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Increase of imipenem resistance among Pseudomonas aeruginosa isolates from a Polish paediatric hospital (1993–2002)
International Journal of Antimicrobial Agents 29 (2007) 153–158
Increase of imipenem resistance among Pseudomonas aeruginosa isolates from a Polish paediatric hospital (1993–2002) Jan A. Patzer ∗ , Danuta Dzier˙zanowska Department of Clinical Microbiology and Immunology, The Children’s Memorial Health Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland Received 31 January 2006; accepted 25 August 2006
Abstract Analysis of the in vitro activity of imipenem and 13 other antibiotics against 2485 Pseudomonas aeruginosa isolates obtained from clinical specimens from children hospitalized during 1993–2002 was performed. In 2002, the percentage of P. aeruginosa isolates susceptible to all tested antibiotics, with the exception of imipenem, increased or remained on nearly the same level as in 1993. An increase of resistance to imipenem from 4.3% to 18.3% was observed. The MIC90 value of imipenem increased from 2 mg/L to 16 mg/L. Simultaneously, a four-fold increase of the usage of carbapenems imipenem and meropenem in the hospital was noted. In 2000–2001, a high incidence of imipenemresistant strains was observed. The imipenem-resistant P. aeruginosa strains of serotype O6 from the general surgery ward and serotype O11 from the intensive care unit were shown to be clonally related by the pulsed-field gel electrophoresis method. © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Pseudomonas aeruginosa; Susceptibility; MIC; Imipenem; Serotypes; PFGE
1. Introduction Pseudomonas aeruginosa is an opportunistic human pathogen and a leading aetiological factor of nosocomial infections particularly in immuno-compromised hosts [1,2]. Included in this broad risk category are neonates and children who previously received antibiotics and those with primary or secondary immunodeficiencies. P. aeruginosa is characterized by inherent resistance to a wide variety of antibiotics. Its resistance to anti-pseudomonal beta-lactams, advanced generation cephalosporins, monobactams and carbapenems is also an increasing clinical problem [3]. Carbapenems, mainly imipenem and meropenem, are potent agents for the treatment of infections due to multi-drug-resistant P. aeruginosa. However, the prevalence of carbapenem-resistant P. aeruginosa has recently been increasing. Mechanisms of low-level resistance to carbapenems (MIC 8–32 mg/L) in P. aeruginosa are associated with reduced uptake as a result of loss of the OprD porin [4] combined with de-repression ∗
Corresponding author. Tel.: +48 22 815 72 71; fax: +48 22 815 72 75. E-mail address: [email protected] (J.A. Patzer).
of the chromosomal ampC beta-lactamase gene [5] or by over-expression of an efflux pump system [6,7,8]. High-level resistance to carbapenems (MIC > 32 mg/L) is still uncommon in P. aeruginosa, but can be caused by the presence of carbapenemases [9]. These beta-lactamases are characterized as enzymes which hydrolyse imipemem and/or meropenem and other penicillins or cephalosporins [10]. The aim of this 10-year study (1993–2002) was to analyse the in vitro activity of imipenem, meropenem and 12 other antibiotics against P. aeruginosa isolates obtained from hospitalized children.
2. Materials and methods 2.1. Bacterial strains The 2485 P. aeruginosa isolates used in this study were recovered from a variety of clinical specimens obtained from children hospitalized in the Children’s Memorial Health Institute (Warsaw, Poland) between 1993 and 2002. All the children admitted to the hospital during this time-frame were included in the study. The isolates were identified by stan-
0924-8579/$ – see front matter © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2006.08.044
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dard laboratory methods. Multiple isolates from individual patients were excluded if they belonged to the same serotype and had the same antimicrobial resistance pattern. 2.2. Serotyping The O serotype of the isolates was determined by slide agglutination [11] using a commercially available antiserum (Bio-Rad, Marnes la Coquette, France). 2.3. Antimicrobial susceptibility testing MICs of imipenem, meropenem, amikacin, gentamicin, netilmicin, tobramycin, carbenicillin, piperacillin + tazobactam, ceftazidime, cefoperazone, cefepime, aztreonam and ciprofloxacin were determined by the agar dilution method on Mueller–Hinton II agar (BD Microbiology Systems, Cockeysville, MD, USA), as recommended by the National Committee for Clinical Laboratory Standards [12,13].
buffer (10 mM Tris–HCl pH 7.2, 50 mM NaCl, 0.2% sodium deoxycholate, 0.5% N-lauroylsarcosine) with lysozyme 25 mg/mL and incubated for 1 h at 37 ◦ C. The plugs were washed with TE buffer (10 mM Tris–HCl, 50 mM EDTA pH 8.0; Bio-Rad), transferred to 0.5 mL proteolysis buffer (100 mM EDTA pH 8.0, 0.2% sodium deoxycholate, 1% lauroylsarcosine; Bio-Rad) supplemented with 50 g proteinase K (Boehringer Mannheim) and incubated for 18 h at 50 ◦ C. The plugs were washed five times for 30–40 min with TE buffer. Chromosomal DNA was restricted with 40 units/mL of SpeI (Bio-Rad) for 18 h at 37 ◦ C. The obtained DNA fragments were separated in a CHEF-DRII apparatus (Bio-Rad). A 1% agarose gel (pulsed field certified agarose, Bio-Rad) was prepared and held in TBE buffer (45 mM Tris–base, 45 mM boric acid, 1 mM EDTA, pH 8.0; Bio-Rad). The electrophoresis was carried out at 6 V/cm at 12 ◦ C for 20 h with pulses ranging from 5 to 45 s. The DNA size marker was a lambda ladder (Bio-Rad). Ethidium bromide-stained gels were examined visually. Isolates showing a difference of less than three bands were considered identical/clonally related [14].
