Special Contribution

Yonago Acta medica 2007;50:1–8

How Can Antimicrobial Resistance in Pseudomonas aeruginosa
Be Controlled?
Toshinobu Horii
Division of Bacteriology, Department of Microbiology and Immunology, School of Medicine, Tottori
University Faculty of Medicine, Yonago 683-8503 and Infection Control Division, Tottori University
Hospital, Yonago 683-8504 Japan

Pseudomonas aeruginosa remains one of the most difﬁcult to treat and to control nosocomial infections. In vitro antimicrobial susceptibility data are required for successful therapy because acquired resistance to such antimicrobials as -lactams, ﬂuoroquinolones andaminoglycosides is so prevalent in P. aeruginosa. Strategies for controlling P. aeruginosa
infections include early detection of P. aeruginosa as the causative pathogen, determination of its antimicrobial susceptibilities, initiation of effective and adequate therapy andstrict infection control practice such as hand hygiene and equipment procedures. Once
antimicrobial therapy has been initiated against a P. aeruginosa infection, its susceptibility
to antimicrobials, especially to carbapenems and ﬂuoroquinolones, should be monitored
during antimicrobial therapy to detect clonal shifts in resistance and microbial substitutions as early as possible. Continued surveillance of nosocomial infections and monitoringof antimicrobial resistance by the infection control staff plays major roles in preventing
nosocomial infections and the spread of antimicrobial resistance. Additional strategies
for controlling antimicrobial resistance in P. aeruginosa include the development of new
methods for rapid detection of antimicrobial resistance and new agents and vaccines
against P. aeruginosa infections in the laboratories and pharmaceuticals, while preserving
the efﬁcacy of currently available antimicrobials for as long as possible in the hospital settings.
Key words: antimicrobial resistance; antimicrobial therapy; infection control; nosocomial infection; Pseudomonas aeruginosa

ter in ﬂower vases, showers and toilets, disinfectant
solutions, uncooked vegetables, skin, respiratory
equipment and other moist environments can act as
reservoirs of P. aeruginosa in the hospital settings(Pier et al., 2004; Rossolini and Mantengoli, 2005).
Consequently, P. aeruginosa is a common nosocomial pathogen and often is the pathogen in cases ofventilator-associated pneumonia, catheter-related
urinary tract infections and catheter-related bloodstream infections. Community-acquired infections
by P. aeruginosa are uncommon.

Pseudomonas aeruginosa as a pathogen
Pseudomonas aeruginosa is a non-fermentative,
aerobic, Gram-negative rod that normally lives inmoist environments. P. aeruginosa is an opportunistic human pathogen and causes pneumonia, urinary tract infections, wound infections and bloodstream infections. P. aeruginosa has minimal
nutritional requirements, which contributes to its
broad ecological adaptability and distribution. Wa-

Abbreviations: AAC, aminoglycoside acetyltransferase; ANT, aminoglycoside nucleotidyltransferase; APH, aminoglycosidephosphoryltransferase; MIC, minimum inhibitory concentration; MDRP, multidrug-resistant Pseudomonas aeruginosa; OMP,
outer membrane protein; QRDR, quinolone resistance-detremining region; RT, reverse transcription; WHO, World Health Organization1

T. Horii

duction of outer membrane proteins (OMPs) and
up-regulation of efﬂux pumps (Szabo et al., 2005).
To date, a number of -lactamases have been
identiﬁed in clinical P. aeruginosa isolates. Most
isolates produce chromosomally encoded AmpCtype -lactamases (molecular class C) (Ambler,1980). Hyperproduction of AmpC-type -lactamases induced by exposure to certain antimicrobials can lead to resistance to penicillins andcephalosporins but not to carbapenems (Livermore,1987). In P. aeruginosa, carbapenem resistance canbe conferred by production of metallo- -lactamases(molecular class B), which hydrolyze all classes of-lactams (Ambler, 1980). Metallo- -lactamases
are not inhibited by -lactamase inhibitors such as
clavulanic acid, sulbactam and tazobactam. Most
metallo- -lactamase genes are located on plasmids and can be transferred to other strains. The
ﬁrst documented instance of transferable metallo-lactamase derived from P. aeruginosa was foundin Japan in 1988 (Watanabe et al., 1991), and transferable metallo- -lactamases such as IMP- andVIM-types have now been found in Japan, Korea,
Europe and USA (Walsh et al., 2005). Additionally P. aeruginosa can produce OXA- and PSEtype -lactamases belonging to molecular class D,
resulting primarily in inactivation of penicillins(Bonomo and Szabo, 2006; Naas and Nordmann,
1999). Recently extended-spectrum -lactamases(ESBLs) derived from molecular class A and D(OXA)-types also have been described (Bonomo
and Szabo, 2006).
Mutational impermeability is one of the majormechanisms responsible for carbapenem resistanceand arises via mutational loss or decrease of OMPs
(Studemeister and Quinn, 1998; Livermore, 2002).
OprD is a porin-forming transmembrane channel(D2 porin), which is accessible to carbapenems
but not to other -lactams. It has been shown thatloss or decrease of OprD production or inactivation of OprD results in carbapenem resistance inP. aeruginosa but does not confer resistance toother -lactams (Pirnary et al., 2002; Horii et al.,
2003; Muramatsu et al., 2003 and 2005; Wolter etal., 2004; Rossolini and Mantengoli, 2005). It wassuggested that carbapenem resistance caused bymutational changes in OprD can emerge during antimicrobial therapy against a P. aeruginosa infec-

