancomycin was 47%, hence
the levels of free drug are responsible for greater ecacy
(Tsuji et al. 2003). Anti-MRSA ecacy of S-3578 may also
be related to structural aspects, such as a new side chain
at position 3 rather than at position 7 on the cephem
nucleus. is compound was suggested as a promising
new cephalosporin for the treatment of polymicrobial
infections or superinfection caused by Gram-positive
and Gram-negative bacteria including MRSA and P. aer-
uginosa, however, similar to RWJ 54428, this compound
does not appear to be in active development.
Ceftobiprole (BAL 9141)
Ceftobiprole, formerly known as BAL 9141 and RO
63-9141, is a member of class of parenteral pyrro-
lidinone -3- ylidemethyl cephalosporin. BAL 9141 has
documented activity against methicillin-resistant (MR)
staphylococci (MIC90 2- 4 mg/L), Enterococcus faecalis
(MIC90 4 mg/L) and penicillin resistant pneumococci
(MIC90 2 mg/L), while preserving anti-Gram-negative
activity of third and fourth generation cephalosporin.
BAL 9141 exhibits in vivo cidal activity with a moderate
post-antibiotic effect (Davies et al. 2006). In addition
to inhibiting normal complement of PBPs in most spe-
cies, it also binds with high affinity to acquired PBP2a
(PBP2’) of MRSA strains, PBP2a of S. epidermidis and
PBP2x of penicillin resistant S.pneumoniae (Hebeisen
et al. 2001; Davies et al. 2006; Lovering et al. 2006)
while it does not bind to PBP5 of E.faecium. Signature
activity of ceftobiprole is its activity against MRSA,
due to combination of potent binding to PBP2a and
stability to Staphylococcal penicillinase (Hebiesen
et al. 2001). Ceftobiprole was also shown by Hebeisen
et al. (2001) to be measurably hydrolysed by TEM-3
and TEM-4 ESBLs. For AmpC producing organisms,
potent activities of ceftobiprole and cefepime were
not directly correlated with B-lactamase stability,
suggesting that ceftobiprole may possess other prop-
erties, such as rapid penetration into periplasm of
Gram-negative organisms. It was found to be refrac-
tory to hydrolysis by common Staphylococcal PC 1,
the classA TEM-1 and class C AmpC B-lactamase but
was labile to hydrolysis by class B and D and class A
ESBLs (Queenan et al. 2007).
In vitro activities, as reported in recent studies,
conrmed the potency and promising activity of BAL
9141 against MR-Staphylococci including multidrug-
resistant strains. MICs of ceftobiprole for MRSA ranged
from 0.12 to 4 mg/L, with MIC90 values of 2mg/L gen-
erally observed (Jones et al. 2002; Bogdanovich et al.
2005; Davies et al. 2006). Ceftobiprole is poorly hydro-
lysed by PC 1 B-lactamase of S. aureus (Hebiesen et al.
2001), resulting in ceftobiprole MIC90 value of 0. 5mg/L
for methicillin-sensitive Staphylococcus aureus (MSSA)
strains (Jones et al. 2002). It also showed excellent
activity against penicillin-resistant S. pneumoniae
(MIC90 0. 25 mg/L), vancomycin susceptible and resist-
ant E. faecalis (MIC90 4 mg/L), penicillin-resistant
viridans group Streptococci (MIC90 1 mg/L), H. inu-
enzae (MIC90 0.06–0.5mg/L) and M. catarrhalis (MIC90
0.5mg/L ) (Queenan et al. 2007). Ceftobiprole was also
found to be active against pathogenic Neisseria (MIC90
0.004–0. 006 mg/L) and Gram-positive anaerobes (MIC90
< 0. 25 mg/L).
BMS-247243
BMS-247243 is a B-lactam with improved anity
for PBP2a (a mecA gene product), the target enzyme
responsible for methicillin resistance in Staphylococci.
It is bactericidal for MRSA, killing the bacteria twice as
fast as vancomycin. BMS-247243 has cephalosporin
nucleus and unique features on C-7 and C-3 (Fung
Tomc et al. 2002). At C-7, dichloro group imparts
lipophilicity (necessary for good anti- MRSA activity).
At C-3, the morpholinium quaternary component is
important for potency and dimethyl group contribute
to improved solubility. As reported in recent study
(Fung Tomc et al. 2002), MIC90 of BMS-247243 for
methicillin-susceptible Staphylococci ranged from <
0.25 to 1 mg/L while MIC90 for B-lactamase produc-
ing S.aureus strains was four fold higher than for
B-lactamase non-producing strains. e anity of
BMS-247243 for PBP 2a was >100-fold better than that
of methicillin or cefotaxime. Hence it may prove to be
useful in the treatment of infections caused by multi-
drug resistant organisms, especially MRSA, S. epider-
midis, and S. haemolyticus.
Ceftaroline (PPI-0903)
Ceftaroline fosamil which was formerly known as PPI-
0903 and TAK-599 is an N-phosphano-type prodrug
cephalosporin and upon hydrolysis of phosphonate
group it is released in its active form in vivo (Iizawa et al.
2004; Sader et al. 2005; Sader et al. 2008). Ceftaroline
has potent activity against MRSA, and many other
Gram-positive organisms, as well as against Gram-
negative bacilli (Sader et al. 2005; Jacqueline et al. 2007;
Mushtaq et al. 2007; Sader et al. 2008). Staphylococcus
spp., including MRSA and MRCoNS were reported to
be susceptible to ceftaroline with an MIC90 ranging
between 0.25- 2mg/L (Iizawa et al. 2004; Sader et al.
2005). Ceftaroline has also been reported to posses
potent activity against vancomycin-resistant MRSA
strains, multidrug-resistant S. pneumoniae strains, and
Haemophilus inuenza (Kurazono et al. 2004; Sader
et al. 2006).
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b-lactams in clinical practice 87
In a more recent study (Sader et al. 2008), ceftaroline
showed an MIC50 and MIC90 of 0.5 mg/L, and the highest
MIC was found as 1 mg/L against community acquired
methicillin-resistant S. aureus (CA-MRSA) strains. In
that study performed on 152 geographically diverse
strains, 97.4% strains had an MIC of 0.5 mg/L, and the
MICs ranged between 0.25 - 1 mg/L in the total study
population (Sanders et al. 2008). us, to conclude, the
preliminary results on this investigational anti-MRSA
cephalosporin demonstrated that it could be a potent
antimicrobial for the treatment of both community- and
hospital-acquired infections.
Mechanism of resistance
e signicantly high consumption of B-lactams
exerts considerable selection for resistance to these
antibiotics. e resistance may occur via one of these
mechanisms:
(a) modication of normal penicillin binding proteins
(PBPs)
(b) bypassing of the normal PBPs
(c) impermeability of outer membranes to drugs
(d) production of B-lactamases
(e) ability to pump out (eux) B-lactams
Eux system
Drug eux system pump out antibiotics from bacteria
in an energy dependent manner using transporters clas-
sied as follows:
- Major Facilitator Superfamily (MFS)
- Adenosine triphosphate binding cassette (ABC)
- Small Multidrug Resistance (SMR)
- Resistance Nodulation Cell Division (RND)
- Multidrug and toxic Compound Extrusion (MATE)
e transporters used could be single component
transport across the cytoplasmic membrane or multi-
component pumps-transport substrate in conjunction
with a periplasmic membrane fusion protein (MFP)
component and an outer membrane protein (OMP)
component across the entire cell envelope (Butaye et al.
2003). In Gram-negative bacteria, RND pumps are most
prominent because of their major role in the develop-
ment of acquired and intrinsic resistance to a number
of antibiotics and the transportation of biocides, dyes,
detergents and organic solvents out of cell. Genome of
E.coli has the AcrAB-Tol C System, P. aeruginosa has the
Mex AB-OprM, Mex CD-Opr J, Mex EF-OprN, Mex XY,
Mex JK, Mex GHI-Opm D and Mex VW Systems. While
other Gram-negative bacteria, such as B.cepacia com-
plex with emerging resistance in cystic brosis patients,
S. maltophilia and Neisseria gonorrhae may have Ceo
AB-Opc M, Amr AB-Opr A, Sme ABC, Sme DEF or Mtr
CDE Systems, respectively (Jo et al. 2003).
Modication of PBPs and bypassing are the most
important mechanisms of resistance in Gram-
positive cocci, but the hydrolysis of B-lactam rings by
B-lactamases is the prevalent mechanism in Gram-
negative bacterial species (Livermore 1998).
b-lactamases and resistance
e common form of resistance is either through lack
of drug penetration [outer membrane protein (OMP)
mutation and eux pumps], ESBL production including
hyperproduction of an AmpC type B-lactamase, and/
or carbapenem hydrolyzing B-lactamases. Increased
clinical use of B-lactams has selected for B-lactamase-
producing organisms. Sequencing of B-lactamases
allows them to be divided into four classes, A to D. Class
A, C, and D function by a serine ester hydrolysis mecha-
nism. Whereas class B B-lactamases function using
a zinc ion to attack B-lactam ring. Attempts to classify
B-lactamases began in the late 1960s and the rst scheme
to achieve wide acceptance was proposed by Richmond
and Sykes (1973). is scheme was based on whether
an enzyme hydrolyzed penicillin more or less rapidly
than cephaloridine, and whether its activity was inhib-
ited by cloxacillin and/or p-chloromercuribenzoate.
Subsequently, Bush proposed a major revision in 1989
and updated in 1995 (Bush et al. 1995). Another type of
B-lactamases called SHV-type ESBLs is found frequently
in clinical isolates than any other type of ESBL. SHV
refers to sulfhydryl variable, and this designation was
made because it was thought that the inhibition of SHV
activity by p-chloromercuribenzoate was substrate-
related and was variable according to the substrate used
for the assay. SHV-type ESBLs have been detected in a
wide range of Enterobacteriaceae and outbreaks of SHV-
producing Pseudomonas aeruginosa and Acinetobacter
spp. have also been reported. TEM-type ESBLs are deriv-
ative of TEM-1 and TEM-2. TEM-1 was reported in 1965
from an E. coli isolate from a patient in Athens, Greece,
named Temoniera (hence the designation TEM). TEM-1
is able to hydrolyze ampicillin at a greater rate than
carbenicillin, oxacillin, or cephalothin, and has negligi-
ble activity against extended-spectrum cephalosporins.