2.4. Pulsed-field gel electrophoresis 2.5. Antibiotic consumption Genotyping was done using pulsed-field gel electrophoresis (PFGE). Imipenem-resistant P. aeruginosa strains were grown overnight on Mueller–Hinton II agar. Bacterial cell suspension was mixed with an equal volume of 2% lowmelting point agarose (Bio-Rad) and dispensed into plug moulds. The resulting plugs were transferred to 0.5 mL lysis
Total antibiotic consumption in the hospital was recorded in the hospital pharmacy on a monthly basis using a computer-based pharmacy documentation system. Gram amounts of antibiotics were converted to ‘defined daily doses’, which were as follows: imipenem, 1 g; meropenem,
Table 1 Antibiotic susceptibility of Pseudomonas aeruginosa clinical isolates collected from children hospitalized in 1993–2002 Year
No. of isolates
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
210 216 258 231 269 232 281 259 277 252
Year
No. of isolates
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
210 216 258 231 269 232 281 259 277 252
N.d., no data.
Percent of susceptible isolates/MIC (mg/L) Amikacin
Gentamicin
Netilmicin
Tobramycin
Azlocillin
Carbenicillin
Piperacillin
Piperacillin/Tazobactam
96.2/8.0 95.4/8.0 93.8/16 95.2/16 92.9/16 92.7/16 93.2/16 91.1/16 93.1/16 97.6/8.0
71.4/128 73.6/128 67.1/64 72.7/32 76.6/32 76.3/32 78.6/128 73.7/128 75.8/128 89.7/8.0
79.5/>128 83.3/128 82.2/128 N.d. 86.8/32 83.2/32 86.1/32 84.9/>128 88.1/64 98.0/8.0
74.3/32 75.9/16 74.4/32 82.2/32 80.3/32 81.5/64 81.5/64 79.5/64 83.4/64 93.2/2
81.4/>128 85.2/>128 78.3/>128 84.0/>128 N.d. 78.9/>128 85.0/128 80.7/>128 88.1/128 88.9/128
76.2/>128 85.2/>128 81.0/>128 85.4/>128 N.d. 89.2/>128 87.5/>128 79.9/>128 88.1/>128 91.7/128
80.9/>128 87.0/>128 81.8/>128 82.7/>128 83.2/>128 81.9/>128 N.d. N.d. N.d. N.d.
N.d. N.d. N.d. N.d. N.d. N.d. 86.8/128 88.4/128 91.7/64 91.3/64
Percent of susceptible isolates/MIC (mg/L) Cefoperazone
Ceftazidime
Cefepime
Aztreonam
Ciprofloxacin
Imipenem
Meropenem
78.6/128 85.2/128 75.6/64 77.5/64 74.3/64 76.3/64 81.8/64 79.2/128 86.6/64 87.3/64
98.1/4.0 95.8/4.0 88.4/16 85.7/16 85.1/16 76.7/32 85.4/16 85.3/16 91.3/8.0 89.3/16
N.d. 92.6/8.0 85.3/16 N.d. N.d. N.d. 79.7/16 83.4/16 89.5/16 92.9/8.0
51.9/32 63.0/16 67.1/32 N.d. N.d. 70.3/32 81.5/16 81.1/16 85.6/16 86.1/16
98.1/0.5 99.1/0.5 96.1/0.5 99.1/0.5 98.1/0.5 N.d. 95.0/0.5 93.8/0.5 97.8/0.5 95.6/0.5
95.7/2.0 97.7/4.0 93.4/4.0 91.8/4.0 90.0/4.0 91.8/4.0 83.6/16 74.1/16 73.6/16 81.7/16
N.d. N.d. N.d. N.d. N.d. N.d. 93.2/4.0 84.9/8.0 87.7/8.0 90.1/4.0
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1 g; ciprofloxacin, 1 g; amikacin, 1 g; gentamicin, 0.24 g; tobramycin, 0.24 g; netilmicin, 0.35 g; aztreonam, 4 g; cefepime, 4 g; ceftazidime, 6 g; azlocillin, 12 g; carbenicillin, 12 g; piperacillin, 14 g; piperacillin/tazobactam, 14 g.
