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HIPPOKRATIA 2012, 16, 4: 303-307

Mechanisms responsible for the emergence of carbapenem resistance in Pseudomo-

nas aeruginosaMeletis G1, Exindari M2, Vavatsi N3, Soanou D4, Diza E5

1Department of Clinical Microbiology, Veroia G. Hospital, Veroia, Greece2Department of Microbiology, Medical School, Aristotle University of Thessaloniki, Greece3Depertment of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, Greece4Department of Clinical Microbiology, Hippokration G. Hospital, Greece5Department of Clinical Microbiology, AHEPA Hospital, Aristotle University of Thessaloniki, Greece

Abstract

Pseudomonas aeruginosa (P. aeruginosa)is an opportunistic pathogen associated with a range of nosocomial infections.

This microorganism is noted for its intrinsic resistance to antibiotics and for its ability to acquire genes encoding resis-

tance determinants. Among the beta-lactam antibiotics, carbapenems with antipseudomonal activity are important agents

for the therapy of infections due to P. aeruginosa. The development of carbapenem resistance among P. aeruginosa

strains is multifactorial. Plasmid or integron-mediated carbapenemases, increased expression of efux systems, reduced

porin expression and increased chromosomal cephalosporinase activity have all been dened as contributory factors.

Phenotypic tests and molecular techniques are used for the characterization of the resistance determinants. The isolation

of carbapenem resistant strains is alarming and requires the implementation of strict infection control measures in order

to prevent the spread of carbapenemase encoding genes to unrelated clones or to other bacterial species. Hippokratia

2012, 16, 4: 303-307

Key wordsPseudomonas aeruginosa, carbapenem resistance, carbapenemases, efux systems, OprD

Corresponding author: Meletis Georgios, Veroia General Hospital, Papagou District, Veroia, Greece, e-mail:meletisg@hotmail.com tel:

+306974282575, fax: +302381022841

REVIEW ARTICLE

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is an im-

portant cause of nosocomial infections that can be par-

ticularly severe in immunocompromised patients. These

pathogens are common causative agents of pneumonia,

bacteremia, urinary tract, skin and soft tissue infections.

The increasing isolation in healthcare settings ofP. aerug-

inosastrains non-susceptible to most anti-pseudomonal

agents is due to a number of factors, including its innate

resistance to a variety of antimicrobial agents, its ability

to acquire resistance determinants and the increased use

of antibiotics, which promotes the selection of resistant

clones. Carbapenems have a broad spectrum of antibacte-

rial activity and are used as last resort drugs for the treat-

ment of infections caused by multiresistantP. aeruginosa

isolates. This extraordinary microorganism however, of-

ten possesses the necessary mechanisms to overcome the

activity of almost all the available antibiotics.

Carbapenem use forP. aeruginosainfections

Carbapenems are the most effective antimicrobial

agents against gram-positive and gram-negative bacteria

including P. aeruginosa. Carbapenems bear a penemic

together with the beta-lactam ring and, like all other be-

ta-lactams, they inhibit bacterial cell wall synthesis by

binding to and inactivating Penicillin Binding Proteins

(PBPs). This unique molecular structure offers them their

exceptional stability to many beta-lactamases including

AmpC and most of the extended spectrum beta-lactama-

ses (ESBLs).

Seven antibiotics belong to the carbapenem family

(Figure 1), each one presenting particular characteristics

that inuence their way of administration and their use-

fulness as anti-pseudomonal agents.

Imipenem, for example, is susceptible to renal dehy-dropeptidase I and is administered in combination with

cilastatin which acts as a dehydropeptidase I-inhibitor.

Panipenem is a carbapenem antibiotic that was devel-

oped in Japan in 1993 and is available to date only in the

Asian market. Similar to imipenem, panipenem is used in

combination with betamipron in order to overcome the

inactivating effect of dehydropeptidase I1.

Meropenem is stable to dehydropeptidase I inactiva-

tion but is less efcient than imipenem against gram-pos-

itive microorganisms. On the other hand, meropenem is

more active against gram-negative bacteria and especial-

ly against P. aeruginosabecause it passes more swiftly

through the OprD porin.

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304 MELETIS G

The Asian biapenem has a similar molecular structure

to imipenem bearing a methyl group in 1B position2. It

shows a broad spectrum of activity especially against an-aerobes and is stable to dehydropeptidase I inactivation.

Ertapenem has longer half-life and has been intro-

duced in Europe and North America as the one-daily-

dose carbapenem for the treatment of community ac-

quired infections. Even though it is efcient against En-

terobacteriaceae, it shows little or no activity against P.

aeruginosaandAcinetobacterspp. The large ertapenem

molecule has higher afnity for theP. aeruginosaefux

systems and cannot pass easily through the porins of non-

fermenters3.

