Susceptibility patterns and cross resistances of antibiotics against Pseudomonas aeruginosa in a teaching hospital of Turkey.

Abstract

Pseudomonas aeruginosa is the third most common pathogen responsible for nosocomial infections and the prevalence of multiple resistant isolates has been increasing. Ninety-nine clinical isolates were studied in order to assess the current levels of susceptibility and cross-resistances of widely used antipseudomonal antibiotics against P. aeruginosa and to determine some resistance mechanisms by phenotypic methods.
MICs of isolates for nine antipseudomonal antibiotics were determined by the E test method.
Thirty-six percent of isolates were resistant to more than one group of antibiotics. The rates of susceptible isolates were ciprofloxacin 75%, amikacin 73%, ceftazidime 65%, meropenem 63%, imipenem 63%, piperacillin/tazobactam 60%, cefoperazone/sulbactam 59%, cefepime 54% and tobramycin 44%. The majority of carbapenem resistant isolates were susceptible to ciprofloxacin and amikacin.
Ciprofloxacin seems to be the most active agent against P. aeruginosa followed by amikacin in our unit. The usefulness of combinations of these antibiotics and beta-lactams should be tested in treating multi-drug resistant P. aeruginosa.

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BioMed Central
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Annals of Clinical Microbiology
and Antimicrobials
Open Access
Annals of Clinical Microbiology and Antimicrobials
2002,
1
Research
Susceptibility patterns and cross resistances of antibiotics against
Pseudomonas aeruginosa in a teaching hospital of Turkey
Serap Gençer*, Öznur Ak, Nur Benzonana, Ays ¸e Bat rel and Serdar Özer
x
Address: Department of Infectious Diseases and Clinical Microbiology, Kartal Dr.Lütfi K rdar Training and Research Hospital, İstanbul, Turkey
E-mail: Serap Gençer* - segencer@turk.net; Öznur Ak - oznurak@turk.net; Nur Benzonana - benzonana@turk.net;
Ays ¸e Bat rel - aysebatirel@yahoo.com; Serdar Özer - eozer@turk.net
*Corresponding author
Abstract
Background: Pseudomonas aeruginosa is the third most common pathogen responsible for
nosocomial infections and the prevalence of multiple resistant isolates has been increasing. Ninety-
nine clinical isolates were studied in order to assess the current levels of susceptibility and cross-
resistances of widely used antipseudomonal antibiotics against P. aeruginosa and to determine some
resistance mechanisms by phenotypic methods.
Methods: MICs of isolates for nine antipseudomonal antibiotics were determined by the E test
method.
Results: Thirty-six percent of isolates were resistant to more than one group of antibiotics. The
rates of susceptible isolates were ciprofloxacin 75%, amikacin 73%, ceftazidime 65%, meropenem
63%, imipenem 63%, piperacillin/tazobactam 60%, cefoperazone/sulbactam 59%, cefepime 54% and
tobramycin 44%. The majority of carbapenem resistant isolates were susceptible to ciprofloxacin
and amikacin.
Conclusion: Ciprofloxacin seems to be the most active agent against P. aeruginosa followed by
amikacin in our unit. The usefulness of combinations of these antibiotics and ?-lactams should be
tested in treating multi-drug resistant P. aeruginosa.
Background
Pseudomonas aeruginosa is an important bacterial pathogen
most frequently responsible for nosocomial infections
and infections in immunocompromised patients. It ac-
counts for 21% of the bacterial isolates from the surgical
intensive care unit and for 14% of the bacterial isolates
from febrile neutropenic patients in our unit, being the
third most common isolate from both groups [1]. The
prevalence of multiple resistant P. aeruginosa isolates has
been increasing, so this study was undertaken in order to
assess the current level of susceptibility and cross-resist-
ances of widely used antipseudomonal antibiotics against
P. aeruginosa in our hospital and an attempt was made to
determine possible resistance mechanisms by phenotypic
methods.
Methods
Bacterial strains
Ninety-nine consecutive, nonduplicate P. aeruginosa iso-
lates were collected between June 2000 and May 2001
from various clinical materials at our hospital, which is a
700-beds teaching hospital in Istanbul, Turkey. Repeated
Published: 9 October 2002
Annals of Clinical Microbiology and Antimicrobials 2002, 1:2
Received: 7 August 2002
Accepted: 9 October 2002
This article is available from: http://www.ann-clinmicrob.com/content/1/1/2
© 2002 Gençer et al; licensee BioMed Central Ltd. This article is published in Open Access: verbatim copying and redistribution of this article are permitted
in all media for any purpose, provided this notice is preserved along with the article's original URL.