Antimicrobial resistance in P. aeruginosa
It is difﬁcult to treat P. aeruginosa infections because acquired resistance to such antimicrobials as-lactams, fluoroquinolones and aminoglycosides
is common. Clinically it has been shown that P.
aeruginosa has the capacity to develop resistance
rapidly during the course of antimicrobial therapy
by several mechanisms (Fish et al., 1995; Hancock,1998; Carmeli et al., 1999; Le Thomas et al., 2001).
It has been also shown that selection of resistanceduring antimicrobial therapy occurs frequently
in P. aeruginosa (Harris et al., 1999). Therefore,
sequential accumulation of resistance may resultin emergence of multidrug resistance in P. aeruginosa.
Notably integron-mediated multidrug resistance frequently found in P. aeruginosa and is amajor clinical problem (Weldhagen, 2004). Integrons are genetic elements that possess the capacity to capture individual antimicrobial resistancegenes, including those encoding -lactamases andaminoglycoside-modifying enzymes, and to promote transcription and expression of these genes.
Integrons include a receptor site, attI, where thecaptured genes are integrated, and a recombinasegene, int. Widespread integron-mediated resistance poses an increasing threat to the treatment
and control of P. aeruginosa infections.
Factors inﬂuencing the emergence and spreadof acquired resistance in P. aeruginosa include
inadequate use and overuse of antimicrobials(Rossolini and Mantengoli, 2005). Previous reports have shown that use of carbapenems and other antimicrobials increases the risk of emergenceof resistant P. aeruginosa, although antimicrobial
rankings differ between studies (Carmeli et al.,
1999; Amari et al., 2001; Harris et al., 2002).

-Lactam resistance-Lactams including carbapenems are commonlyused to treat P. aeruginosa infections in Japan.
The predominant mechanisms conferring -lactam
resistance in clinical P. aeruginosa isolates include
production of -lactamases, loss or decreased pro2

Antimicrobial resistance in P. aeruginosation (Horii et al., 2003). Mutations leading to upregulation of efﬂux systems such as MexA-MexBOprM, MexC-MexD-OprJ and MexX-MexY-OprM
can variably result in decreased susceptibility
to -lactams and ﬂuoroquinolones (Rossolini andMantengoli, 2005).

in terms of minimum inhibitory concentrations(MICs). Techniques for detection of speciﬁc antimicrobial resistance alleles, including both clinically available and those limited to research laboratories, are summarized in Table 1.

-Lactam resistance

Fluoroquinolone resistance

Genetic techniques to determine types of -lactamases include PCR, cloning, DNA probes andnucleotide sequencing, although the nongenetic
gold standard remains examination for ability ofbacterial crude extracts to hydrolyze -lactams.
Hyperproduction of AmpC-type -lactamases canbe detected by RNA-based techniques such as realtime reverse transcription (RT)-PCR (Quale et al.,2006). Metallo- -lactamases can be detected easily using microbiological methods (Walsh et al.,2005). Metallo- -lactamase activity is inhibitedby the removal of zinc from the active site. Microbiological laboratories usually apply disk diffusion
methods using ceftazidime (substrate) and 2-mercaptoproprionic acid (inhibitor), microdilution
methods using imipenem (substrate) and EDTA or
1,10-phenanthroline (inhibitor) and Etest methods
using imipenem and EDTA to the examination.
Metallo- -lactamases can also be detected by PCR
for the specific bla IMP and bla VIM genes, DNAprobes and/or nucleotide sequencing.
Alterations in OprD result from decreased
production or inactivation of OprD due to deletions, substitutions or insertions in the oprD geneor regulatory mutations (Pirnary et al., 2002;
Rossolini and Mantengoli, 2005). The alterations can be detected by SDS-PAGE following the
preparation of OMPs, RNA-based techniques or
nucleotide sequencing of the oprD and relevant
genes (Pirnary et al., 2002; Horii et al., 2003;
Muramatsu et al., 2003).