It is inhibited by clavulanic acid (readers are encouraged
to read Paterson and Bonomo 2005, for description of
TEM- and SHV-ESBLs in greater detail). In recent years,
CTX-M types ESBLs are expanding rapidly. e name
CTX reects the potent hydrolyzing activity of these
B-lactamases against cefotaxime. Organisms produc-
ing CTX-M type B-lactamases typically have cefotaxime
MICs in the resistant range (>64mg/L), while ceftazi-
dime MICs are usually in the apparently susceptible
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88 Shahid et al.
range (2 to 8 mg/L). However, some CTX-M type ESBLs
may actually hydrolyze ceftazidime and confer resist-
ance to this cephalosporin (MICs as high as 256 mg/L)
(Baraniaket al. 2002; Poirel et al. 2002). CTX-M type
B-lactamases hydrolyze cefepime with high eciency,
and cefepime MICs are higher than observed in bacteria
producing other ESBL types. It appears that CTX-M type
B-lactamases are closely related to B-lactamases of kluy-
vera spp. ( Decousser et al. 2001; Di Conza et al. 2002;
Humeniuk et al. 2002; Poirel et al. 2002). For example, a
chromosomal B-lactamases gene of Kluyvera georgiana
encoded an ESBL, KLUG-1, which shares 99% amino
acid identity with CTX-M-8 (Poirel et al. 2002). CTX-M-
type B-lactamases have 40% or less identity with TEM
and SHV-type ESBLs.
Two types of carbapenem-hydrolyzing enzymes have
been described, (1) serine enzymes possessing a serine
moiety at the active site, (2) and metallo-B-lactamases
(MBLs), requiring divalent cations, usually zinc, as
metal cofactor for enzyme activity (Bush et al. 1995;
Bush 2001). e serine carbapenemases are invariably
derivative of class A or class B enzymes and usually
mediate carbapenem resistance in Enterobacteriaceae
or Acinetobacter spp. Carbapenemases characterized
from Enterobacteriaceae include Nmc A, Sme-1, Sme-2,
Sme-3, Imi-1, KPC-1, KPC-2, KPC-3, and GES-2 (Yang
et al. 1990; Nordmann et al. 1993; Rasmussen et al. 1994;
Queenan et al. 2000; Poirel et al. 2001; Yigit et al. 2001).
Despite the avidity of these enzymes for carbapenems,
they do not always mediate high-level resistance, and
clavulanate inhibits not all of them. All MBLs hydrolyze
imipenem, but their ability to achieve this varies consid-
erably and rate of hydrolyses may or may not correlate
with the bacterium’s level of resistance to carbapenem
(Massidda et al. 1991). e zinc ions, in MBLs, in turn
usually coordinate two water molecules necessary for
hydrolysis (Rasmussen and Bush 1997). e principal
zinc-binding motif is histidine-x-histidine-x-aspartic
acid (HXHXD) (Rasmussen and Bush 1997) and as they
are zinc dependent, their activities are inhibited by
EDTA. Unlike serine B-lactamases, MBLs possess a wide
plastic active site groove and accordingly can accom-
modate most B-lactam substrate, facilitating their very
broad spectrum of activity.
IMP-1, IMP-2, IMP-4, IMP-7, IMP-9, IMP-10, IMP-11,
IMP-16, and IMP-18 has been identied in P. aeruginosa
isolates while A. baumanii strains were also found to pos-
sess variety of IMP type MBLs including IMP-1, IMP-2,
IMP-4, IMP-5, IMP-6, and IMP-11. Other isolates in
which IMP MBLs were detected include Shigella exneri,
Serratia marcescens, Pseudomonas putida, Alcaligenes
spp., Enterobacter cloacae, Klebsiella oxytoca, K. pneu-
moniae, Proteus spp., and Providencia spp. etc. (Walsh
et al. 2005). IMP-3 may actually be the progenitor of
IMP-1 rather than being a variant of IMP-1 (Iyobe et al.
2000). Another dominant group of acquired MBLs is the
VIM type enzymes. VIM-1 (Varonese Imepenemase)
was described rst in Verona, Italy from a P. aeruginosa
isolates (Lauretti et al. 1999). Later VIM-1 has also been
detected in E. coli (Scoulica et al. 2004) and in several K.
pneumoniae isolates in Greece (Giakkoupi et al. 2003),
and very recently VIM-1 positive K. pneumoniae isolates
has been detected in France (P. Nordmann, unpublished
data) that was associated with the ESBL SHV-5. VIM-2
producing P. aeruginosa strains have also been reported
from other countries such as Japan, South Korea,
Portugal, Spain, Poland, Croatia, Chile, Venezuela,
Argentina, Belgium, and recently in the United States
(Prats et al. 2002; Yum et al. 2002; Mendes et al. 2004).
VIM-3, a novel variant of VIM series has been identied
inP. aeruginosa isolates in Taiwan (Yan et al. 2001). VIM-4
was reported from P. aeruginosa from Greece. VIM-5,
VIM-6 and VIM-7 have also been characterized. VIM-7
has been isolated from carbapenem-resistant P. aerugi-
nosa isolate from Houston, Texas (Toleman et al. 2004),
and shares 77% identity with VIM-1 and 74% with VIM-2
type B-lactamase. Although its sequence is very dierent
from that of other VIMs and probably arose from a dif-
ferent ancestral source (Toleman et al. 2004). Moreover,
new MBL-types, including SPM, GIM, SIM have also
been described in recent years (Walsh 2002; Castanheira
et al. 2004; Lee et al. 2005). SPM-1 (Sao Paulo MBL) was
isolated from P. aeruginosa isolate in 1997 (Walsh 2002),
showed highest identities with IMP-1 (35.5%), ImiS
(32.2%), CphA (32.1%), BCII (30.0%), and Ccr (27.0%)
(Walsh 2002). Sequence of SPM-1 diers signicantly
from that of IMP and VIM, not least due to presence of an
insertion of 24 amino acids just after the active site. is
insertion has been shown to be very exible and acts as a
“loop”, probably augmenting the binding and hydrolysis
of B-lactams (T. R. Walsh, unpublished data). Preferred
substrates of SPM are penicillins, ampicillin, piperacil-
lin, carbenicillin, azlocillin, and cephalothin (Livermore
1995; Koh et al. 2001; Docquier et al. 2003; Giakkoupi
etal. 2003). Like IMP-1 and VIM-1, SPM-1 does not hydro-
lyze clavulanate or aztreonam. In general, SPM-1 binds
cephalosporins more tightly than penicillins. GIM-1
(German Imipenemase), a novel class B B-lactamase,
was isolated from P. aeruginosa (Castanheira etal. 2004)
and displayed identity with IMP-1, IMP-4 and IMP-6
(43.1%, 43.1%, and 43.5%, respectively) while it showed
31.2% identity to VIM-7, 28.8% to VIM-1, VIM-4, and
VIM-5 and only 28.0% similarity with SPM-1. It possess
major consensus feature of MBL class B1 family, such as
principal zinc-binding motif (HXHXD) and possess two
zinc at its active site (Rasmussen and Bush 1997). GIM-1
demonstrates a hydrolytic prole similar to IMP-1 but is
a weaker enzyme.
AmpC B-lactamases, belonging to Ambler’s molec-
ular class C and group 1 of Bush-Jacoby-Medieros
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b-lactams in clinical practice 89
functional classication (Bush et al. 1995), are a large
group of enzymes of broad substrate specicity. ey can
degrade penicillins and most cephalosporins (including
7-A-methoxy derivatives) and are poorly inhibited by
serine B-lactamase inactivators. AmpC B-lactamases
are usually not active or very poorly active on aztre-
onam, the zwitterionic oxyimino-cephalosporins (like
cefepime and cefpirome), and imipenem, but can
contribute to decreased susceptibility or even resist-
ance to these compounds especially when enzyme
overproduction is associated with permeability defects
or drug eux (Hanson 2003; Toleman et al. 2006). A
number of AmpC B-lactamase are encoded by chromo-
somal genes resident in some Gram-negative pathogens
(P. aeruginosa, Acinetobacter spp., and several members
of Enterobacteriaceae), while others are encoded by
genes associated with mobile DNA elements that can
be acquired by horizontal gene transfer (Hanson 2003).
ISCR1 is recognized as a very important element in the
mobilization of many dierent antibiotic resistance
genes, including plasmid-mediated AmpC B-lactamase
genes (Toleman et al. 2006).
Other important types of ESBLs are OXA-type ESBLs.
ese are named because of their oxacillin-hydrolyzing
abilities and they were characterized by hydrolysis
rates for cloxacillin and oxacillin being greater than
50% in comparison to that for benzyl penicillins (Bush
et al. 1995). OXA-1, which is most common OXA-type,
has been found in 1-10% of E. coli isolates (Hanson
2003). Most OXA-type-B-lactamases do not hydrolyze
the extended-spectrum cephalosporins to a signicant
degree and are not regarded as ESBLs however OXA-10
hydrolyze cefotaxime, ceftriaxone and aztreonam.
Other OXA-type ESBLs include OXA-11, -14, -16, -17,
-19, -15, -18, -28, -31, -32, -35 and -45 (Toleman et al.
2003). e oxacillinases that have been character-
ized from A. baumanii includes OXA-23 to OXA-27,
OXA-40, and OXA-48 (Livermore and Woodford 2006).
ese enzymes confer resistance to cefotaxime and
sometimes ceftazidime and aztreonam (Danel et al.
1998; Danel et al. 1999). e evolution of OXA-type
B-lactamases from parent enzymes with narrower
spectra has many parallels with evolution of SHV- and
TEM-type ESBLs.
Eorts to combat resistance due to
b- lactamases
As the resistance has spread, pharmaceutical chem-
ists have responded by developing compounds with
increased stability, or by protecting labile agents by
inhibiting the B-lactamases. Stability to B-lactamases
can be achieved by attaching to the B-lactam ring a
substituent that hinders access of the active-site serine
or that displaces water from preventing completion of
hydrolysis (Frere 1995).
Strategies to achieve these include
(a) attaching a bulky acyl group to the amino group of
6-aminopenicillanic acid, or 7-aminocephalospo-
ranic acid (see Fig. 5 for the acyl groups attached
in some of the representative B-lactams)
(b) replacing the hydrogen on carbon 6 (penicillins)
or 7 (cephalosporins) with an A-methoxy group
(Abraham 1987).
Although responded by the pharmaceutical chemists,
these new agents, in turn, have selected for new forms of
B-lactamase-mediated resistance. us, it appeared to be
as a cyclic process of drug development and the emergence
of resistance. Some of the representative B-lactamases and
their preferred substrate are shown in Table 2.
B-lactamase stability was more dicult to achieve in
B-lactams that have activity against Gram-negative spe-
cies than in those directed against Gram-positive because
Gram-negative organisms produce a wider variety of
B-lactamases than Gram-positive organisms (Tomasz
1994). Incorporating broad-spectrum stability proved
particularly dicult with penicillins, the only one with
such stability being temocillin, the 6-alpha-methoxy
analogue of ticarcillin. Benzylpenicillin and cephalothin
CH2
Benzyl penicillin
Methicillin Cefotaxime
Cephalothin
S
CH2
OCH3
CH2
OCH3
N
S
N
NH2
OCH3
S
N
O
S
X
CH
2
CONH
CH
2
O.CO.R
2
COOH
X=H, R2=CH3 Cephalothin
X=OCH3, R2=NH2 Cefoxitin
Figure 5. Side chain substitution to confer B-lactamase stability.
e gure above shows bulky acyl groups attached to amino group of
6-APA or 7-ACA of some of the representative B-lactam antibiotics.