3. Results 3.1. Antibiotic susceptibility The results of antimicrobial susceptibility testing of 2485 P. aeruginosa isolates are shown in Table 1. Among the aminoglycosides, amikacin was characterized by the highest percentage of susceptible strains for the whole 10-year period. The average susceptibility to amikacin was equal to 94%. Netilmicin, tobramycin and gentamicin were active against 85.9, 80.8 and 75.7% of isolates, respectively. From 1993 to 2002 the percentage of susceptible P. aeruginosa isolates for all tested aminoglycosides increased. In this timeperiod the MIC90 values for aminoglycosides varied, but in 2002 they had the lowest values. The percentage of isolates susceptible to ciprofloxacin remained on a similar level (93.8% in 2000 compared with 99.1% in 1994), and the MIC90 was equal to 0.5 mg/L for the whole period studied. This preferential and stable activity of ciprofloxacin against P. aeruginosa is a consequence of the low levels of fluoroquinolones given to hospitalized children. The percentage of P. aeruginosa isolates susceptible to azlocillin increased from 78.3% in 1995 to 88.9% in 2002, and those susceptible to carbenicillin increased from 76.2% in 1993 to 91.7% in 2002. In the period 1993–1998 the percentage of isolates susceptible to piperacillin varied from 80.9% in 1993 to 87% in 1994. After this time piperacillin–tazobactam was introduced, and the susceptibility to this agent increased from 86.8% in 1999 to 91.3% in 2002. Among the tested cephalosporins, in the period 1993–2002, the highest percentage of susceptible P. aeruginosa isolates were found for ceftazidime (87.9%) and cefepime (87%). The percentage of isolates susceptible to ceftazidime varied from 98.1% in 1993 to 89.3% in 2002 and to cefepime from 79.7% in 1999 to 92.9% in 2002. The average susceptibility to cefoperazone was equal to 80.3%, and increased from 74.3% in 1997 to 87.3% in 2002. The monobactam aztreonam was active against 74.3% of iso-
Fig. 1. Comparison of the imipenem MIC values for the P. aeruginosa population isolated from hospitalized children in 1993 and 2002.
lates and the percentage of isolates susceptible to this agent increased from 51.9% (1993) to 86.1% (2002). In the same 10-year period the percentage of P. aeruginosa isolates susceptible to imipenem, in contrast to all the above mentioned antibiotics, showed a decrease from 95.7% to 81.7%. The percentage of isolates susceptible to meropenem, in 1999–2002, varied between 84.9% and 93.2%. The activity of meropenem was higher than that of imipenem: the MIC90 value for imipenem was equal to 16 mg/L and for meropenem it was 4–8 mg/L. During 10 years of study an increasing resistance of P. aeruginosa to imipenem was noted. The imipenem MIC values for the P. aeruginosa population in 2002 were higher than those in 1993 (Fig. 1). This could possibly be connected with the high usage of imipenem in our hospital. 3.2. Antibiotic consumption in hospital The usage of some antibiotics in our hospital in 1993 to 2002, calculated as defined daily doses, is shown in Table 2. From 1993 to 2001 the usage of amikacin decreased, whereas that of netilmicin increased two-fold. In 2002, amikacin was more frequently used because the supply of netilmicin was stopped. The usage of ceftazidime showed a small increase from 1101 to 1569 defined daily doses. Small quantities of piperacillin with tazobactam were used in therapy. In contrast to the above a four-fold increase in the usage of two carbapenems, imipenem and meropenem, was noted in our hospital. In the first period of our study imipenem only was
Table 2 Antibiotic consumption in hospital in the period 1993–2001 (in DDD) Antibiotic
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
Amikacin Netilmicin Ceftazidime Piperacillin/tazobactam Imipenem Meropenem
1777 2031 1101 0 981 0
1300 1365 691 0 938 0
986 1973 602 0 1120 0
N.d. N.d. N.d. N.d N.d. N.d.
1208 2521 926 273 2209 155
1101 3218 1241 188 1737 512
1377 3444 1411 414 2061 890
894 3389 1296 245 2365 1000
859 4855 1569 334 2473 1311
2508 1173 1567 455 3225 1040
DDD, defined daily dosage; N.d., no data.
