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DOI 10.1099/jmm.0.058354-0
Carbapenem-resistance mechanisms of
multidrug-resistant Pseudomonas aeruginosa
Ester Fuste,1 Ldia Lopez-Jimenez,1 Concha Segura,2 Eusebio Gainza,3
Teresa Vinuesa1 and Miguel Vinas1
1
Correspondence
Miguel Vinas
mvinyas@ub.edu
INTRODUCTION
Pseudomonas aeruginosa frequently causes infections that
develop in wound and burn patients and in the severe
respiratory tract infections commonly seen in cystic fibrosis
patients. In many countries, a major cause of concern is the
increasing prevalence of infections caused by isolates of
multidrug-resistant P. aeruginosa (MDRPA). Controlling
the spread of MDRPA, enlarging the therapeutic arsenal
used to fight these infections and reducing the mortality of
infected individuals (Woodford et al., 2011; Cabot et al.,
2012) are currently important goals of microbiological
surveillance. However, a serious challenge to these efforts is
that some of the classic antimicrobial drugs as well as
Abbreviations: AadB, aminoglycoside adenyltransferase B; CCCP,
carbonyl cyanide m-chlorophenylhydrazone; EPI, efflux pump inhibitor;
IVR, integron variable region; MDRPA, multidrug-resistant Pseudomonas
aeruginosa; OM, outer membrane; OMP, outer membrane protein; OprD,
outer membrane porin D; PAbN, Phe-Arg-b-naphthylamide; PFGE,
pulsed-field gel electrophoresis.
The GenBank/EMBL/DDBJ accession numbers for the nucleotide
sequences of class 1 integrons are JF714996.1, JF718319.1,
JF727876.1 and JF742758.1.
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METHODS
Bacteria, media, antibiotics and EPIs. MDRPA isolates 459, 133,
Ceftazidime, ciprofloxacin, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and PabN were purchased from Sigma Chemical Co.
Meropenem was from Astra Zeneca UK Ltd, imipenem from Merck
and tobramycin from Fagron Iberica. All bacteriological culture
media were from Scharlab.
Drug susceptibility testing. Susceptibility to the antimicrobial
Table 1. MICs (mg l1) of ATCC 27853 and MDRPA strains determined by a microdilution method
Clinical breakpoints, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2011): imipenem, susceptible 4 mg
l21, resistant .8 mg l21; meropenem, susceptible 2 mg l21, resistant .8 mg l21; ciprofloxacin, susceptible 0.5 mg l21, resistant .1 mg l21;
tobramycin, susceptible 4 mg l21, resistant .4 mg l21; ceftazidime, susceptible 8 mg l21, resistant .8 mg l21.
Strain
459
Drug
Imipenem
Meropenem
Ciprofloxacin
Tobramycin
Ceftazidime
133
162
527
ATCC 27853
8
8
64
64
64
1
2
0.5
0.5
4
Family
Carbapenem
Carbapenem
Fluoroquinolone
Aminoglycoside
Cephalosporin
8
8
64
32
128
8
8
64
64
64
8
8
64
64
64
kDa
ATCC
459
133
162
527
97.4
66.7
45
31
RESULTS
Drug susceptibility testing
MIC data for the ATCC 27853 and MDRPA isolates for
several antimicrobial agents are shown in Table 1.
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Fig. 2. Effects of a high-concentration of meropenem on P. aeruginosa cell shape. (a) P. aeruginosa rods grown without antibiotic. (b)
P. aeruginosa rods grown with 4 MIC of meropenem. (c) P. aeruginosa rods after removal of antibiotic pressure. Scale bar, 10 mm.
from these strains were identical and carried only one resistance
gene, for aminoglycoside adenyltransferase B [aadB, also
called ant(20)-Ia], contained in a cassette. The enzyme encoded
by this gene is involved in aminoglycoside resistance (Fig. 3b),
specifically to gentamicin, kanamycin and tobramycin.