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90 Shahid et al.
are labile to many B-lactamases but methicillin and
cefotaxime, with bulkier substituents, are more stable.
e development of B-lactamase-stable cephalosporins
has been more successful. An oxyimino-aminothiazolyl
7-acyl side chain makes cephalosporin stable to TEM-1
and SHV-1 enzymes. Such groups are incorporated in
most third-generation cephalosporins. A 7-A-methoxy
group (as in cefoxitin, cefmetazole, and latamoxef) con-
fers stability to TEM-1 and SHV-1 enzymes and to the
CepA chromosomal B-lactamase of Bacteroides fragilis
(Livermore 1998). Similarly, addition of an A-methoxy
group at position 6 (penicillins) or 7 (cephalosporins)
provides stability against many B-lactamases. Ticarcillin
and cephalothin, which lack this substituent, are labile
to many B-lactamases but their A-methoxy analogues
temocillin and cefoxitin are more stable.
During the mid 1980s, a threat to B-lactamase sta-
ble oxyimino aminothiazolyl cephalosporins became
apparent, with the emergence of extended-spectrum
B-lactamases (ESBLs). Most of the ESBLs are mutants of
the classic TEM-1, TEM-2 and SHV-1 types (Jacoby and
Medieros 1991; Du Bois et al. 1995). ese ESBLs have
one to four amino acid replacements compared with
their parent enzymes, and these changes suciently
remodel the active site to allow attack on aminothiazolyl
compounds (Livermore 1998).
Other B-lactam antibiotics with broader spectrum of
activity are described in the following section.
Carbapenems
Carbapenems (Imipenem and meropenem) are
B-lactams that contain a fused B-lactam ring and a
5-membered ring system that diers from the penicil-
lins in being unsaturated and containing a carbon atom
instead of the sulphur atom.
Chemistry of carbapenems
Imipenem (Fig. 6a) is derived from a compound
(thienamycin) produced by Streptomyces catt-
leya. ienamycin is unstable, but imipenem, the
Table 2. Classications of B-lactamases.
Structural
class(Ambler)
Functional
class (Bush
et al.**)
Richamond and
Sykes class#Preferred substrate
Inhibited by
Clavulanate;Aztreonam;EDTA Representative enzymes
Serine B-lactamases
A 2a NL Penicillin ++ - - Staphylococcal
penicillinase
2b II and III Penicillin, Cephalosporin ++ - TEM-1,TEM-2,SHV-1
2be III and IV Penicillin, Cephalosporin,
Aztreonam, Monobactam
++ - - TEM-3 to TEM-29,SHV-2
to SHV-7 K.oxytoca K1,
PER1
2br NL Penicillin - - - TEM-30 to TEM-41,TRC-1,
2c II and V Carbenicillin, Penicillin + - - PSE-1,PSE-3,PSE-4,BRO-
1,BRO-2
2e IC Penicillin, Carbenicillin
Aztreonam, Cephalosporin
++ - - Cephalosporinases
from P.vulgaris FEC-I,
Bacteroides Cep A
2f NL Penicillin, Aztreanem,
Imipenem
+ - - Carbapenemase Imi-1,
NMC-A, Sme-I
C 1 I, except Ic Cephalosporin, Penicillin - ++ - AmpC enzymes from
Gram-negative bacteria,
MIR-I.
D 2d V Oxacillin, Penicillin V - - Oxa-1 to Oxa-11,13-15
Aeromonas OXA-12,PSE-2
Undetermined* 4* NL Penicillin, Carbenicillin,
Oxacillin
- - - Penicillinase from
Burkholderia cepacia,,
SAR-2
Zinc
B-lactamasesB
3 NL Penicillin, Carbenicillin,
Oxacillin Cephalosporin,
Imipenem
- - ++ L1 from
Stenotrophomonas
maltophilia, IMP-I,
Aeromonas hydrophilia
A 2h, Aeromonas A2s,
B. fragilis Cer A, P.
aeruginosa pMS 350.
NL = Not listed* None of Bush’s group 4 enzymes has yet been sequenced. V = Varies within group. ** For a comprehensive listing of dierent
B-lactamase types, see Bush et al. 1995. # For detailed classication, see Richmond and Sykes 1973.
Downloaded By: [INFLIBNET India Order] At: 08:16 20 June 2009
b-lactams in clinical practice 91
Figure 6. Newer carbapenems.
N
HH
C
O
COOH
SCH2CH2NHCH
H
HO
H3C
NH
(a) Imipenem
S
N
O
N
H
H
N
O
HO CH3
CO2Na
(b) Ertapenem
CO2Na
NSNH
SNH2
O
O
H
H
CH3
H
HH
H
HO
O
H2O
H
N
H3C
CO2H
(c) Doripenem (formerly S-4661)
R
S
N
H
H
O
R:
HO Me
NH2
NH
NH-Me
CONH2
NH2
NH2
J-114 ,871
CONH2
(d) trans-3,5 disubstrituted 1B-methyl carbapenems
J-114,870
J-111,225
J-111,347 (Prototype)
COOH
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92 Shahid et al.
(e) 1B-methyl-2-(thiazol-2-ylthio) carbapenem
S
N
H
H
O
N
S
R
R:
Me
HO Me
COOH
OH
Me
NH
NH
H
N
SM-232724SM-197436 SM-232721
(f) CS-023.
N
S
O
H
NN
H
HO
CON
NHCO NH2
NH
CO2H
(g) Faropenem
N
SO
OHO
O
H
H
OH
H
(h) Tebipenem
NS
CH3
H
HH
O
COOH
N
N
S
H
OH
H3C
N
O
H
O
O
H
S
NN
N
O
OH
NH2
(i) ME-1036
(j) Sanfetrinem
N
O
H
H
OH
O
OCH3
OH
Figure 6. Continued.
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b-lactams in clinical practice 93
N-formimidoyl derivative, is stable. Imipenem is mar-
keted in combination with cilastatin, a drug that inhibits
the degradation of imipenem by a renal tubular dehy-
dropeptidase. Meropenem is a dimethyl-carbamoyl-
pyrolidinyl derivative of thienamycin. It does not require
co-administration with cilastatin as it is not sensitive to
renal dehydropeptidase. Its in vitro activity and toxicity
is very much similar to imipenem.
Activity and stability to b-lactamases
Imipenem is active against a wide variety, including pen-
icillinase-producing strains, and Listeria. Some strains
of methicillin-resistant staphylococci are susceptible
but many strains are not. Activity is excellent against the
Enterobacteriaceae and most strains of Pseudomonas
and Acinetobacter are inhibited. Meropenem is active
against some imipenem-resistant P.aeruginosa but
less active against Gram-positive cocci. Imipenem and
meropenem have wider B-lactamase stability than
other B-lactams, being eectively resistant to all the
common plasmid encoded class A, C, and D enzymes,
including extended-spectrum TEM and SHV deriva-
tives (Heritage et al. 1999), PER-1 and AmpC enzymes
(Livermore 1998). is stability partly depends on the
presence of a simple trans-6-hydroxyethyl group; ana-
logues with acyl substituents or with 6-hydroxyethyl
in the cis conguration are less stable to B-lactamases
(Christensen 1981).
Unlike new cephalosporins, they are not prone to
attack by B-lactamases types that are widely prevalent,
so B-lactamase-mediated resistance has been slow to
emerge and spread. Nevertheless, carbapenems are
attacked by few B-lactamases, most notably the zinc
(molecular class B) enzymes (Livermore 1998). A seri-
ous concern is the emergence of plasmid-mediated class
B enzymes in Enterobacteriaceae and Pseudomonas.
Such enzymes, including IMP, VIM, and KPC, have been
found in Serratia marcescens,K. pneumoniae,P. aerugi-
nosa,Acinetobacter, and other bacterial species (Osano
et al. 1994; Livermore and Woodford 2006). is enzyme
confers resistance to all B-lactams except monobactams.
Carbapenemase activity also occurs in three closely
related class A B-lactamases, Sme-I (Yang et al. 1990),
Imi (Rasmussen et al. 1994), and NMC-A (Nordmann
et al. 1993), found in Enterobacter cloacae and Serratia
marcescens strains.
ere have been reports of multiple B-lactamase pro-
ductions in a single Gram-negative pathogen (Bradford
et al. 2004; Bratu etal. 2005). Isolates of Klebsiella spp.
producing KPC-2, SHV, and inhibitor-resistant TEM-30
B-lactamases have been reported as endemic in New York
city (Moland et al. 2007). Recently, Moland et al (2007)
reported a K. pneumoniae isolate from New York city that
produced up to eight dierent B-lactamases including
FOX-like plasmid-mediated AmpC B-lactamase, SHV-
1-like, SHV-12-like ESBL, a KPC-like carbapenemase,
OXA-like, TEM-1-like, TEM-30-like, and PSE-1-like
B-lactamase. Currently rising problem includes CTX-M
ESBLs, plasmid-mediated AmpC B-lactamases, and KPC
carbapenemases in Enterobacteriaceae, while OXA- and
metallo-carbapenemases are of growing importance
in Acinetobacter spp. Clones of K. pneumoniae and
E. cloacae with KPC enzymes have spread in multiple
hospitals around New York since 2003 (Livermore and
Woodford 2006).
Newer carbapenems
Carbapenem development continues to discover agents
with greater potency or improved pharmacokinetic
properties (Tsuji et al. 1998). Various newer carbapen-
ems have been discovered, some in recent years, and are
really novel.
Ertapenem
Ertapenem (formerly MK-0826 and L-749, 345) (Fig. 6b)
is a carbapenem that shares the broad spectrum of imi-
penem and meropenem against Enterobacteriaceae,
Gram-positive organisms, and anaerobes, but is less
active against non-fermenters. Ertapenem diers from
meropenem in its 2` substituents, where it carries a
meta-substituted benzoic acid group. Carbon 1 car-
ries a B-methyl group, protecting against hydrolysis
by renal dehydropeptidase I (Livermore et al. 2003).
Ertapenem has a trans hydroxyethyl on the 6-position
and this conguration is critical to B-lactamase stability
in carbapenems.
Like meropenem, ertapenem binds most strongly
to penicillin binding protein (PBP-2) of E.coli, as com-
pared to PBP-3, and has good anity also for PBP-1a
and 1b (Kohler et al. 1999). However in contrast, imi-
penem binds primarily to PBP-2, then 1a and 1b, and
has only weak anity for PBP-3 (Sumita et al. 1990).