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Table 3 Distribution of imipenem-resistant Pseudomonas aeruginosa isolates among the most frequently encountered serotypes Year
No. (%) of imipenem-resistant isolates in particular serotypes
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
O1
O3
O6
O11
O12
Other
Total
1 (11) 1 (20) 1 (5.9) 1 (5.3) 5 (18.5) 2 (10.5) 2 (4.3) 6 (8.9) 1 (1.4) 2 (4.3)
0 1 (20) 1 (5.9) 0 1 (3.7) 3 (15.8) 5 (10.9) 1 (1.5) 1 (1.4) 1 (2.2)
0 2 (40) 4 (23.5) 8 (42) 5 (18.5) 6 (31.6) 18 (39) 26 (38.8) 33 (45.2) 25 (54.3)
0 0 4 (23.5) 5 (26.3) 8 (29.6) 3 (15.8) 8 (17.4) 14 (20.9) 24 (32.9) 11 (23.9)
3 (33.3) 0 3 (17.6) 1 (5.3) 4 (14.8) 4 (21) 5 (10.9) 2 (3.0) 2 (2.7) 4 (8.7)
5 (55.5) 1 (20) 4 (23.5) 4 (21) 4 (14.8) 1 (5.3) 8 (17.4) 18 (26.9) 12 (16.4) 3 (6.5)
9 (100) 5 (100) 17 (100) 19 (100) 27 (100) 19 (100) 46 (100) 67 (100) 73 (100) 46 (100)
used, but from 1997 meropenem was introduced to antibiotic therapy in our hospital. 3.3. Distribution of imipenem-resistant P. aeruginosa strains among different serotypes During the period 1993–2002 the most frequently encountered serotypes among P. aeruginosa isolates were O6 (30.8%), O11 (16.1%), O1 (13.4%), O3 (8.5%) and O12 (6.5%). From 1993 the number of imipenem-resistant P. aeruginosa strains increased, and a high incidence of imipenem-resistant strains was observed in 2000–2001. Table 3 shows the distribution of imipenem-resistant P. aeruginosa strains among the most frequently encountered serotypes. The highest number of imipenem-resistant strains was found to be in the isolates of serotypes O6 and O11. 3.4. Distribution of imipenem-resistant P. aeruginosa strains among hospital wards The highest number of P. aeruginosa isolates was collected from children hospitalized in the intensive care unit, general surgery and urology wards. The analysis of imipenem-resistant strains from the intensive care unit isolated in 1993–2002 showed different resistance patterns (Table 4). The most frequently encountered were P. aerug-
inosa strains characterized by imipenem-resistance and susceptibility to other antibiotics. The highest number of strains in this group was of serotype O11 and they were characterized by resistance to imipenem (MIC90 = 16 mg/L) and intermediate resistance to meropenem (MIC90 = 8 mg/L). Imipenem-resistant P. aeruginosa strains of serotype O11 isolated in 2000 and 2001 were clonally related, as shown by the PFGE results (Fig. 2). In 2001, the imipenem-resistant P. aeruginosa strains consisted of 35% of all the isolates from the general surgery ward. The analysis of resistance patterns of imipenem-resistant isolates showed that the most frequently encountered strains were resistant to imipenem only (Table 5). They were all of serotype O6, characterized by the MIC values 16 mg/L for imipenem and 4 mg/L for meropenem. Genotyping of the imipenem-resistant P. aeruginosa strains of serotype O6 by the PFGE method showed that strains isolated in 2001 and 2002 were clonally related and had nearly identical restriction patterns, whereas strains from 2000 had different restriction patterns (Fig. 3). 4. Discussion The hospital’s antibiotic-giving policy plays a key role in the increase and spread of antibiotic resistant strains. Some
Table 4 Antibiotic resistance of Pseudomonas aeruginosa isolates obtained from children hospitalized in the intensive care unit in 1993–2002 Year
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Total
No. of isolates
24 35 37 31 31 21 30 32 50 42 333
No. (%) antibiotic resistant isolates A
B
A+B
IPM
A + IPM
B + IPM
A + B + IPM
Total
1 (4.2) 6 (17.1) 2 (5.4) 1 (3.2) 2 (6.4) 2 (9.5) 2 (6.7) 3 (9.4) 5 (10) 1 (2.4) 25 (7.5)
0 1 (2.9) 7 (18.9) 1 (3.2) 1 (3.2) 1 (4.8) 2 (6.7) 0 1 (2) 6 (14.3) 20 (6)
2 (8.3) 5 (14.3) 10 (27.0) 2 (6.4) 4 (12.9) 2 (9.5) 3 (10) 2 (6.2) 0 2 (4.8) 32 (9.6)
1 (4.2) 1 (2.9) 2 (5.4) 4 (12.9) 1 (3.2) 4 (19) 4 (13.3) 8 (25) 18 (36) 4 (9.8) 47 (14.1)
0 0 1 (2.7) 2 (6.4) 2 (6.4) 0 1 (3.3) 2 (6.2) 2 (4) 0 10 (3)
1 (4.2) 0 0 0 4 (12.9) 3 (14.3) 3 (10) 3 (9.4) 1 (2.0) 4 (9.8) 19 (5.7)
2 (8.3) 0 1 (2.7) 1 (3.2) 1 (3.2) 3 (14.3) 3 (10) 2 (6.2) 1 (2) 3 (7.1) 17 (5.1)
7 (29.2) 13 (37.1) 23 (62.2) 11 (35.5) 15 (48.4) 15 (71.4) 18 (60.0) 20 (62.5) 28 (56.0) 20 (47.6) 170 (51.1)
A, aminoglycosides; B, beta-lactams; IPM, imipenem.
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Table 5 Antibiotic resistance of Pseudomonas aeruginosa isolates obtained from children hospitalized in the general surgery ward in 1993–2002 Year
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Total
No. of isolates
17 21 22 22 28 31 33 34 40 40 288
No. (%) antibiotic resistant isolates A
B
A+B
IPM
A + IPM
B + IPM
A + B + IPM
Total
1 (5.9) 5 (23.8) 2 (9.1) 3 (13.6) 2 (7.1) 3 (9.7) 1 (3.0) 0 0 1 (2.5) 18 (6.3)
1 (5.9) 1 (4.8) 1 (4.5) 0 1 (3.6) 4 (12.9) 2 (6.1) 2 (5.9) 2 (5.0) 1 (2.5) 15 (5.2)
0 0 1 (4.5) 3 (13.6) 4 (14.3) 1 (3.2) 4 (12.1) 0 0 0 13 (4.5)
0 0 0 1 (4.5) 1 (3.6) 1 (3.2) 2 (6.1) 6 (17.6) 14 (35.0) 10 (25.0) 35 (12.1)
0 0 1 (4.5) 0 2 (7.1) 0 2 (6.1) 3 (8.8) 1 (2.5) 1 (2.5) 10 (3.5)
0 0 0 0 3 (10.7) 1 (3.2) 1 (3.0) 1 (2.9) 0 0 6 (2.1)
1 (5.9) 0 2 (9.1) 1 (4.5) 0 1 (3.2) 3 (9.1) 8 (23.5) 4 (10.0) 3 (7.5) 23 (8.0)
3 (17.6) 6 (28.6) 7 (31.8) 8 (36.4) 13 (46.4) 11 (35.5) 15 (45.5) 20 (58.8) 21 (52.5) 16 (40.0) 120 (41.7)
A, aminoglycosides; B, beta-lactams; IPM, imipenem.