Doripenem like the other carbapenems, has a broad

spectrum of activity and has been proven to perform bet-

ter againstP. aeruginosathan imipenem and the same as

well as meropenem3. Studies showed that the in vitro ef-

fect of doripenem againstP. aeruginosais more efcient

than that of the other carbapenems4; however, carbapen-

em-resistantP. aeruginosastrains are non-susceptible to

all antibiotics of this category3.

Tebipenem, the newest carbapenem is under develop-ment in Japan. Tebipenem is the active form of tebipenem

pivoxil and is formed by the addition of a new side chain

in position 2C of the biapenem molecule5. Tebipenem is

the carbapenem with the highest biocombatibility and can

be administered per os. Despite its advantages, tebipenem

like ertapenem, is not active againstP. aeruginosa.

From just beta-lactam resistance to carbapenem

resistance

P. aeruginosa exhibits intrinsic resistance to sev-

eral beta-lactam antibiotics and may acquire additional

resistance mechanisms due to mutational events or the

acquisition of transferable genetic elements. Intrinsically,P. aeruginosaexpresses chromosomally-encoded induc-

ible AmpC beta-lactamase6and several important efux

pump systems7 that export antibiotics,biocides, dyes, de-

tergents, metabolic inhibitors, organic solvents and mol-

ecules involved in bacterial cell to cell communication.

Carbapenem resistance mechanisms have immerged

under the pressure of carbapenem use in clinical settings

and may be classied as enzymatic, mediated by carbap-

enemases (beta-lactamases hydrolyzing carbapenems

among other beta-lactams), and non enzymatic8. Carbap-

enem resistance however, develops frequently due to the

concomitant presence of more than one mechanisms9,10.

Carbapenemases inP. aeruginosaisolates

Beta-lactamases are classied according to a structur-

al11and a functional12classication. Amblers structural

classication comprises four molecular classes: A) The

Extended Spectrum Beta-Lactamases (ESBL) that are

inhibited by clavulanic acid, B) the Metallo-Beta-Lac-

tamases (MBL), C) the Cefalosporinases (AmpC) and

D) the Oxacillinases (OXA). Classes A, C and D share

a common characteristic: Their enzymes bear serine in

their active center whereas the MBLs bear Zn2+. Serine

enzymes cleave the amide bond of the beta-lactam ring

thus inactivating the antibiotic, while MBLs catalyze the

same chemical reaction, using one or two divalent Zn

2+

cations13. More precisely, the MBL active site orients and

polarizes the beta-lactam bond to facilitate nucleophilic

attack by zinc-bound water hydroxide.

All types of transferable carbapenemases, except

SIM-114 (Seoul imipenemase), have been detected in P.

aeruginosaisolates around the world. Among them, the

MBLs are considered as the most clinically important for

P. aeruginosa. Most genes encoding MBLs are found as

gene cassettes in integrons and are transferable15. Further-

more, more resistance genes for other antibiotic classes

can be present in the same integrons contributing thus in

the development of a multi-drug resistant phenotype16.

IMP (active on imipenem) and VIM (Verona in-

Figure 1: Carbapenem antibiotics.

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tegron-encoded metallo--lactamase) type MBLs are

spread through all continents after their rst identica-

tion in Japan17and Italy18respectively while other metal-

lo-enzymes have been detected sporadically. SPM-1 (Sao

Paolo metallo--lactamase) has caused serious outbreaks

in Brazil19

and has also been recently found in Basel20

in an isolate recovered by a patient previously hospital-

ized in Brazil. GIM-1 (German imipenemase) and AIM-1

(Australian imipenemase) have been reported from Ger-

many21 and Australia22 and did not spread elsewhere

whereas the rst and to date only report for NDM-1 (New

Delhi Metallo--lactamase) in P. aeruginosawas made

from Serbia23.

Beta-lactamases of P. aeruginosa with carbapen-

emase activity are classied- apart from class B- in the

remaining three enzymatic classes as well. Ambler class

A carbapenemase KPC (Klebsiella pneumoniaecarbap-

enemase)was rst reported in P. aeruginosa isolates in

Colombia24

and unlikely to KPC-producing Klebsiellapneumoniae, KPC-producingP. aeruginosa did not reach

other continents except Latin America. Due to their high

rates of carbapenem hydrolysis, KPCs make powerful re-

sistance determinants that do not need additional mecha-

nisms such as efux or impermeability. Enzymes GES/

IBC (Guiana extended spectrum) / (Integron-borne cefa-

losporinase) of the same enzymatic class possess carbap-

enemase activity which may become clinically important

when combined with diminished outer membrane per-

meability or efux over-expression. For P. aeruginosa,

GES-2 has been reported in South Africa25and IBC-2 in

Greece26.

Class C beta-lactamases are not carbapenemases.They posses however a low potential of carbapenem hy-

drolysis and their overproduction combined with efux

systems over-expression and/or diminished outer mem-

brane permeability has been proven to lead to carbap-

enem resistance27.