Inactivation of PBP-1a and b achieves rapid bactericidal
action without the prior lamentation that occurs with
agents such as third-generation cephalosporins, which
binds primarily to PBP-3. In vitro activities, as reported
in recent studies, showed that MICs of ertapenem for
Enterobacteriaceae fall between 0.008 and 0. 12mg/L
(Livermore et al. 2003). ese values are similar to those
of meropenem, and eight to 16-fold below those of imi-
penem. Like imipenem and meropenem, ertapenem is
active against methicillin-susceptible S. aureus (MSSA)
but not against MRSA. However, ertapenem has only
marginal activity against important non-fermenters
(Livermore et al. 2001; Fuchs et al. 2001). Ertapenem
overcomes most resistance to other B-lactams in
Enterobacteriaceae, but unlike earlier carbapenems,
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94 Shahid et al.
lacks signicant activity against non-fermenters, limit-
ing its empirical role in nosocomial infections.
Doripenem
Doripenem (Fig. 6c), which was formerly S-4661, is a novel,
broad-spectrum parenteral carbapenem with a chemical
formula (+)-(4R,5S,6S)-6-[(1R)-1-1hydroxyethyl]-4-me-
thyl-7-oxo-3[[3S,5S)-S-(sulfamoylaminomethyl)-pyr-
rolidin-3-yl]thio]-1-azabicyclo[3.2.0]hept-2-ene-2-
carboxylic acid monohydrate. is structure confers
B-lactamase stability and resistance to inactivation by
renal dehydropeptidases. Recent studies have indicated
that doripenem has a spectrum and potency against
Gram-positive cocci most similar to imipenem or ertap-
enem (Tsuji et al. 1998; Curran et al. 2001; Jones 2001;
PanKuch et al. 2002; Hoellman et al. 2003; Livermore
et al. 2003) and a Gram-negative activity like meropenem
but two- or four-fold superior to imipenem (Wiseman
et al. 1995).
Earlier reports on the microbiological/pharmacologi-
cal features of doripenem demonstrated:
1. high anity for PBP targets that are species-specic
(PBP3 in P.aeruginosa, PBPs 1, 2 and 4 in S. aureus,
PBP2 in E.coli (Hanaki et al. 1996)
2. bactericidal action (Inoue et al. 1996; Miwa et al.
1996; Nishino et al. 1996; Sasaki et al. 1994)
3. a post antibiotic eect of 1.8 h (in vitro) to 4.3 h
(in vivo) for P.aeruginosa (Nishino et al. 1996;
Totsuka et al. 1996)
4. inux in Gram-negative species by OprD channels
with eux sensitivity via the MexAB-OprM system
(Yamano et al. 1997)
5. pharmacokinetic parameters resembling mero-
penem (Nakashima et al. 1994)
6. low risk of convulsive side eects (Hori et al. 1997)
7. stability to wide variety of B-lactamases (Inoue et al.
1996; Sasaki et al. 1994)
8. high level stability to human recombinant dehy-
dropeptidase-I (Mori et al. 1996)
9. clinical success from human trials in Japan (Saito
et al. 1997; Arakawa et al. 1997)
Early in vitro development trials clearly placed dorip-
enem as a very broad-spectrum B-lactam, having a
compromised spectrum when used alone (Kobayashi
et al. 1997) against oxacillin-resistant staphylococci, E.
faecium, some Burkholderia spp. and Stenotrophomonas
maltophilia. is spectrum was similar to imipenem
and meropenem (Wiseman et al. 1995; Tsuji et al.
1998) but markedly superior to ertapenem (Jones 2001;
Pankuch et al. 2002; Curran et al. 2003; Hoellman et al.
2003; Livermore et al. 2003 ). In a recent clinical study,
doripenem exhibited potency and /or spectrum advan-
tages compared with both imipenem and meropenem
against the entire group of non-fermentative Gram-
negative bacilli, and for Enterobacteriaceae. is newer
carbapenem was four-to 32 fold more active than imi-
penem (MIC range, < 0.015–0.5mg/L).
trans-3,5-disubstituted Pyrrolidinylthio-1b-
methylcarbapenems
In the course of derivatisation, study of 1B-methyl
carbapenems, a novel trans-3,5 –disubstituted
Pyrrolidinylthio-1-B methyl carbapenem, J-111, 347,
was identied as a broad spectrum agent with activ-
ity against MRSA, as well as other Gram-positive and
Gram-negative organisms, including P.aeruginosa.
Subsequently, the 1B-methylcarbapenems, J -111, 225,
J-114, 870 and J-114, 871 (Fig. 6d) were synthesized from
the prototype J-111, 347 (Hashizume et al. 2000). e
stereochemistry of the side chains in these compounds
is novel. Known Pyrrolidinylthio-1B-carbapenems like
meropenem (Sunagawa et al. 1990), SM-17466 (Sumita
et al. 1995), BO-2727 (Nakagawa et al. 1993), MK-826,
and E1010 (formerly ER-35786) (Ohba et al. 1997) share a
cis-counterpart at the side chains. In a recent study, 90%
isolates of methicillin-resistant S. aureus (MRSA) and
methicillin-resistant coagulase-negative Staphylococci
(MRCoNS) were inhibited by J-111,347 (prototype), as
well as by, J-111,225, J-114,870, and J-114,871 at con-
centrations of 2,4,4, and 4mg/L (MIC-90) respectively
indicating that these agents were 32- to 64-fold more
potent than imipenem, which had MIC90 of 128 mg/L.
Although these drugs were less active in vitro than van-
comycin, which had MIC90s of 1 and 2 mg/L for MRSA
and MRCoNS respectively. However, these new carbap-
enems displayed better kinetics than vancomycin.
2- (iazol – 2 – ylthio) - 1b – methyl carbapenems
SM-197436, SM-232721, and SM-232724 are new
1B–methyl carbapenems with a unique 4- substituted
thiazol –2– ylthio moiety at the C-2 side chain (Fig. 6e).
ese newer carbapenems are subsequent modica-
tion of 2–(4-arylthiazol-2-ylthio)-1 B-methyl carbap-
enems. SM-17466 and its derivatives exhibited potent
anti-MRSA activity corresponding to a high anity for
PBP 2a. Although SM-17466 showed good ecacy in a
murine systemic infection model of MRSA, it did not
show sucient activity against E. faecium and thus
prompted for the search of new carbapenems (Sumita
et al. 1995). ese newer carbapenems listed above
are novel carbapenems having a ve or six member
cyclic amino group instead of the pyridium moiety of
SM-17466 and showed improved activity against E.fae-
cium. In recent studies (Ueda and Sunagawa 2003), the
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b-lactams in clinical practice 95
novel carbapenems were found active against MRSA and
MRSE with MIC90 of < 4mg/L. e MICs of SM-197436,
SM-232721, and SM-232724 for Streptococci including
penicillin resistant S.pneumoniae strains, ranged from
< 0.063 to 0. 5mg/L. ese drugs were the most active
B-lactams tested against E.faecium, and the MIC90S
for ampicillin resistant E.faecium ranged between 8
and 16 mg/L, which were slightly higher than the value
for linezolid. However, time-kill assays revealed the
superior bactericidal activity of SM-232724 compared to
those of quinupristin / dalfopristin and linezolid against
E. faecium strain with a 4-log reduction in CFU at four
times the MIC after 24h of exposure to antibiotics. Among
the Gram-negative bacteria, these carbapenems were
highly active against Haemophilus inuenzae,Moraxella
catarrhalis,Bacteroides fragilis, and showed antibacte-
rial activity equivalent to that of imipenem for E.coli,
K.pneumoniae, and Proteus spp. ough require further
evaluation, these carbapenems seem to be promising
to treat nosocomial infections caused by Gram-positive
and Gram-negative bacteria including MRSA or VRE.
CS-023 (RO 4908463)
CS-023 (RO 4908463, formerly known as R-115685)
(Fig. 6f ) is a new parenteral 2- substituted 1 B-methyl
carbapenem that has unique guanidine–pyrrolidine side
chain and binds with high anity to PBP1, PBP2 and
PBP4 from S. aureus ATCC 6538P and PBP1a, PBP1b,
PBP2 and PBP3 from E. coli NIHJ (Spratt 1980). CS-023 is
more stable to hydrolysis by human renal dehydropepti-
dase-I than meropenem or imipenem and is bactericidal
against P.aeruginosa and MRSA and also has broad
spectrum of activity against Gram-positive and Gram-
negative bacteria, including MRSA, penicillin-resistant
Streptococcus pneumoniae, H.inuenzae, members
of Enterobacteriaceae, and P. aeruginosa (Jacoby and
Medieros 1991; Kuwamoto et al. 2003; omson and
Moland 2004; Koga et al. 2005).
In recent studies, it was noted that, overall, CS-023
exhibited activity comparable to that of imipenem
against most isolates of Gram-positive pathogens,
whereas it was similar to meropenem against Gram-
negative pathogens (omson and Moland 2004).
However, the CS-023 was signicantly more potent than
imipenem and meropenem against oxacillin-resistant
S. aureus at MIC of 4mg/L, compared with 32mg/L for
meropenem and > 32mg/L for imipenem. CS-023 was
highly potent against B-lactamase–producing or non-
fermenting Moraxella catarrhalis (MIC 0. 008mg/L),
Haemophillus inuenzae (MIC 0. 06 mg/L) and against
most isolates of Enterobacteriaceae, including E.coli,
Shigella,Salmonella, and Klebsiella (MIC 0. 06mg/L). It
was interesting to note that the CS-023 was 4- to 8-fold
more potent against the isolates of meropenem resistant
P.aeruginosa. e broad spectrum of in vitro activity of
CS-023, in recent studies, suggest potential for therapy
of a wide range of infections. However, further studies
are warranted to determine its detailed clinical ecacy.
CS-023 is predominantly distributed to the extracel-
lular uid in body after intravenous administration
(Davies and Morris 1993). is limited distribution
would be favorable property of an antimicrobial agent,
since most infecting microorganisms resides in extra
cellular space (Shentag 1990). A low protein binding
ratio would also be favorable property for CS-023, since
the protein unbound, free form of CS-023 in plasma is a
pharmacologically active fraction, which achieve rapid
equilibrium with drug in extracellular uid (Shibayama
et al. 2007).
Faropenem
Faropenem medoxil (formerly faropenem daloxate)
(Fig. 6g) is an oral prodrug that is rapidly cleaved, releas-
ing the microbiologically active faropenem following
absorption in plasma. Faropenem is active against the
primary respiratory pathogens, including non–penicillin
susceptible S.pneumoniae and M.catarrhalis (Critchley
et al. 2002). Faropenem is most active B-lactam against
S. pneumoniae with MIC90 of 0. 5mg/L and is four fold
active than amoxicillin clavulanate (MIC90, 2mg/L)
eight-fold more active than cefdinir and cefuroxime
(MIC90, 4 mg/L). Activity of faropenem was aected
by penicillin susceptibility status of isolates, with faro-
penem MIC90 increasing from 0.015mg/L for penicillin
susceptible isolates to 1mg/L for penicillin resistant
strains (Stone et al. 2007). Although faropenem exhib-
ited higher MIC against penicillin resistant S. pneumo-
niae it had activity equivalent to that of telithromycin
and levooxacin (MIC90, 1mg/L) and was more active
than all other B-lactam tested, including amoxicillin-
clavulanate, cefdinir and cefuroxime, with MIC90 of 2, 4,
and 8 mg/L .