Fig. 2. PFGE typing of the imipenem-resistant P. aeruginosa strains isolated from children hospitalized in the intensive care unit. Lines: , marker; 1–13, strains from 2001 (serotype O11); 14–16, strains from 2000 (serotype O11); 17–19, strains from 2001 (serotype O6).
Fig. 3. PFGE typing of the imipenem-resistant P. aeruginosa strains isolated from children hospitalized in the general surgery ward. Lines: , marker; 1–2, strains (serotype O6) from 2000; 3–14, strains (serotype O6) from 2001; 15–18, strains (serotype O6) from 2002.
antimicrobials, such as imipenem, are more susceptible to the development of resistance during treatment, leading to failure in therapy [15]. Imipenem therapy in patients with P. aeruginosa infections caused an increase in imipenem resistance as well as resistance to piperacillin with tazobactam and ceftazidime [16]. The results of a 3-year study (1997–2000) on the usage of beta-lactams and P. aeruginosa resistance indicated that periods of intensive imipenem consumption correlated with periods of increased P. aeruginosa resistance to imipenem, ceftazidime and piperacillin with tazobactam [17]. The epidemiological situation of our hospital cannot be compared with other paediatric hospitals in Poland. Our specificity is connected with the treatment of children from the whole country with serious diseases, often with defects of the immunology system, and often colonized or infected with hospital bacterial strains as a result of earlier hospitalization in other hospitals. Furthermore these patients had been treated many times with a broad spectrum of different antimicrobials, which results in the only therapeutic option for our hospital to be the use of carbapenems. The susceptibility of Gram-negative bacterial strains isolated from clinical specimens of children hospitalized in the intensive care unit of our hospital from 1997 to 2000 was studied [18]. Extended spectrum beta-lactamases and AmpC beta-lactamase producers among Enterobacteriaceae isolates from this intensive care unit continued to be a serious therapeutic problem, although the carbapenems were highly active against these phenotypes. This epidemiological situation was the cause of a high consumption of carbapenems, both imipenem and meropenem, in our hospital. In fact, there was a four-fold increase in the period 1993–2002. In 2003, the usage of carbapenems increased further and was five-fold higher in comparison to 1993. Therefore, P. aeruginosa strains resistant to imipenem and susceptible to other antibiotics appeared in our hospital as a direct consequence of this high usage of carbapenems. The percentage of imipenem-resistant P. aeruginosa strains increased from 4.3% in 1993 to 26.4% in 2001. Results obtained in the SENTRY program for the period 1997–1999 indicated a decrease in carbapenem susceptibility of P. aerug-
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inosa isolates from three regions: in Canada for imipenem, in South America for meropenem and in Europe for both drugs [3]. Imipenem had probably been a selective agent, because in the majority of countries meropenem was just introduced to therapy or its usage was limited at this time. The analysis of data from the time-period 1992–2000 obtained in different programs of antibiotic susceptibility studies showed that resistance of P. aeruginosa to imipenem in Europe varied between 21 and 31%, but in Greece reached 64% [19]. On a molecular level the imipenem resistance of the majority of these selected P. aeruginosa strains was connected with mutation in the oprD gene. In our studies, the analysis of P. aeruginosa strains of the most frequently encountered serotypes showed that the number of strains resistant to imipenem and simultaneously susceptible to other antibiotics reached a peak in our hospital in 2000–2001. Meropenem was characterized by higher activity than imipenem and about 10% of imipenem-resistant strains were susceptible to meropenem. The clonally related, imipenem-resistant P. aeruginosa strains of serotype O11, from the intensive care unit, showed only intermediate resistance to meropenem. The clonally related imipenem-resistant P. aeruginosa strains of serotype O6, from the general surgery ward, were meropenem-susceptible. The mechanism of imipenem-resistance in some of the P. aeruginosa strains was connected with the efflux system (unpublished data). Furthermore, among the imipenem-resistant P. aeruginosa isolates were found 11 multi-resistant strains which produced metallo-beta-lactamase VIM-4 [20]. The first strain appeared in 1998 and P. aeruginosa strains harbouring this metallobeta-lactamase have become endemic in our hospital since then.
Acknowledgment The excellent technical assistance of Teresa Guzik and Małgorzata Walus is gratefully appreciated.