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isolates from the same patient were excluded. All isolates
were identified by a positive reaction to oxidase and pro-
duction of pyocyanin. Of these isolates, 25 were from sur-
gical wound or abscess, 25 from urine, 13 from blood, 12
from transtracheal aspirates, 9 from pleural fluid, 9 from
cerebrospinal fluid, 5 from ear and 1 from peritoneal flu-
id. Forty-five % of them were isolated on the surgical in-
tensive care unit.
Susceptibility tests
MICs for imipenem, meropenem, cefepime, ceftazidime,
cefoperazone/sulbactam, piperacillin/tazobactam, cipro-
floxacin, tobramycin, amikacin were determined by the E
test method (AB Biodisk, Sweden). After overnight incu-
bation, MIC breakpoints were interpreted according to the
NCCLS guidelines [2]. MIC breakpoints of cefoperazone/
sulbactam were interpreted according to the firm's recom-
mendations. P. aeruginosa ATCC 27853 was used as refer-
ence strain.
Detection of group 1 inducible ?-lactamases
The prevalence of inducible ?-lactamases was investigated
by disk approximation test method [3]. Ceftazidime (30
?g) disk was placed 20 mm. (centre to centre) from the
imipenem (10 ?g) disk on Mueller-Hinton agar plate in-
oculated with the test organism. After overnight incuba-
tion, distinct flattening of the inhibitory zone around the
ceftazidime disk on the side nearest to the imipenem disk
was regarded as the presence of inducible ?-lactamase.
Detection of extended-spectrum ?-lactamase (ESBL) ac-
tivity
ESBL activity was investigated by double disk synergy
method [4]. Ceftazidime (30 ?g), cefepime (30 ?g), az-
treonem (30 ?g) and cefotaxime (30 ?g) disks (Oxoid)
were placed 25 mm. (centre to centre) from the amoxicil-
lin/clavulanic acid (20/10 ?g) disk on Mueller-Hinton
agar plate inoculated with the test organism. After over-
night incubation, enhancement of the inhibition zone
around one of these disks toward the clavulanate-contain-
ing disk indicated the presence of ESBLs.
Detection of metallo-?-lactamase activity
The presence of metallo-?-lactamase was investigated by
the modified Hodge test [5]. An imipenem disk (10 ?g)
was placed at the centre of the Mueller-Hinton agar plate
inoculated with an overnight culture suspension of imi-
penem-sensitive E. coli, which was adjusted to one-tenth
turbidity of the McFarland no.0.5 tube. Imipenem-resist-
ant isolates from the overnight culture plates were
streaked heavily from the edge of the disk to the periphery
of the plate. The presence of a distorted inhibition zone
after overnight incubation was interpreted as the presence
of metallo-?-lactamase.
Statistical analysis
Susceptibility data were compared by using a chi-square
test. Correspondence analysis were performed by Cohen's
kappa measurement.
Results
Table 1 shows the minimum inhibitory concentrations
(MICs) of the tested antibiotics. Ciprofloxacin was the
most in vitro active antibacterial agent followed by ami-
kacin and ceftazidime (p < 0.05). Thirty-six % of isolates
were resistant to more than one group of antibiotics.
There were cross-resistances between antibiotics (Table 2).
About all of meropenem resistant isolates were also resist-
ant to imipenem (? = 0.93, p < 0.001) and 75% of them
were resistant to piperacillin/tazobactam (? = 0.61, p <
0.001). Seventy-five % of carbapenem resistant isolates
were susceptible to ciprofloxacin and amikacin. An im-
portant part (50–86%) of ceftazidime resistant isolates
were also resistant to other ?-lactams, especially cefepime
(? = 0.68, p < 0.001). Thirty-five -65% of ciprofloxacin re-
sistant isolates were also resistant to ?-lactams and 65% of
them were susceptible to amikacin.
Table 1: Comparative MIC values of antibiotics tested for P.aeruginosa isolates.
MIC (?g/mL) IPMP PM TZ CFSPTc Cl TM AK
MIC50
MIC90
1,5
>32
35
1
63
0,75
>32
36
0
63
82 12/6 8/4 0,25
>32
20
4
75
168
>256
28
17
54
>256
22
12
65
>256/128
32
8
59
>256/4
36
3
60
>256
51
4
44
48
20
6
73
R
I
S
IP: imipenem, MP: meropenem, PM: cefepime, TZ: ceftazidime, CFS: cefoperazone/sulbactam, PTc: piperacillin/tazobactam, Cl: ciprofloxacin, TM:
tobramycin, AK: amikacin, R: resistant, I: intermediate, S: susceptible.