Tebipenem
Tebipenem (TBM)-pivoxil (PI) is an oral carbapenem
agent with a 1-(1,3-thiazolin-2-yl) azetidin-3-yl thio
group at C-2 position (Fig. 6h). e agent (previously
reported to be LJC 11,036; Miyazaki et al. 2001) shows high
degree of stability to dehydropeptidase-I and absorption
of the active metabolite, which is converted by esterase,
into blood from intestine. e prominent feature of TBM
is its potent activity against the main causative microor-
ganisms, except metallo B-lactamases-producing path-
ogens and MRSA strains causing RTIs and UTIs (Hikida
et al. 1999; Miyazaki et al. 2001). TBM shows higher
anities for PBP1A and PBP1B and for PBP2X (Hikida
et al. 1999). Inhibition of cell wall synthesis towards long
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96 Shahid et al.
axis and subsequent cell lysis was observed after treat-
ment with TBM. It showed excellent bactericidal activity
against penicillin-resistant Streptococcus pneumoniae
(PRSP) (Hikida et al. 1999; Miyazaki et al. 2001).
ME 1036 (CP5609)
ME 1036, formerly known as CP5609, is a parenteral
carbapenem with a 7- acylated imidazo [5,1-b] thiazole
-2-yl group directly attached to the carbapenem skel-
eton at C-2 position (Fig. 6i). It showed excellent activity
against MRSA and MRCoNS and is unique in that it also
showed potent activity against penicillin resistant S.
pneumoniae, E. faecalis, H. inuenzae, and members of
family Enterobacteriaceae (Kurazono etal. 2004). e ME
1036 MICs (ranging 0.5 to 4mg/L) for ceftriaxone non-
susceptible strains (ceftriaxone MICs >8 mg/L) were
higher than those for ceftriaxone susceptible strains
(ranging, 0.063 to 1 mg/L) (Kurazono et al. 2004). In
a more recent study, ME 1036 showed an MIC50 of
0. 12mg/L, an MIC90 of 0.25 mg/L, and a highest MIC
of only 0.5mg/L against community-acquired methi-
cillin-resistant S. aureus (CA-MRSA) (Sader et al. 2008).
ME 1036 may be slightly less stable than imipenem and
meropenem, in the presence of AmpC B-lactamases.
Like most other carbapenems, ME 1036 was found to
be inactive against E. faecium and also it had no activ-
ity against P.aeruginosa (ME 1036 might not reach
the targets due to low permeability or the presence
of eux pumps, B–lactamases, and other factors in P.
aeruginosa). ME 1036 inhibited the growth of MRSA
at one-fourth the MIC and this may occur because of
its binding to PBPs (except PBP 2a) as also observed in
case of imipenem.
Recently Hirai et al. (2007) investigated the bind-
ing anity of ME 1036 for various PBPs of E. faecalis
ATCC 29212, E. coli NIHJ JC-2 and H. inuenzae MSC
07274. As compared to other carbapenems, ME 1036
showed much stronger anity to PBPs especially
PBP3 and PBP4 of E. faecalis (IC50 of ME 1036 for PBP3
and PBP4 were 0.09 and 0. 03 mg/L while that of mero-
penem, a comparator drug, for same PBPs were 9.76
and 0. 20mg/L). Binding of ME 1036 to PBPs in turn
indicates its antibacterial activity as MICs of ME 1036
and meropenem for E. faecalis were 0.12 and 4. 0mg/L,
respectively. For H. inuenzae, binding of ME 1036 to
almost all PBPs, more precisely PBP3, was shown to be
much stronger than that of meropenem (IC5 0 of PBP3a
and PBP3b were 0.07 and 0. 06 mg/L, respectively
and that of meropenem for PBP3a and PBP3b were
0. 67 mg/L and 0.44 mg/L, respectively). Antibacterial
activity of ME 1036 can be compared with mero-
penem in terms of their MICs (MICs of ME 1036 and
meropenem were found to be 0. 12 mg/L and 1 mg/L,
respectively). For E. coli, a correlation was observed
between binding anity of ME 1036 for PBP2, PBP4,
PBP7/8, and its antibacterial activity. Hence, ME
1036 is supposed to be highly promising carbapenem
against E. faecalis, E. coli and H. inuenzae due to its
high anity for dierent PBPs.
SMP-601 (PZ-601)
PZ-601 is a novel broad-spectrum carbapenem. High
anity of PZ-601 (SMP-601) for penicillin-binding pro-
teins enhanced its activity against multidrug-resistant
Gram-positive organisms including MRSA, and it
retained the typical carbapenem-like spectrum against
Gram-negative bacteria including Pseudomonas spp.
Paterson et al. (2007) investigated the activity of PZ-601
against ESBL producers, which included (1) wild type
ESBL producers (K. pneumoniae and E. coli isolates
which were known by PCR/sequencing to harbor genes
coding for ESBL but which do not posses currently
known mechanisms of carbapenem resistance. ey
posses various combinations of CTX-M, SHV and TEM
type ESBLs), (2) carbapenem-resistant ESBL producers
(K. pneumoniae and E. coli isolates that were ESBL pro-
ducers as well as carbapenem resistant) and, (3) ESBL
producing E. cloacae. e MIC50 and MIC90 for PZ-601
were found to be 1 mg/L and 4 mg/L, respectively,
while carbapenem resistant-ESBL producers showed
elevated MICs (ranging from 4 mg/L to > 32 mg/L).
ESBL-producing E. cloacae seems to be less susceptible
to PZ-601. Hence PZ-601 was found to be more active
against ESBL-producing K. pneumoniae and E. coli
which are susceptible to currently used carbapenems.
Bouchillon et al. (2007) showed in vitro activity of PZ-601
against various Gram-positive, Gram-negative and
anaerobic microorganisms. MIC50 and MIC90, respec-
tively, were 0.25 mg/L and 1 mg/L (against staphylococci
including MRSA), 1mg/L and 2 mg/L (against E. faecalis
including VRE) and 0.06mg/L and 0.25 mg/L (against
S. pneumoniae including penicillin-resistant strains).
For E. coli and K. pneumoniae MIC50/MIC90 were found
to be 0.25/ 1mg/L and 1/4mg/L, respectively, for ESBL-
producers. PZ-601 also showed better activity against
E. cloacae (MIC50 4mg/L; MIC90 8mg/L) and peptostrep-
tococci (MIC50 < 0.015mg/L; MIC90 0.06mg/L).
In vitro antimicrobial activity of SMP-601 was also
shown by Kanazawa et al. (2007). ey showed that
SMP was broadly active against Gram-negative iso-
lates including BLNAR and CF-R-Proteus spp. MIC90
of E. coli, K. pneumoniae, M. morganii, Proteus spp.
(including cefepime-resistant strains), H. inuenzae
BLNAS (including BLNAR), M. catarrhalis, and N. gon-
orrhoeae were 0.5, 0.5, 2, 0.25, 0.06 (0.12 for BLNAR),
0.03, and 0. 12mg/L respectively. MIC90 of SMP against
S. marcescens (8 mg/L), P. aeruginosa ( 32 mg/L) and B.
cepacia (32 mg/L) were found to be higher than those
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b-lactams in clinical practice 97
of meropenem and imipenem. MICs of SMP against
E. coli, K. pneumoniae, M. morganii, and Proteus spp.
were comparable or superior to those of imipenem,
whereas M. catarrhalis and N. gonorrhoeae were highly
susceptible to SMP. us SMP proves to be a promising
candidate of a broad-spectrum anti-MRSA carbapenem
agent for serious nosocomial infections.
SMP had potent activity against wide range of Gram-
positive and Gram-negative bacteria however it showed
relatively weak activity against some Gram-negative
organisms. Its activity can be improved by combining it
with dierent antibacterial agents. Fujimoto et al (2007)
showed in vitro activity of SMP-601 in combination
with other antimicrobial agents against Burkholderia
cepacia and Serratia marcescens. MIC range of SMP
was 0.15– 16 mg/L for B. cepacia and 0.25– 16 mg/L for
S. marcescens. Combination of SMP and meropenem or
ciprooxacin was found to be synergistic (in 4% of tested
strains) (Min FIC ≤ 0.5) or additive (0.5< to ≤1) (92% of
tested strains) against B. cepacia. For S. marcescens, the
combination of SMP and amikacin showed synergis-
tic (19% of tested strains) or additive eect. But it was
observed that there occurs antagonism (in 4% of total
strains of B. cepacia tested) in combination of SMP and
minocycline and hence it should not be recommended,
while other combinations could represent an eective
treatment of serious infections caused by Gram-negative
and multidrug-resistant Gram-positive pathogens.
Trinems
Trinems (previously tribactams) have a Carbapenem
related structure but with a Cyclohexane ring attached
across carbon 1 and 2 (Fig. 6j). Sanfetrinem (GV 104326),
which is a trinem B-lactam, can be administered orally
as a hexatil ester. Like imipenem, sanfetrinem was
found to be stable to TEM-1 and TEM-10 enzyme, and
like imipenem and cexime but unlike cefpodoxime,
N
SCH3
CH3
O
OOH
X
X=I or Br
(a) Halogenated penicillanic acid
(b) Olivanic acid
(f) Oxapenems
(g) AVE-1330
(c) Penems (BRL 42715) (d) Clavulanic acid
(e) Sulbactam and Tazobactam
N
O
S
N
H
O
OSO3H
CH3
COOH
CH3
N
N
N
CH3
N
S
O
COOH
H
CH2OH
N
O
OCOOH
H
H
N
S
O
OO
CH3
R
OOH R = CH3Sulbactam
CH2N
N
N
Tazobactam
N
O
O
H
R
1
R
2
R
3
COO
Oxapenem compound
AM-112
AM-113
AM-114
AM-115
R1
OH
OH
H
H
R2
H
H
OH
OH
R3
(CH3)2NH3
CH3
CH3
(CH3)2NH3
N
N
O
S O
O
O
OCONH2
Na
Figure 7. B-Lactamase inhibitors.