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Increase of imipenem resistance among Pseudomonas aeruginosa isolates from a Polish paediatric hospital (1993–2002) Jan A. Patzer ∗ , Danuta Dzier˙zanowska Department of Clinical Microbiology and Immunology, The Children’s Memorial Health Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland Received 31 January 2006; accepted 25 August 2006
Abstract Analysis of the in vitro activity of imipenem and 13 other antibiotics against 2485 Pseudomonas aeruginosa isolates obtained from clinical specimens from children hospitalized during 1993–2002 was performed. In 2002, the percentage of P. aeruginosa isolates susceptible to all tested antibiotics, with the exception of imipenem, increased or remained on nearly the same level as in 1993. An increase of resistance to imipenem from 4.3% to 18.3% was observed. The MIC90 value of imipenem increased from 2 mg/L to 16 mg/L. Simultaneously, a four-fold increase of the usage of carbapenems imipenem and meropenem in the hospital was noted. In 2000–2001, a high incidence of imipenemresistant strains was observed. The imipenem-resistant P. aeruginosa strains of serotype O6 from the general surgery ward and serotype O11 from the intensive care unit were shown to be clonally related by the pulsed-field gel electrophoresis method. © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Pseudomonas aeruginosa; Susceptibility; MIC; Imipenem; Serotypes; PFGE
1. Introduction Pseudomonas aeruginosa is an opportunistic human pathogen and a leading aetiological factor of nosocomial infections particularly in immuno-compromised hosts [1,2]. Included in this broad risk category are neonates and children who previously received antibiotics and those with primary or secondary immunodeficiencies. P. aeruginosa is characterized by inherent resistance to a wide variety of antibiotics. Its resistance to anti-pseudomonal beta-lactams, advanced generation cephalosporins, monobactams and carbapenems is also an increasing clinical problem [3]. Carbapenems, mainly imipenem and meropenem, are potent agents for the treatment of infections due to multi-drug-resistant P. aeruginosa. However, the prevalence of carbapenem-resistant P. aeruginosa has recently been increasing. Mechanisms of low-level resistance to carbapenems (MIC 8–32 mg/L) in P. aeruginosa are associated with reduced uptake as a result of loss of the OprD porin [4] combined with de-repression ∗
Corresponding author. Tel.: +48 22 815 72 71; fax: +48 22 815 72 75. E-mail address: [email protected] (J.A. Patzer).
of the chromosomal ampC beta-lactamase gene [5] or by over-expression of an efflux pump system [6,7,8]. High-level resistance to carbapenems (MIC > 32 mg/L) is still uncommon in P. aeruginosa, but can be caused by the presence of carbapenemases [9]. These beta-lactamases are characterized as enzymes which hydrolyse imipemem and/or meropenem and other penicillins or cephalosporins [10]. The aim of this 10-year study (1993–2002) was to analyse the in vitro activity of imipenem, meropenem and 12 other antibiotics against P. aeruginosa isolates obtained from hospitalized children.
2. Materials and methods 2.1. Bacterial strains The 2485 P. aeruginosa isolates used in this study were recovered from a variety of clinical specimens obtained from children hospitalized in the Children’s Memorial Health Institute (Warsaw, Poland) between 1993 and 2002. All the children admitted to the hospital during this time-frame were included in the study. The isolates were identified by stan-
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dard laboratory methods. Multiple isolates from individual patients were excluded if they belonged to the same serotype and had the same antimicrobial resistance pattern. 2.2. Serotyping The O serotype of the isolates was determined by slide agglutination [11] using a commercially available antiserum (Bio-Rad, Marnes la Coquette, France). 2.3. Antimicrobial susceptibility testing MICs of imipenem, meropenem, amikacin, gentamicin, netilmicin, tobramycin, carbenicillin, piperacillin + tazobactam, ceftazidime, cefoperazone, cefepime, aztreonam and ciprofloxacin were determined by the agar dilution method on Mueller–Hinton II agar (BD Microbiology Systems, Cockeysville, MD, USA), as recommended by the National Committee for Clinical Laboratory Standards [12,13].
buffer (10 mM Tris–HCl pH 7.2, 50 mM NaCl, 0.2% sodium deoxycholate, 0.5% N-lauroylsarcosine) with lysozyme 25 mg/mL and incubated for 1 h at 37 ◦ C. The plugs were washed with TE buffer (10 mM Tris–HCl, 50 mM EDTA pH 8.0; Bio-Rad), transferred to 0.5 mL proteolysis buffer (100 mM EDTA pH 8.0, 0.2% sodium deoxycholate, 1% lauroylsarcosine; Bio-Rad) supplemented with 50 g proteinase K (Boehringer Mannheim) and incubated for 18 h at 50 ◦ C. The plugs were washed five times for 30–40 min with TE buffer. Chromosomal DNA was restricted with 40 units/mL of SpeI (Bio-Rad) for 18 h at 37 ◦ C. The obtained DNA fragments were separated in a CHEF-DRII apparatus (Bio-Rad). A 1% agarose gel (pulsed field certified agarose, Bio-Rad) was prepared and held in TBE buffer (45 mM Tris–base, 45 mM boric acid, 1 mM EDTA, pH 8.0; Bio-Rad). The electrophoresis was carried out at 6 V/cm at 12 ◦ C for 20 h with pulses ranging from 5 to 45 s. The DNA size marker was a lambda ladder (Bio-Rad). Ethidium bromide-stained gels were examined visually. Isolates showing a difference of less than three bands were considered identical/clonally related [14].