The major mechanisms of ﬂuoroquinolone resistancein clinical isolates of P. aeruginosa include alterations
in DNA gyrase and/or topoisomerase IV caused bymutations in the quinolone resistance-determining
regions (QRDRs) of GyrA and ParC (Akasaka et al.,
2001; Muramatsu et al., 2005). Other reports havesuggested that mutations of GyrB are associated withﬂuoroquinolone resistance (Mouneimnè et al., 1999;
Le Thomas et al., 2001). A secondary mechanism,
active efﬂux systems, contributes to reduced susceptibility to ﬂuoroquinolones (Le Thomas et al., 2001;
Livermore, 2002). Of the known efﬂux pumps in P.
aeruginosa, only MexA-MexB-OprM is expressed
constitutively at sufﬁcient levels to result in intrinsic
ﬂuoroquinolone resistance (Zhanel et al., 2004).

Aminoglycoside resistanceMechanisms conferring aminoglycoside resistancein P. aeruginosa include enzymatic modificationof aminoglycosides, active efﬂux systems and impermeability (Poole, 2005). Aminoglycosides areinactivated by enzymatic phosphorylation (aminoglycoside phosphoryltransferase [APH]), acetylation (aminoglycoside acetyltransferase [AAC]) andadenylation (aminoglycoside nucleotidyltransferase[ANT]). These modifying enzymes are locatedon chromosome or plasmids. Individual aminoglycoside-resistant isolates of P. aeruginosa carry
multiple modifying enzymes, resulting in broadspectrum aminoglycoside resistance (Poole, 2005).

Fluoroquinolone resistance
Detection of antimicrobial resistancein P. aeruginosa

Our understanding of fluoroquinolone resistanceis based on nucleotide sequencing of the QRDRsof the gyrA, gyrB and parC genes (Akasaka et al.,
2001; Le Thomas et al., 2001; Muramatsu et al.,2005). Additional contributions to ﬂuoroquinolone
resistance by efflux systems can be detected by

Antimicrobial therapy against individual P. aeruginosa infections should be based on in vitro antimicrobial susceptibility data generally expressed3

T. Horii

Table 1. Detection techniques of antimicrobial resistance alleles in Pseudomonas aeruginosa
Antimicrobial
Resistance mechanism
Resistance gene

Gene product-Lactam
Production of -LactamaseblaTEM-, SHV-,OXA-type(class A and D)
ampC
AmpC-type(class C)
bla

IMP-, VIM-type(class B)

Detection techniques

Reference

PCR, real-time RT-PCR,
cloning, DNA probes,
nucleotide sequencing,enzymatic analysis

Bauernfeind et al., 1996;
Nordmann and Polrel, 2002;
Lee et al., 2005;
Bonomo and Szabo, 2006;
Quale et al., 2006

PCR, cloning, DNA probes,
cloning, nucleotide
sequencing, microbiologic
methods (disk diffusion
and Etest), enzymatic analysis

Lauretti et al., 1999;
Franceschini et al., 2000;
Shibata et al., 2003;
Nordmann and Polrel, 2002;
Wash et al., 2002

Loss or decreased OprD production, inactivation of OprD
oprD
OprD
OMP analysis, realtime RT-PCR,
RNA-based techniques,
nucleotide sequencing
Fluoroquinolone
Alteration of in DNA gyrase and topoisomerase IV
gyrA, gyrB, parC
GyrA, GyrB, ParC
Nucleotide sequencing
Aminoglycoside
Enzymatic modiﬁcation of aminoglycosideaac(6’)-I, aac(6’)-II, AAC(6’)-I, AAC(6’)-II,aac(3)-I, aac(3)-II,AAC(3)-I, AAC(3)-II,aph(3’)-II, ant(2’)-I
APH(3’)-II, ANT(2’)-I
Production of 16S rRNA methylase
rmtA
RmtA
Multidrug
Up-regulation of efﬂux systems
mexA, mexB, oprM
MexA-MexB-OprM
mexC, mexD, oprJ
MexC-MexD-OprJ
mexX, mexY, oprM
MexX-MexY-OprM