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98 Shahid et al.
also retained activity against P. vulgaris and K. oxy-
toca that hyperproduced potent chromosomal class A
B-lactamase (Livermore 1998). Sme1, NMC-A and an
unnamed enzyme from Acinetobacter spp. increased
the Sanfetrinem MICs by up to 64-fold.
b-Lactamase Inhibitors
Mechanism of action: General
B-lactamases can be found either extracellularly
or within the periplasmic space; in general active
B-lactamases from Gram-positive bacteria are excreted
into the medium. B-lactamase activity in Gram-negative
organism is found primarily in the periplasmic space,
although some leakage of enzyme into the medium can
occur. B-lactamase production lead to resistance to com-
mon B-lactam antibiotics. Protection of B-lactam with an
inhibitor is used as an alternative strategy to overcome
resistance due to B-lactamases. Enzyme inhibitors can
be classied as reversible or irreversible. Reversible
inhibitors are those that bind to an enzyme in such a
manner that enzyme activity may be restored. Inhibitors
of B-lactamases that bind at or close to the active site are
often B-lactams. Although these molecules may act as
inhibitor, they also can be hydrolyzed as substrates. us,
many reversible inhibitors are really poor substrates that
are bound with high anity but are hydrolyzed at low
rates. Irreversible inhibitors may be more eective than
reversible inhibitors in that the eventual result is destruc-
tion of enzymatic activity. e preferred terminology for
inhibitor that renders their targets useless is “inactivator”.
A special subclass is the suicide inactivator, a molecule
that must bind initially at the enzyme active site, but
which is converted into an inactivator through catalytic
action of enzyme itself (Abeles and Maycock 1976).
Several classes of successive inhibitors were found in
the mid 1970s, including clavams, penicillanic acid sul-
phones, halogenated penicillanic acids (Fig. 7a), olivanic
acids (Fig. 7b), and various penems (Fig. 7c), and many
more are in experimental stages. Of these beta-lactamase
inhibitors, clavulanic acid, tazobactam, and sulbactam
are now established molecules that are used in clinic,
and other molecules are still under experimental stages
which include oxapenems, NXL 104 (AVE 1330A), Mono-
and bicyclic-bridged monobactams, BAL 30376, LK-157,
BLI-489, and so on.
Clavulanic acid
Clavulanic acid was the rst suicide inactivator of
B-lactamases (Brown et al. 1976) (Fig. 7d). is natural
product was isolated from Streptomyces clavuligerus on
the basis of its potent inhibitory activity against the broad-
spectrum B-lactamase from K. pneumoniae. Clavulanic
acid was quite eective in preventing the destruction of
substrates of enzymes that could eectively hydrolyze
penicillins. Inhibition of cephalosporinases from a variety
of sources was considerably weaker. e mechanism for
inactivation was studied in detail for the PC 1 (Cartwright
and Coulson 1979), TEM-2 (Charnas et al. 1978; Fisher
etal. 1978),Bacillus cereus I (Durkin and Viswanatha 1978),
K1, and Proteus mirabilis (Reading and Farmer 1981)
B-lactamases. Although interaction of clavulanic acid with
these B-lactamase showed enzymes inactivation, but the
individual mechanism varied somewhat among dierent
systems. Results from Knowles and coworkers provided
the rst evidence that clavulanic acid behaved like a sui-
cide inactivator (Charnas et al. 1978; Fisher et al. 1978).
After interacting with TEM-2 B-lactamases, clavulanic
acid forms acyl enzyme. Once acyl enzyme was formed,
there were several potential fates for this acyl enzyme, (1)
the acyl enzyme could yield a transiently inhibited form,
which is not permanently inactivated. is type of inhibi-
tion would be similar to reversible inhibition in that inhi-
bition can be reversed with addition of substrate and free
enzyme can eventually be recovered, (2) the acyl enzyme
can simply act as enzyme-substrate complex, with even-
tual hydrolyses of clavulanic acid, again resulting in the
release of free enzyme, (3) there occurs permanent loss
of enzymatic activity, a desirable property for a bacterial
enzyme inhibitor. Hence the extent to which inactivation
occurs depends upon the interrelationship among vari-
ous pathways.
Tazobactam
Tazobactam is a compound with chemical formula (2S,
3S,5R)-3-methyl-4,4,7-trioxo-3-(triazol-1-ylmethyl)-4-
$1^{6}-1-azabicyclo[3.2.0]heptane-2-carboxylic acid
(Fig. 7e) which inhibit action of bacterial B-lactamases.
It exhibits an almost 10-fold greater inhibitory activity
than clavulanic acid against CTX-M type B-lactamases
(Bush et al. 1993). Movement of Ser130 in the inhibi-
tor resistant TEM-30 (Arg244Ser), TEM-32 (Met69Ile-
Met182r), and TEM-34 (Met69Val) B-lactamases
emphasizes the strategic role of Ser130 (Wang et al.
2002). Kuzin et al. (1999) illustrated the importance
of Ser130 in their structural analysis of the SHV-1 and
tazobactam inactivated SHV-1 (Kuzin et al. 1999; Kuzin
et al. 2001). e crystal structure of the SHV-1- tazo-
bactam complex showed the formation of an acyclic
form of tazobactam attached to Ser70 (assigned to the
imine on the basis of dihedral angles) and a 5-atom
vinyl carboxylic acid fragment attached to Ser130-OH
in the inactive species. As in the case with clavulanic
acid inactivation of TEM-2, the amino acids Ser70
and Ser130 were again the tazobactam-modied
residues. Evidence for an aldehyde and hydrated
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b-lactams in clinical practice 99
aldehyde attached to the Ser130 residue was also
detected. Hence the critical involvement of Ser130 in
the inactivation process, as rst emphasized by Imtiaz
et al. (1994), is central to the studies of the inhibition
of class A B-lactamases by clavulanic acid and tazo-
bactam. Tazobactam undergoes fragmentation while
still attached to the active site Ser70 in Ser130Gly
B-lactamase. After acylation of Ser130Gly B-lactamase
by tazobactam, inactivation proceeds independent of
any additional covalent interaction.
Sulbactam
Sulbactam was developed as potential B-lactamase
inhibitor with chemical formula (2R,5R)-3,3-dimethyl-
4,4,7-trioxo-4L6-thia-1-azabicyclo[3.2.0]heptane-2-
carboxylic acid (Fig.7e). It exhibits some of the same
properties as clavulanic acid. Cephalosporinases were
inhibited less eectively than were penicillinases or
broad-spectrum B-lactamases. However, the dierential
between the enzyme classes was not as great as observed
with clavulanic acid in that sulbactam was slightly more
active against cephalosporinases than clavulanic acid. In
addition to having similar inhibition prole, they exhib-
ited a time dependent mode of inhibition. Mechanism
of action of sulbactam was examined, it was apparent
that the same kind of inhibitory pathway was operative
for both clavulanic acid and sulbactam. In extensive
studies of sulbactam is the E. coli TEM-2 B-lactamase,
the sulfone was observed to form a transiently inhibited
complex with the enzyme; hydrolysis of sulbactam was
also observed before irreversible inactivation resulted.
Sulbactam is able to inhibit the most common forms of
B-lactamase but is not able to interact with AmpC cepha-
losporinase. us it confers little protection against bac-
teria such as P. aeruginosa,Citrobacter, Enterobacter, and
Serratia, which often express this gene. It does possess
some antibacterial activity when administered alone,
but it is too weak to have any clinical importance.
Oxapenem
Oxapenem a ve membered, oxygen-containing ring
fused to B-lactam ring with a double bond between C2
and C3, were found to be potent B-lactamase inhibitor
but have poor stability (Fig. 7f). Oxapenems were potent
B-lactamase inhibitors however their activity varied
within the group for eg. AM-113 and its stereoisomer
AM-114 proving to be most active compounds (Lambert
et al. 2003). MIC50 of these agents were up to 105 than
that of clavulanate against class C and class D enzymes.
Oxapenems reduced MICs for ceftazidime against
class C hyperproducing, TEM, and SHV derived ESBL
producing strains however these compounds failed to
reduce MICs of ceftazidime against derepressed Amp C
producing P. aeruginosa strains. AM-112 had anity for
PBPs of E. coli DCO, with PBP2 being inhibited by lowest
concentration of AM-112 tested (0. 1mg/L). It was found
to be eective at protecting ceftazidime and reduces
ceftazidime MICs by 16- and 2048-fold (Jamieson et al.
2003). Protection of ceftazidime with AM-112 was
maintained against E. cloaceae P99 and K. pneumoniae
SHV-5. e activity of the combination of ceftazidime
and AM-112 against ESBL producing E. coli strains was
similar to that of piperacillin and tazobactam. AM-114
and AM-115 were found to be most potent inhibitor of
class A enzymes, AM-113 and AM-114 of class C enzymes
while AM-112 and AM-113 were most active oxapenems
against class D enzymes. e orientation of hydroxyl
ethyl group at C-6 position and nature of C-2 side chain
appears to be responsible for extent of inhibitory activity
against dierent B-Lactamases.
NXL 104 (AVE 1330 A)
NXL 104, previously designated as AVE 1330 A, is a
bridged diazabicyclo [3.2.1]octanone (Fig. 7g) and is
B-lactamase inhibitor able to inhibit both class A and
CB-lactamase. Its combination with ceftazidime exhib-
ited broad-spectrum activity against Ambler class A-
and class C- producing Enterobacteriaceae members.
Two to ve AVE 1330 A molecules only were needed
to inactivate one molecule of TEM-1 or P99 enzyme
and is far less than reported for clavulanate (Bonnefoy
et al. 2004). Beta-lactam-based B-lactamase inhibitors
generally act as a substrate of enzymes, giving an unsta-
ble acyl enzyme intermediate, which is responsible for
transient inhibition. is acyl enzyme then undergoes
a rearrangement, leading to a more stable inhibition.
MICs of ceftazidime/AVE 1330 A for Enterobacteriaceae
were at least eight fold lower than those of ceftazidime
alone (Bonnefoy et al. 2004). All of E. coli, K. pneumo-
neae,Citrobacter, and Proteus mirabilis strains, includ-
ing ceftazidime-resistant isolates, were inhibited at
4- 8 mg/L. Only 2 mg/L were required to inhibit other
Proteus, Enterobacter, Salmonella, and Serratia.
Miossec et al. (2007) evaluated the in vitro ecacy of
combination of ceftazidime and NXL 104 against carbap-
enem resistant Enterobacteriaceae strains, either medi-
ated by class A or D carbapenemases, or by class A or C
enzymes along with membrane impermeability. NXL 104
was found eective in restoration of activity of ceftazi-
dime, cefotaxime or imipenem against class A carbapen-
emase-producers (IMI-1, NMC-A, GES-2, GES-3, GES-4,
KPC-2, and KPC-3) however, activity of ceftazidime/NXL
104 against KPC-2 or KPC-3 enzymes was noticeable with
MIC ≤ 0.015 to 0.5 mg/L as compared to 64 to >128mg/L
for ceftazidime alone. OXA-48 (class D carbapenemase)
was also inhibited eectively by NXL 104 (IC50 value was
0.2 µM) and ceftazidime activity was found to be fully
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100 Shahid et al.
restored against these strains. OXA-23, OXA-40, and
OXA-58, which mostly occur in Acinetobacter spp., were
poorly inhibited by NXL 104. Mushtaq et al. (2007) dem-
onstrated the activity of NXL 104/cephalosporins against
CTX-M ESBL-producers as CTX-M B-lactamase are
increasingly becoming the most prevalent ESBLs world-
wide (though the predominant types vary with dierent
geographic areas, CTX-M-15 are frequently encountered
in Europe, CTX-M-2 in South America, and CTX-M-9/
CTX-M-14 in East Asia). MICs of cefotaxime/NXL 104
were ≤ 1mg/L for all members of Enterobacteriaceae with
128-8000-fold potentiation, irrespective of CTX-M type,
co-production of OXA-1 and/or ertapenem resistance.