2.4. Pulsed-field gel electrophoresis 2.5. Antibiotic consumption Genotyping was done using pulsed-field gel electrophoresis (PFGE). Imipenem-resistant P. aeruginosa strains were grown overnight on Mueller–Hinton II agar. Bacterial cell suspension was mixed with an equal volume of 2% lowmelting point agarose (Bio-Rad) and dispensed into plug moulds. The resulting plugs were transferred to 0.5 mL lysis
Total antibiotic consumption in the hospital was recorded in the hospital pharmacy on a monthly basis using a computer-based pharmacy documentation system. Gram amounts of antibiotics were converted to ‘defined daily doses’, which were as follows: imipenem, 1 g; meropenem,
Table 1 Antibiotic susceptibility of Pseudomonas aeruginosa clinical isolates collected from children hospitalized in 1993–2002 Year
No. of isolates
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
210 216 258 231 269 232 281 259 277 252
Year
No. of isolates
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
210 216 258 231 269 232 281 259 277 252
N.d., no data.
Percent of susceptible isolates/MIC (mg/L) Amikacin
Gentamicin
Netilmicin
Tobramycin
Azlocillin
Carbenicillin
Piperacillin
Piperacillin/Tazobactam
96.2/8.0 95.4/8.0 93.8/16 95.2/16 92.9/16 92.7/16 93.2/16 91.1/16 93.1/16 97.6/8.0
71.4/128 73.6/128 67.1/64 72.7/32 76.6/32 76.3/32 78.6/128 73.7/128 75.8/128 89.7/8.0
79.5/>128 83.3/128 82.2/128 N.d. 86.8/32 83.2/32 86.1/32 84.9/>128 88.1/64 98.0/8.0
74.3/32 75.9/16 74.4/32 82.2/32 80.3/32 81.5/64 81.5/64 79.5/64 83.4/64 93.2/2
81.4/>128 85.2/>128 78.3/>128 84.0/>128 N.d. 78.9/>128 85.0/128 80.7/>128 88.1/128 88.9/128
76.2/>128 85.2/>128 81.0/>128 85.4/>128 N.d. 89.2/>128 87.5/>128 79.9/>128 88.1/>128 91.7/128
80.9/>128 87.0/>128 81.8/>128 82.7/>128 83.2/>128 81.9/>128 N.d. N.d. N.d. N.d.
N.d. N.d. N.d. N.d. N.d. N.d. 86.8/128 88.4/128 91.7/64 91.3/64
Percent of susceptible isolates/MIC (mg/L) Cefoperazone
Ceftazidime
Cefepime
Aztreonam
Ciprofloxacin
Imipenem
Meropenem
78.6/128 85.2/128 75.6/64 77.5/64 74.3/64 76.3/64 81.8/64 79.2/128 86.6/64 87.3/64
98.1/4.0 95.8/4.0 88.4/16 85.7/16 85.1/16 76.7/32 85.4/16 85.3/16 91.3/8.0 89.3/16
N.d. 92.6/8.0 85.3/16 N.d. N.d. N.d. 79.7/16 83.4/16 89.5/16 92.9/8.0
51.9/32 63.0/16 67.1/32 N.d. N.d. 70.3/32 81.5/16 81.1/16 85.6/16 86.1/16
98.1/0.5 99.1/0.5 96.1/0.5 99.1/0.5 98.1/0.5 N.d. 95.0/0.5 93.8/0.5 97.8/0.5 95.6/0.5
95.7/2.0 97.7/4.0 93.4/4.0 91.8/4.0 90.0/4.0 91.8/4.0 83.6/16 74.1/16 73.6/16 81.7/16
N.d. N.d. N.d. N.d. N.d. N.d. 93.2/4.0 84.9/8.0 87.7/8.0 90.1/4.0
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1 g; ciprofloxacin, 1 g; amikacin, 1 g; gentamicin, 0.24 g; tobramycin, 0.24 g; netilmicin, 0.35 g; aztreonam, 4 g; cefepime, 4 g; ceftazidime, 6 g; azlocillin, 12 g; carbenicillin, 12 g; piperacillin, 14 g; piperacillin/tazobactam, 14 g.