Finally, class D carbapenemases like OXA-19828are

rare forP. aeruginosaand do not have the same clinical

impact as forAcinetobacter baumannii29.

Detection of carbapenemases may be performed by

polymerase chain reaction (PCR) using specic primers

for each carbapenemase-encoding gene. Phenotypic as-

says however are also used for class A and B enzymes.

Ethylene-diamine-tetraacetic acid (EDTA) is a polyami-no carboxylic acid that binds metal ions like Zn2+and can

inactivate the metallo-beta-lactamases. EDTA is used

for the detection of class B enzymes by a carbapenem-

EDTA disc synergy test or the comparison of the inhi -

bition halo of a carbapenem and a carbapenem+EDTA

disc. For the detection of class A carbapenemases a dou-

ble disc synergy test may be performed using clavulanic

acid with carbapenems, aztreonam and third generation

cefalosporins. Especially for P. aeruginosa cloxacillin

(200g cloxacillin/1ml) is dissolved into Mueller-Hinton

agar to eliminate any possible AmpC beta-lactamase in-

terference30.

Efux pumps over-expression

Efux pump systems are an extremely important

cause of multi-drug resistance for P. aeruginosa. They

are tripartite systems that are composed by 1) a protein

transporter of the cytoplasmic membrane that uses en-

ergy in the form of proton motif force to transport drugsand other substances through the inner membrane, 2) a

periplasmic connective protein and 3) an outer membrane

protein component with a barrel conguration. This way,

the most commonly observed pump system, MexAB-

OprM (Multidrug efux system AB- Outer membrane

protein M), consists of the MexB pump, the MexA linker

lipoprotein and the OprM exit portal31.

MexAB-OprM together with MexXY-OprM act syn-

ergistically with low outer membrane permeability con-

ferring intrinsic multi-drug resistance to P. aeruginosa.

In addition to this intrinsic resistance, these and some

other additional systems (MexCD-OprJ, MexEF-OprN,

MexJK-OprM, MexVW-OprM) promote acquired multi-drug resistance as a consequence of hyper expression of

the efux genes by mutational events32. Substrates for

these efux pumps are commonly quinolones, antip-

seudomonal penicillins, cefalosporins, aminoglycosides

and meropenem while imipenem and ceftazidime are not

usually affected. However, when efux pump over-ex-

pression coexists with low outer membrane permeability

resistance to other carbapenems develops as well33.

The CCCP-test is used for the phenotypic detection

of efux pumps over-expression34. Carbonyl cyanide m-

chlorophenyl hydrazone (CCCP) is a pump inhibitor that

can be added in Mueller-Hinton agar during its prepara-

tion. The strain is inoculated in an agar plate with CCCPas well as in a plate without CCCP, and a meropenem disc

is placed for both inoculations. The result is considered

positive if the inhibition zone of meropenem is wider in

the agar plate with CCCP than the one in the plate with-

out pump inhibitor.

In PCR, primers for mexB and mexY can be used

to detect MexAB-OprM and MexXY-OprM systems re-

spectively although the efux pumps over-expression is

certied performing reverse transcriptase real time PCR

that shows the presence and also the quantitative expres-

sion of mRNAs from these genes35.

Diminished outer membrane permeabilityCarbapenems enter into the periplasmic space of P.

aeruginosathrough the OprD outer membrane porin for-

merly known as D2 porin. The porin loss probably by a

mutational event of the OprD gene leads to imipenem re-

sistance36. Furthermore, in strains with OprD downregu-

lation37, reduced susceptibility to meropenem is observed

while other beta-lactams are not affected31. Diminished

expression or loss of the OprD porin is rather frequent

during imipenem treatment38.

A carbapenem resistant, beta-lactam susceptible

phenotype is characteristic for diminished expression

of OprD even though such phenotypes will not be ob-

served among multi-resistant isolates. Traditionally, the

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306 MELETIS G

loss of OprD is determined by Sodium Dodecyl Sulfate-

Polyacrylamide Gel Electroforesis (SDS-PAGE) for the

separation of the membranes proteins39while later stud-

ies use reverse transcriptase real time PCR with specic

primers for the OprD gene40.

Concluding remarks

Nosocomial infections due to multi-drug resistant

P. aeruginosa are a signicant cause of morbidity and

mortality in hospital settings. P. aeruginosastrains har-

boring carbapenem resistance mechanisms compromise

severely the selection of appropriate treatments because

of the fact that carbapenem resistance is commonly asso-

ciated with resistance to other antibiotic classes. Conse-

quently, the detection of carbapenem-resistant strains and

the implementation of strict infection control measures

become critical for limiting the spread of the underlying

resistance mechanisms.

Conict of interest

The authors declare no conicts of interest.

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