MICs of ceftazidime/NXL 104 were found to be ≤ 1mg/L
for all ertapenem-susceptible CTX-M producers and
were ≤ 4 mg/L for ertapenem-resistant isolates. Hence,
ceftazidime/NXL 104 combinations could provide useful
alternative for treatment of Enterobacteriaceae strains
that are carbapenem-resistant. It can also be noted that
NXL 104/cephalosporin combinations successfully over-
come oxyimino-cephalosporin resistance mediated by
CTX-M B-lactamases even when they were co-expressed
with OXA-1 enzymes or impermeability. Within class
A, KPCs represent a new family having a potential for
wide dissemination and its production confers resist-
ance to all B-lactams including carbapenems. Stachyra
et al. (2007) evaluated activity of NXL 104 against KPC
B-lactamases and NXL 104 showed an extremely potent
inhibitory activity against KPC-2 B-lactamase, with
IC50 of 38nM. Clavulanic acid and tazobactam, the two
potent comparators, were found to less active with IC50
in µM range. Resistant isolates (KPC producers) showed
MICs > 128mg/L for ceftazidime and ceftriaxone and 16
to > 128mg/L for imipenem, while after adding NXL 104
MICs were <1mg/L each for ceftriaxone/NXL 104 and
imipenem/NXL 104, while MICs ranged between 0.25
to 8mg/L for ceftazidime/NXL 104. NXL 104 was found
to have remarkable activity against KPC-2 enzyme and
eciently restored antimicrobial activity of ceftazidime,
ceftriaxone and imipenem against KPC-2 and KPC-3
B-lactamase-producing members of Enterobacteriaceae.
Mono- and bicyclic-bridged monobactams
Monobactams, such as aztreonam and bridged mono-
bactams, are B-lactamase inhibitors and showed potent
inhibitory activities against AmpC B-lactamase. Gaucher
et al. (2007) synthesize three series of novel bridged
monobactams using parallel chemistry approaches
from benzyl (1S, 5R)-7-oxo-2,6-diazabicyclo[3.2.0]
heptane-2-carboxylate. e siderophore monobactam
BAL0019764 (which is stable to hydrolysis by class B met-
allo B-lactamases and many class D B-lactamases and
has enhanced uptake by many species) reacted with AZT
with AmpC and ESBL enzymes and resisted attack by
metallo-B-lactamases. BAL 0030072 had a stronger inter-
action with ESBLs but found to be a weaker inhibitor of
AmpC enzymes. e bridged monobactam B-lactamases
were potent inhibitors of class C B-lactamases but had no
interaction with ESBLs or metallo- B-lactamases. It has
been noted that by combining bridged monobactams
(BMBs) with either BAL 0019764 or BAL 0030072, elevated
MICs of compounds with strains of Enterobacteriaceae
and P. aeruginosa that hyperproduce AmpCs, can be
depressed. BMBs were able to inhibit the enzymes and
thus aord protection to antibiotics. ey can be used in
combination with monobactam to improve the activity
of monobactam against Enterobacteriaceae and P. aeru-
ginosa. Recent in vitro activities showed the ecacy of
these inhibitors. IC50 of BAL 30072 is 0.09 µM and that of
clavulanate is >100 µM against AmpC enzyme from C.
freundii, while IC50 of aztreonam was found to be 0.01 µM
for same AmpC enzyme (Gaucher et al. 2007).
BAL 30376
BAL 30376 is a novel B-lactamase inhibitor and is a com-
bination of BAL 0019764 (a siderophore monobactam
which is stable to hydrolysis by class B B-lactamases),
BAL 0029880 (a bridged monobactam which is class C
inhibitor), and clavulanic acid. All the three components
are used in the ratio of 1:1:1 by weight. In vitro activity of
BAL 30376 against Gram-negative bacilli was evaluated
by Bowker et al. (2007) and they observed that MIC90
of BAL 30376 for E. coli, K. pneumoniae, and E. cloacae
were 4, 1, and 4mg/L, while MIC50 were found to be 0.5,
0.5, and 1 mg/L for respective organisms. e strains
which had CMY-2, CTX-M, multiple combinations of
SHV, CTX-M, CMY, OXA, and TEM-type enzymes, BAL
30376 had an MIC50 of 1-4mg/L while meropenem, a
most potent comparator drug, had MIC50 of 0. 03 mg/L.
MICs of BAL 30376 for >90% of such strains was found
to be ≤ 8 mg/L and for meropenem all MICs were also
< 8 mg/L. Against IMP containing Gram-negative
bacilli, MIC50 of BAL 30376 was 0.5- 2mg/L and that of
meropenem was 2- ≥64 mg/L. MIC50 of this novel inhib-
itor was 1- 4 mg/L for VIM, GIM and SPM producing
strains. It was observed that BAL 30376 was most potent
against IMP-4, IMP-13 or IMP-16, VIM-1, VIM-2, VIM-4,
GIM-1, and SPM-1 producers. However, meropenem/
imipenem were found to be more potent as compared
N
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ONa
O
OMe
H
Figure 8. LK-157.
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b-lactams in clinical practice 101
to BAL 30376 against recent ESBLs as well as CMY-2 and
CTX-M producers. BAL 30376 had superior activity than
aztreonam, ceftazidime, cefepime and piperacillin/
tazobactam. Page et al. (2007) demonstrated the in vitro
activity of BAL 30376 against various Gram-negative
bacteria and the MICs were observed in a range of
≤0.06 – 4 mg/L, including most carbapenem-resistant
strains. Higher MICs were observed for a few strains of
Acinetobacter spp., C. freundii, Enterobacter spp., and P.
aeruginosa. To test ecacy of this novel inhibitor, Hujer
et al. (2007) demonstrated its activity against ESBL
producing K. pneumoniae and MDR Acinetobacter
baumanii. ey have used BAL 30376-f (concentration
of BAL 19764 was varied while that of BAL 29880 and
clavulanic acid was xed at 4 mg/L and 2mg/L respec-
tively) and BAL 30376-v (concentration of BAL 19764,
BAL 29880 and clavulanic acid were varied in a xed
proportion of 5/3/1 respectively). ey observed that
based on MICs ≤ 8mg/L, BAL 30376-f and BAL 30376-v
demonstrated signicantly better activity (as compared
to imipenem) against Acinetobacter baumanii strains
possessing AmpC, blaADC and the carbapenemase
blaOXA-23 like genes. Similarly BAL 30376-f and BAL
30376-v were found to be more active against MDR K.
pneumoniae (especially CTX-M producing strains) as
compared to cefepime but slightly less active than imi-
penem. However, it has been observed that blaKPC bear-
ing strains of K. pneumoniae remain resistant to BAL
30376 but it oers promising activity against a wider
range of MDR strains bearing serine B-lactamases. It
also showed good activity against Enterobacteriaceae
and non-fermentive Gram-negative bacteria express-
ing AmpC B-lactamases, however it was found to be
compromised by certain carbapenemase when co-
expressed with other B-lactamases.
LK-157
LK-157 (Fig. 8) is a novel tricyclic carbapenem-inhibitor
of serine B-lactamases and is for parenteral use as an
IV agent. Recently, Kresken et al. (2007) investigated in
vitro activity of LK-157 to assess its potency. ey showed
that MIC90 of the novel inhibitor ranged between 16 and
> 128 mg/L for MRSA, penicillin-resistant S. pneumo-
niae, B. fragilis, P. aeruginosa, and Enterobacteriaceae
however, it was found to be ≤ 2mg/L for MSSA, S. pyo-
genes, penicillin-susceptible S. pneumoniae, and H.
inuenzae. When LK-157 was combined with cefotaxime
or cefepime, its spectrum of activity was found compara-
ble to that of tazobactam. LK-157 decreased the MICs of
aztreonam, ceftazidime, and cefuroxime for B. fragilis (8-
to ≥128-fold) and a wide range of B-lactamase-producing
Enterobacteriaceae members (up to ≥64-fold). It also
results in decreased MICs of cefuroxime for P. vulgaris
(≥32-fold) and those of aztreonam for MSSA (≥16-fold),
S. pyogenes (≥32-fold) and PSSP (≥64-fold) and those of
ceftazidime for MSSA (≥8-fold). It was noted that LK-157
did not aect (to a greater extent) the MICs of aztre-
onam, ceftazidime or cefuroxime against CTX-M pro-
ducing members of Enterobacteriaceae. LK-157 showed
potent in vitro activity against Gram-positive cocci and
also showed synergistic activity with B-lactams against
a wide range of Enterobacteriaceae that produce dier-
ent B-lactamases, and hence it can be a potent inhibitor
active against various pathogens including B-lactamase
producing several members of Enterobacteriaceae.
Prezelj et al. (2007) evaluated the activity of LK-157
against class A and C B-lactamase producing strains.
ey observed that IC50s of LK-157 (in nM range) were
superior to comparator drug, tazobactam, however its
activity in combination with cephalosporins against
TEM-, SHV- and BRO-type enzymes were found to be
comparable to cephalosporin/tazobactam combina-
tion. LK-157 was superior to tazobactam against AmpC-
producers and synergy was observed also against PC-type
B-lactamases. Prezelj et al. (2007) also demonstrated the
stability of 10-ethylidene trinems. As LK-157 is a very
low permeability drug, several drugs were designed
for per oral application. ey observed that among the
series of LK-157 analogues, LK-157E1 was most soluble
compound with low intestinal permeability. Pro-drug
ester of LK-157E1 exhibited improved solubility but per-
meability across rat jejunum was low. Hence LK-157 can
be a promising broad-spectrum tricyclic carbapenem
inhibitor of class A and C B-lactamase.
BLI-489
e novel penem B-lactamase inhibitor, BLI-489, has
shown activity against molecular class A or D enzymes,
including ESBLs as well as class C B-lactamases. Petersen
et al. (2007) demonstrated the in vitro activity of pipera-
cillin in combination with BLI-489 against B-lactamase-
producers. ey tested activity of combination of pip-
eracillin/BLI-489 in the ratio of 1:1, 2:1, 4:1, and 8:1 and a
constant concentration of either 2 or 4mg/L of BLI-489.