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Inducible ?-lactamases were detected in 53% of isolates.
Forty-eight (80%) of 65 isolates non-resistant to ceftazi-
dime were positive for inducible beta-lactamase. ESBLs
were detected in only 4 isolates by DDS method. Metallo-
?-lactamases were detected in none of the isolates.
Discussion
P. aeruginosa is inherently resistant to many antimicrobial
agents mainly due to the synergy between multi-drug ef-
flux systems or a type 1 AmpC ?-lactamase and low outer
membrane permeability [6–8]. Inducible AmpC ?-lacta-
mase was detected in 53% of our isolates. The presence of
inducible ?-lactamase in 80% of isolates susceptible to
ceftazidime suggests that resistance may emerge during
treatment via selection of derepressed mutants from in-
ducible populations. Resistance can also arise by the ac-
quisition of plasmids encoding ?-lactamases. The rate of
strains with acquired resistance to ceftazidime has been
estimated to range from 10% to 40% [9]. Our rate of
ceftazidime resistance was 22%. In addition, various ex-
tended spectrum ?-lactamases have been found in P. aer-
uginosa[9]. In our study, ESBLs were detected in only 4
isolates by DDS method. However, clinical laboratory de-
tection may be difficult due to a combination of resistance
mechanisms. The presence of AmpC system and class D
ESBLs confer resistance to ?-lactamase inhibitors, so syn-
ergy can not be detected.
The only ?-lactam active against derepressed mutants and
ESBLs are carbapenems but resistance to carbapenems has
been increasing. The development of antibiotic resistance
during the treatment is more frequent in imipenem than
in ciprofloxacin and ceftazidime [7,10]. Our results dem-
onstrate that carbapenems are second in affectivity after
ceftazidime among other ?-lactams with the resistance
rate of 36%. Although still rare, isolates with metallo-?-
lactamases have been reported from around the world
[8,11]. In this study, we used the modified Hodge test,
sensitivity of which was 100%, to screen metallo-?-lacta-
mase-producing isolates and found no producers [5].
One third of our isolates were multiple resistant. There
were cross-resistances between amikacin and/or cipro-
floxacin and/or ?-lactams. These data indicate that a high
number of isolates probably have resistance due to imper-
meability or multi-drug efflux or a combination of multi-
ple unrelated resistance mechanisms. Ciprofloxacin
showed the highest in vitro antibacterial activity followed
by amikacin in our centre. The major resistance mecha-
nisms to quinolones are mutations in the target genes,
which confer resistance only to quinolones, and muta-
tions in regulatory genes for drug efflux pumps. The latter
one, called Mar (multiple antibiotic resistance) mutation,
results in cross-resistance to chemically unrelated antibi-
otics [6,8,12].
Although comparison between studies is difficult since
the patient populations of the centres and the methods of
studying differ, interestingly, we found a higher level of re-
sistance to the ?-lactams and a lower level of resistance to
ciprofloxacin in contrast to other surveys and our 1995–
1999 survey [13–16]. The incidence of resistance is de-
pendent on the patterns of antibiotic usage. The relation-
ship between the emergence of resistance of group 1 ?-
lactamase-producing organisms and the prior use of ex-
tended-spectrum cephalosporins is clearly proven [17].
The reduction of ceftazidime resistance had been ob-
served after restricted use of it [18]. Our high level of sus-
Table 2: Cross resistances of P.aeruginosa isolates.
Isolates
resistant
number of isolates resistant to
Ton
IPMP PM TZCFS PTcClTM AK
IP
MP
PM
TZ
CFS
PTc
Cl
TM
AK
35
36
28
22
32
36
20
51
20
34 (97)17 (49)
18 (50)
11 (31)
12 (33)
19 (68)
22 (63)
23 (64)
18 (64)
13 (59)
25 (71)
27 (75)
19 (68)
15 (68)
22 (69)
8 (23)
9 (25)
13 (46)
11 (50)
9 (28)
7 (19)
30 (86)
32 (89)
23 (82)
16 (73)
30 (94)
29 (81)
17 (85)
9 (26)
10 (28)
13 (46)
8 (36)
11 (34)
13 (36)
7 (35)
18 (35)
34 (94)
17 (61)
11 (50)
22 (69)
25 (69)
8 (40)
30 (59)
9 (45)
18 (64)
12 (55)
23 (72)
27 (75)
9 (45)
32 (63)
10 (50)
19 (86)
18 (56)
19 (53)
13 (65)
23 (45)
13 (65)
13 (41)
15 (42)
11 (55)
16 (31)
8 (40)
22 (61)
9 (45)
30 (59)
11 (55)
7 (35)
29 (57)
13 (65)
17 (33)
7 (35)18 (90)
IP: imipenem, MP: meropenem, PM: cefepime, TZ: ceftazidime, CFS: cefoperazone/sulbactam, PTc: piperacillin/tazobactam, Cl: ciprofloxacin, TM:
tobramycin, AK: amikacin. *The numbers in parentheses denote percent of isolates

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ceptibility to ceftazidime and the low level of
susceptibility to cefepime may reflect the increased use of
cefepime and the decreased use of ceftazidime in recent
years in our unit.
Conclusions
Resistance of P. aeruginosa to ?-lactams seems common in
our centre. About two-thirds of isolates were resistant to
one or more antibiotics tested, with ciprofloxacin show-
ing the highest in vitro antibacterial activity. Our findings
suggest that ciprofloxacin may be of significant value for
the treatment of severe infections caused by P. aeruginosa
and may be more useful than ?-lactams for combined
treatment with amikacin. However, in order to confirm
this suggestion antibiotic combinations should be tested
in vitro and in vivo. In addition, these isolates from only
one centre may be predominantly clonal, so isolates from
different centres should be tested.
Authors' contributions
S Gençer planned and carried out the design of the study,
the laboratory studies and coordinated all procedures; Ö
Ak, N Benzonana, A Bat rel participated in the laboratory
studies; S Özer participated in the design and coordina-
tion of the study.
Acknowledgements
The authors wish to thank Merck, Sharp & Dohme, Astra-Zeneca, Bristol-
Myers-Squibb, Pfizer and Wyeth for their provision of E test strips.
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