Efflux-pump-mediated fluoroquinolone
resistance
All MDRPA strains had a ciprofloxacin MIC of 64 mg l21
and were thus interpreted as resistant. The efflux pump
overexpression phenotype was defined based on at least a
twofold decrease in the ciprofloxacin MIC when determined in the presence of PAbN and was observed in all
MDRPA strains (Fig. 4).
(a)
(b)
kb
10
8
6
5
4
3.5
3
2.5
2
1.5
Variable region
3-CS
5-CS
intl1
aadB
1
0.75
0.5
Fig. 3. (a) PCR amplification, using the 5-CS and 3-CS primers, of variable regions of integrons from MDRPA isolates. The
PCR products were separated by electrophoresis in 0.7 % agarose. Lane 1, 1 kb DNA ladder; lane 2, P. aeruginosa 459; lane
3, P. aeruginosa 162; lane 4, P. aeruginosa 527; lane 5, P. aeruginosa 133. (b) Schematic representation of a class 1 integron
with an aadB gene cassette; arrows show direction of transcription of genes.
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60
50
40
30
20
10
0
459 Cip
459 Cip
+PAb N
162 Cip
162 Cip
+PAb N
Fig. 4. Effects of inhibition of efflux by the EPI PAbN on ciprofloxacin susceptibility; all the strains were tested over the same
range of ciprofloxacin concentrations (0.125128 mg l1) with and without PAbN, and showed significant decreases in MIC
with the EPI. Error bars indicate SD of three experiments.
DISCUSSION
antimicrobial agents, mainly aminoglycosides, carbapenems, antipseudomonal penicillins, quinolones and cephalosporins (Falagas et al., 2006). The increasing emergence
of MDRPA strains is a cause for concern as it compromises
the therapeutic options for treating infections with these
bacteria. At the same time, it highlights the importance of
developing alternative approaches, such as the use of drug
combinations or EPIs, and of identifying novel antipseudomonal agents (Lomovskaya & Watkins, 2001).
The addition of PAbN resulted in only negligible reductions in the ceftazidime and meropenem MICs, in contrast
to the antagonistic effects of tobramycin and ceftazidime or
tobramycin and meropenem regardless of the b-lactam
concentration (Table 2).
(a) 2.5
2.0
OD550
1.5
1.0
0.5
0
4
Time (h)
(b) 2.5
2.0
OD550
1.5
1.0
0.5
0
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4
Time (h)
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250
200
150
100
50
0
Fig. 6. Effects of the efflux pump inhibitor PAbN on tobramycin susceptibility; all the strains were tested over the same range of
tobramycin concentrations (0.25256 mg l1), and showed antagonism with PAN (40 mg l1). Error bars indicate SD of three
experiments.
0
0
8
8
16
16
32
32
64
64
128
128
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0
40
0
40
0
40
0
40
0
40
0
40
Strain
Strain
459
133
162
527
459
133
162
527
8
4
0.5
8
0.125
8
0.125
8
0.125
8
0.125
8
8
8
1
8
0.5
8
0.125
8
0.125
8
0.125
4
8
4
1
8
0.25
8
0.125
8
0.125
8
0.125
8
8
4
1
16
0.5
8
0.125
8
0.125
16
0.125
8
128
32
4
32
0.125
32
0.125
8
0.125
16
0.125
16
64
32
2
32
0.5
32
0.125
32
0.125
32
0.125
32
64
32
4
64
1
32
0.125
32
0.125
32
0.125
32
64
32
4
32
1
32
0.125
32
0.125
16
0.125
16
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ACKNOWLEDGEMENTS
Dedicated to Professor Miguel Regue in memoriam. E. F. was the
recipient of an IDIBELL pre-doctoral fellowship. This work was
partially supported by the Spanish Ministerio Economia y
Competitividad/FEDER project IPT-2011-1402-900000.
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