Piperacillin/tazobactam was used for comparison (tazo-
bactam was taken at a constant concentration of 4 mg/L).
Numbers of strains were found to be falsely reported
as susceptible or intermediate to piperacillin/BLI-489
N
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CO2Na
O
Ph
O
O2
Figure 9. SA-1-204.
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102 Shahid et al.
when tested at 1:1 and 2:1 ratio. e constant 2mg/L of
BLI-489 and 8:1 ratio had over-predict resistance. Some
strains that were class C or ESBL producers, classied
as non-susceptible (MICs ranging 32 to >256 mg/L) to
piperacillin/tazobactam, were found to be susceptible to
piperacillin/BLI-489 at a xed concentration of 4mg/L of
BLI-489 (MICs 1 to 64mg/L). Piperacillin/BLI-489 com-
bination showed potent in vitro activity against diverse
B-lactamase producers.
CP3242
It is a metallo- B-lactamase (MBL) inhibitor and com-
petitively inhibits both IMP-1 and VIM-2. Morinaka et al.
(2007) showed inhibitory mechanism of CP3242 along
with its in vitro activity. It specically lowered the MICs of
B-lactams against MBL-producing E. coli transformants.
It was noticed that combination ecacy of CP3242 with
carbapenems was not aected by existence of resistance
factors in P. aeruginosa (such as MexAB-OprM, and outer
membrane protein OprD-deciency). It signicantly low-
ered the MICs of biapenem in a concentration depend-
ent manner (2 to 32 mg/L) against MBL-producing P.
aeruginosa. MIC90 of biapenem was lowered from 512
to 16mg/L in the presence of 32mg/L of CP3242. MIC
lowering by CP3242 was also shown for IMP or VIM
producing E. coli, P. putida, S. marcescens, A. baumanii,
and K. pneumoniae. Hence, this novel competitive MBL
inhibitor can be eective against MBL-producers (Gram-
negative bacteria including P. aeruginosa) in combina-
tion with B-lactam antibiotics. It was also shown by Muto
et al. (2007) that the reducing eects of carbapenem on
a viable cell count of MBL-producing P. aeruginosa were
reduced in the presence of CP3242. us CP3242 co-
administered with carbapenem could be promising in
treatment of MBL-producing P. aeruginosa.
SA-1-204
It is a 6-alkylidiene penam sulphone B-lactamase
inhibitor (Fig. 9) that was found eective against SHV-
producing E. coli strains. Buynak et al. (2007) showed
ecacy of this B-lactamase inhibitor and found that
piperacillin/SA-1-204 was more potent than piperacil-
lin/tazobactam against strains bearing blaSHV-1 (MIC
1024/4 vs 64/ 4 mg/L) and blaSHV-5 (MIC 128/4 vs 16/4
mg/L). Both the inhibitors were found to be equally
eective against E. coli bearing SHV-2 (MIC 2-8/4mg/L)
and SHV-30 (MIC 1-2/4mg/L) ESBLs. Also piperacillin/
SA-1-204 was more eective against inhibitor-resistant
SHV-49 variant (128/4 vs 32/ 4mg/L). Alkylidene at the
6 position and 2’ B-substituent increases inhibitor e-
cacy. Hence, SA-1-204 can be considered as an eective
B-lactamase inhibitor against SHV-1, other ESBL vari-
ants of SHV, and SHV-49.
Synergistic activity of inhibitors
Although the B-lactamase-inhibitors act as eective
inactivators of isolated enzymes, a most important ques-
tion is whether these molecules can act to protect sus-
ceptible B-lactam antibiotic from hydrolysis in growing
cells. It is imperative that these inhibitors penetrate the
periplasm of Gram-negative organisms rapidly so as to
intercept the B-lactamase before all labile antibiotics
has been destroyed. Enzyme inhibition occur faster than
synthesis of new protein; otherwise, succeeding genera-
tions of cells will simply continue to elaborate additional
B-lactamase capable of hydrolyzing antibiotic. To evalu-
ate synergy between a susceptible B-lactam antibiotic
and a B-lactamase inhibitor MICs of antibiotic were
determined in the presence and absence of this xed
inhibitor concentration, and synergy was evaluated. In
other studies equal concentrations of inhibitor and anti-
biotic were varied stepwise by twofold dilution, and MICs
of combination were compared with those observed in
the absence of inhibitor. Synergy is dened as a fourfold
reduction in MIC for both components. As might be
predicted from the isolated enzyme studies, synergy in
B-lactamase-producing E. coli strains was much greater
with clavulanic acid than with sulbactam. is would be
expected, if one assumes that majority of these strains
produce plasmid mediated, TEM-type, broad-spectrum
B-lactamases. When a plasmid is present in a high copy
number, the elevated level of B-lactamase hydrolyze
sulbactam more quickly than the enzyme will become
inactivated because of reasonably high turnover number.
is was demonstrated in studies by Easton and Knowles,
who observed good synergy with both sulbactam and
clavulanic acid in a Proteus species that produced a
moderate level of TEM B- lactamase. Another factor that
will contribute to a dierential in activity is the ease with
which the inhibitor can penetrate the cell. As indicated
by Li et al. (1981), clavulanic acid may have less of a
penetration problem than sulbactam in P. aeruginosa,
Citrobacter, and Enterobacter strains.
Among the B-lactams, third generation cepha-
losporins, such as ceftazidime, cefotaxime, and ceftriax-
one are routinely used in our clinical setting, and resist-
ance to these drugs, due to B-lactamase production, is
rampant (Mahapatra et al. 2003). e combination of
cefoperazone/sulbactam shows marked degree of syn-
ergy against organisms that are resistant to cefoperazone
alone (Wexler and Finegold 1988). Broad-spectrum
B-lactams, such as imipenem, cefdinir, cefepime, and
cefpirome, and B-lactamase-inhibitor combination, such
as piperacillin/tazobactam, cefoperazone/sulbactam
and ticarcillin/clavulanate, have been introduced to
overcome the resistance. Piperacillin/tazobactam
exhibited greater in vitro activity only against E. coli and
P. vulgaris as compared to piperacillin/sulbactam. Both
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b-lactams in clinical practice 103
combinations were found equally eective against other
Enterobacteriaceae and P. aeruginosa isolates. Sulbactam
may have higher intrinsic activity against Acinetobacter
isolates and thus may be considered as drug of choice
where Acinetobacter is a suspected pathogen.
e combination of piperacillin and tazobactam
is active against many piperacillin-resistant strains of
Staphylococci, Enterobacteriaceae and Bacteroides
species (Eliopoulos et al. 1989). However the two
piperacillin-B-lactam inhibitor combinations (pipera-
cillin/sulbactam and piperacillin/tazobactam) did not
display signicant dierences in antimicrobial suscepti-
bility of Gram-positive isolates (methicillin-susceptible
S. aureus and E. faecium) (Kuck et al. 1989).In vitro stud-
ies have demonstrated that antimicrobial activity does
not predominantly depend on equal proportions, but on
critical concentration of the inhibitor (Lister et al. 1997).
e combination of cefoperazone/sulbactam shows a
marked degree of synergy against organisms that are
resistant to cefoperazone alone, including Acinetobacter
species and Enterobacter species (Dias et al. 1986).
Piperacillin/tazobactam and cefoperazone/sulbactam
were usually much more active than ticarcillin/clavu-
lanate against strains producing elevated levels of non-
class I B-lactamase. However increased resistance to all
three combinations was associated with increased pro-
duction of certain B-lactamases. Ticarcillin/clavulanate
would be expected to be the most eective combina-
tion against TEM-1 producing strains. Yoshimura and
Nikiado (1985) reported that cefoperazone penetrate
the outer membrane of E. coli faster than piperacillin
and carbenicillin. If ticarcillin has penetration rate simi-
lar to that of carbenicillin, then greater potency of cef-
operazone/sulbactam could be consequence of faster
penetration by cefoperazone. is, however, does not
explain the greater potency of piperacillin/tazobactam
over ticarcillin/clavulanate.
e dierence between these combinations may be
due to greater anity of piperacillin for PBP3, the prin-
cipal target of both drugs. is is suggested by a 20-fold-
greater anity of ureidopenicillins-piperacillins, mezlo-
cillin and azlocillin- for PBP3 of E. coli compared with
carboxypenicillins-carbenicillin (omson et al. 1990).
e enhanced activity of ceftriaxone observed in the pres-
ence of B-lactamase inhibitor-sulbactam seems promis-
ing for the treatment of serious infections due to members
of Enterobacteriaceae producing an ESBL such as SHV-2
(Fantin et al. 1990). Recent In vitro studies indicated that
among available B-lactam/B-lactamase inhibitor combi-
nations, piperacillin/tazobactam has best activity against
nosocomial Gram-negative pathogens followed by cef-
operazone/sulbactam. However, very recently, ceftriax-
one-sulbactam was found even better than piperacillin-
tazobactam and the activity was demonstrated at par with
carbapenem, the imipenem (Shahid 2007a).
Conclusions
Resistance to third and fourth generation cephalosporins
has become a major concern worldwide. Even more
alarming is the emergence of carbapenem resistance;
the carbapenems are often considered to be a “drug
of choice” and increasingly used in empirical therapy.
Against this rising resistance the role of B-lactam-B-
lactamase inhibitor combinations needs to be consid-
ered. Sulbactam has been approved recently in many
countries to be combined with B-lactam antibiotics
including, recently, in India, and ceftriaxone-sulbactam
proved to be eective in vitro against diverse CTX-M-15-
producing E. coli strains. e third and fourth genera-
tion cephalosporins and combination of cephalosporins
(especially third generation) with new B-lactam inhibitor
can be used alternately to avoid selection pressure and
it can be an eective therapy to avoid drug resistance.
Since, B-lactams are often combined with aminogly-
cosides, in empirical therapy (Shahid 2007b), another
alternate approach to prevent selection pressure could
be periodical prescription rotation between B-lactam-
aminoglycoside combination therapy, B-lacatm-
B-lactamase-inhibitor, and the carbapenems.
Acknowledgements
e authors wish to thank Prof. Gopal Nath, Department
of Medical Microbiology, Banaras Hindu University,
Varanasi, and Prof. N. P. Singh, Department of
Microbiology, University College of Medical Sciences,
Delhi for critical suggestions during preparation of this
manuscript. M. Shahid is grateful to Department of
Science & Technology, Ministry of Science & Technology,
Government of India, for the award of Young Scientist
Project (SR/FT/L-111/2006), and to the University
Grants Commission, India, and the Association of
Commonwealth Universities and British Council, UK,
for awarding a Commonwealth Academic Fellowship
(INCF-108-05). e authors are also grateful to T.
Tripathi and M. Arif for graphical assistance especially
the preparation of chemical structures.
Declaration of interest: e authors report no conicts
of interest. e authors alone are responsible for the
content and writing of the paper.
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