Pirnary et al., 2002;
Horii et al., 2003;
Muramatsu et al., 2003;
Dumas et al., 2006;
Quale et al., 2006
Akasaka et al., 2001;
Le Thomas et al., 2001;
Muramatsu et al., 2005

PCR, cloning, DNA probes,
nucleotide sequencing

Shaw et al., 1991;
Vliegenthart et al., 1991;
Mendes et al., 2004

PCR

Yokoyama et al., 2003

Real-time PCR, nucleotide
sequencing, RNA-based
techniques, biological assay

Mortimer and Piddock, 1991;
Quale et al., 2006

OMP, outer membrane protein; RT, reverse transcription.

biological assays that measure active intracellularconcentrations of ﬂuoroquinolones with and without carbonyl cyanide m-chlorophenylhydrazone(CCCP) (Hirai et al., 1986). Nucleotide sequencing
and RNA-based techniques are used to characterize disruption and overexpression of the genes coding for efﬂux pumps such as MexA-MexB-OprM(Zhanel et al., 2004; Quale et al., 2006).

Aminoglycoside resistance
Although the mechanisms of amionoglycosideresistance are multiplexed, the genes encoding
the aminoglycoside-modifying enzymes such
as AAC(6’)-I, AAC(6’)-II, AAC(3)-I, AAC(3)II, APH(3’)-II and ANT(2’)-I can be detected by4

Antimicrobial resistance in P. aeruginosa

PCR, DNA probes and/or nucleotide sequencing (Shaw et al., 1991; Vliegenthart et al., 1991;
Mendes et al., 2004).

nosocomial pneumonia and bacteraemia, combination therapy with an antipseudomonal penicillin, an
antipseudomonal cephalosporin or aztreonam plus
an aminoglycoside is administered. The questionof whether combination therapy prevents the emergence of resistance remains highly controversial(Paul et al., 2004; Paterson, 2006).
Carbapenems are widely used in the treatment of P. aeruginosa infections, and a strong
association between use and resistance has been
documented for these antimicrobials (Rossolini
and Mantengoli, 2005). In some cases, prior useof a particular antimicrobial predicts developmentof resistance in P. aeruginosa (El Amari et al.,2001). Resistance emerges during antimicrobial
therapy in as many as 50% of patients treated for aserious P. aeruginosa infection with imipenem. In
this context, resistance is most likely attributable tomutational loss of OprD (Livermore, 2002). Casesof clonal shifts in carbapenem resistance resulting
in loss or decreased production of OprD during
antimicrobial therapies have been reported (Horii
et al., 2003). In addition, it was reported thatemergence of resistance to both fluoroquinolones
and -lactams such as cefsulodin and aztreonam
during monotherapy with ciproﬂoxacin was caused
by amino acid mutations of QRDRs in GyrB plus
overexpression of the active efﬂux system, MexAMexB-OprM, and required combination therapy
with ceftazidime and amikacin (Le Thomas et al.,2001). Accordingly, it is critical to monitor antimicrobial susceptibility, especially to carbapenems
and ﬂuoroquinolones, during antimicrobial therapy
against a P. aeruginosa infection.

Nosocomial infections caused byantimicrobial-resistant P. aeruginosa
Antimicrobial-resistant P. aeruginosa are a major
cause of nosocomial infections (Table 2). Nosocomial transmission of antimicrobial-resistant P.
aeruginosa has been associated with endoscopes,
tap water and other environmental surfaces(Muscarella, 2004). Contaminated environmental
surfaces in the hospital settings can lead to nosocomial transmission via contact with contaminatedhands (or gloves) of the healthcare staff, patientsthemselves or visitors. Of particular concern is
the increasing isolation of P. aeruginosa from
intensive and high care units (Trautmann et al.,2005). The risk factors for nosocomial infections
in critically ill patients include length-of-stay andextent of exposure to invasive devices such asmechanical ventilators, urinary bladder catheters
and intravenous and intraarterial catheters, as wellas inadequate use of antimicrobials (Trilla, 1994;
Obritsch, 2005).

Antimicrobial therapy against infections
caused by P. aeruginosa
The prevalence of antimicrobial resistance in P.
aeruginosa leads to limitation in efficacious antimicrobial therapies. Acquired resistance thatdevelops during the course of treatment, especiallywith carbapenems and fluoroquinolones, is another serious problem (Fish et al., 1995; Hancock,1998; Carmeli et al., 1999; Amari et al., 2001; Le
Thomas et al., 2001; Harris et al., 2002). Therefore, in vitro susceptibility data are essential in the
choice of antimicrobials: the alternatives include
antipseudomonal penicillins (piperacillin and tazobactam/piperacillin), antipseudomonal cephalosporins (cefoperazone, ceftazidime and cefepime),
aztreonam, carbapenems, aminoglycosides (tobramycin and amikacin) and fluoroquinolones.
In some types of infections such as endocarditis,

Table 2. Recent cases of nosocomial transmission of antimicrobial-resistant Pseudomonas aeruginosa in Japan
Case

Year

1† 2000–2001
2 2001–2002
3†
2004
4†
2004
5†
2005

Number ofpatients*

Environmental
source of bacteria

18 (1)30
9 (3)
11 (2)
6 (5)Urinary catheter
Urinary catheter
Transesophageal endoscope
Endoscope
Cup for urine examination*Number of deceased patients is shown in the parenthesis.
†Case was reported from a university hospital.

5

T. Horii

Multidrug-resistant P. aeruginosa (MDRP)
in Japan is defined as a strain showing MICs of≥ 16 mg/L for imipenem, ≥ 4 mg/L for ciproﬂoxacin and ≥ 32 mg/L for amikacin. Risk factors for
MDRP infection include prolonged hospitalization, protracted and broad-spectrum antimicrobial therapy and an immunocompromised state(Obritsch et al, 2005). The frequency of MDRP
infections in Japan is reported in the Infectious
Diseases Weekly Report (IDWR, see http://idsc.
nih.go.jp/kanja/idwr/idwr-j.htm) and varies between hospitals. The intrinsic susceptibility of P.
aeruginosa is already limited to only several antimicrobials, and the emergence of multidrug resistance compromised most antipseudomonal therapies except colistin and synergistic combinationsof antimicrobials (Obritsch et al., 2005). Colistin
is a multicomponent polypeptide antimicrobial,
comprised mainly of colistin A and B. Colistin
became available for clinical use in the 1960s,
but is not currently available in Japan. There are
no recommended breakpoints for susceptibility
testing of colistin for P. aeruginosa. Strategies
against MDRP infections include combination
therapy with cefepime plus amikacin, continuousinfusion meropenem (not applicable in Japan) andparenteral colistin therapy (Obritsch et al., 2005).
Concomitantly, strict compliance with recommended infection control practices and isolation
procedures is required to prevent the spread of
MDRP clones within the hospital settings.

new drugs and vaccines (http://www.who.int/csr/
resources/publications/drugresist/WHO_CDS_CS
R_DRS_2001_2_EN/en/).
In hospital settings, strategies for control of P.
aeruginosa infections include early detection of P.
aeruginosa as the causative pathogen, determination of its antimicrobial susceptibilities, effective
therapy against the infection with adequate use of
antimicrobials and strict infection control practices. Once antimicrobial therapy has been initiatedagainst a P. aeruginosa infection, its susceptibility to antimicrobials, especially to carbapenems
and fluoroquinolones, should be monitored during antimicrobial therapy to detect clonal shifts inresistance and microbial substitutions as early as
possible. Continued surveillance of nosocomial infections and monitoring of antimicrobial resistanceby the infection control staff will help prevent
nosocomial infections and antimicrobial resistance.
In the future, the development of methods forthe rapid detection of antimicrobial resistance, especially to carbapenem and ﬂuoroquinolone, willlead to early detection of clonal shifts in resistanceduring antimicrobial therapy and identificationof resistance alleles associated with nosocomial
dissemination of antimicrobial-resistant P. aeruginosa. The spread of MDRP represents an increasing threat and efforts should be made to develop
new agents and vaccines against P. aeruginosa
infections in the laboratories and pharmaceuticals, while preserving the efﬁcacy of the currently
available antimicrobials for as long as possible in
the hospital settings.

Strategies for controlling infections
caused by antimicrobial-resistantP. aeruginosa

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Corresponding author: Toshinobu Horii, MD, PhD

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