3. Results 3.1. Antibiotic susceptibility The results of antimicrobial susceptibility testing of 2485 P. aeruginosa isolates are shown in Table 1. Among the aminoglycosides, amikacin was characterized by the highest percentage of susceptible strains for the whole 10-year period. The average susceptibility to amikacin was equal to 94%. Netilmicin, tobramycin and gentamicin were active against 85.9, 80.8 and 75.7% of isolates, respectively. From 1993 to 2002 the percentage of susceptible P. aeruginosa isolates for all tested aminoglycosides increased. In this timeperiod the MIC90 values for aminoglycosides varied, but in 2002 they had the lowest values. The percentage of isolates susceptible to ciprofloxacin remained on a similar level (93.8% in 2000 compared with 99.1% in 1994), and the MIC90 was equal to 0.5 mg/L for the whole period studied. This preferential and stable activity of ciprofloxacin against P. aeruginosa is a consequence of the low levels of fluoroquinolones given to hospitalized children. The percentage of P. aeruginosa isolates susceptible to azlocillin increased from 78.3% in 1995 to 88.9% in 2002, and those susceptible to carbenicillin increased from 76.2% in 1993 to 91.7% in 2002. In the period 1993–1998 the percentage of isolates susceptible to piperacillin varied from 80.9% in 1993 to 87% in 1994. After this time piperacillin–tazobactam was introduced, and the susceptibility to this agent increased from 86.8% in 1999 to 91.3% in 2002. Among the tested cephalosporins, in the period 1993–2002, the highest percentage of susceptible P. aeruginosa isolates were found for ceftazidime (87.9%) and cefepime (87%). The percentage of isolates susceptible to ceftazidime varied from 98.1% in 1993 to 89.3% in 2002 and to cefepime from 79.7% in 1999 to 92.9% in 2002. The average susceptibility to cefoperazone was equal to 80.3%, and increased from 74.3% in 1997 to 87.3% in 2002. The monobactam aztreonam was active against 74.3% of iso-
Fig. 1. Comparison of the imipenem MIC values for the P. aeruginosa population isolated from hospitalized children in 1993 and 2002.
lates and the percentage of isolates susceptible to this agent increased from 51.9% (1993) to 86.1% (2002). In the same 10-year period the percentage of P. aeruginosa isolates susceptible to imipenem, in contrast to all the above mentioned antibiotics, showed a decrease from 95.7% to 81.7%. The percentage of isolates susceptible to meropenem, in 1999–2002, varied between 84.9% and 93.2%. The activity of meropenem was higher than that of imipenem: the MIC90 value for imipenem was equal to 16 mg/L and for meropenem it was 4–8 mg/L. During 10 years of study an increasing resistance of P. aeruginosa to imipenem was noted. The imipenem MIC values for the P. aeruginosa population in 2002 were higher than those in 1993 (Fig. 1). This could possibly be connected with the high usage of imipenem in our hospital. 3.2. Antibiotic consumption in hospital The usage of some antibiotics in our hospital in 1993 to 2002, calculated as defined daily doses, is shown in Table 2. From 1993 to 2001 the usage of amikacin decreased, whereas that of netilmicin increased two-fold. In 2002, amikacin was more frequently used because the supply of netilmicin was stopped. The usage of ceftazidime showed a small increase from 1101 to 1569 defined daily doses. Small quantities of piperacillin with tazobactam were used in therapy. In contrast to the above a four-fold increase in the usage of two carbapenems, imipenem and meropenem, was noted in our hospital. In the first period of our study imipenem only was
Table 2 Antibiotic consumption in hospital in the period 1993–2001 (in DDD) Antibiotic
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
Amikacin Netilmicin Ceftazidime Piperacillin/tazobactam Imipenem Meropenem
1777 2031 1101 0 981 0
1300 1365 691 0 938 0
986 1973 602 0 1120 0
N.d. N.d. N.d. N.d N.d. N.d.
1208 2521 926 273 2209 155
1101 3218 1241 188 1737 512
1377 3444 1411 414 2061 890
894 3389 1296 245 2365 1000
859 4855 1569 334 2473 1311
2508 1173 1567 455 3225 1040
DDD, defined daily dosage; N.d., no data.
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Table 3 Distribution of imipenem-resistant Pseudomonas aeruginosa isolates among the most frequently encountered serotypes Year
No. (%) of imipenem-resistant isolates in particular serotypes
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002
O1
O3
O6
O11
O12
Other
Total
1 (11) 1 (20) 1 (5.9) 1 (5.3) 5 (18.5) 2 (10.5) 2 (4.3) 6 (8.9) 1 (1.4) 2 (4.3)
0 1 (20) 1 (5.9) 0 1 (3.7) 3 (15.8) 5 (10.9) 1 (1.5) 1 (1.4) 1 (2.2)
0 2 (40) 4 (23.5) 8 (42) 5 (18.5) 6 (31.6) 18 (39) 26 (38.8) 33 (45.2) 25 (54.3)
0 0 4 (23.5) 5 (26.3) 8 (29.6) 3 (15.8) 8 (17.4) 14 (20.9) 24 (32.9) 11 (23.9)
3 (33.3) 0 3 (17.6) 1 (5.3) 4 (14.8) 4 (21) 5 (10.9) 2 (3.0) 2 (2.7) 4 (8.7)
5 (55.5) 1 (20) 4 (23.5) 4 (21) 4 (14.8) 1 (5.3) 8 (17.4) 18 (26.9) 12 (16.4) 3 (6.5)
9 (100) 5 (100) 17 (100) 19 (100) 27 (100) 19 (100) 46 (100) 67 (100) 73 (100) 46 (100)
used, but from 1997 meropenem was introduced to antibiotic therapy in our hospital. 3.3. Distribution of imipenem-resistant P. aeruginosa strains among different serotypes During the period 1993–2002 the most frequently encountered serotypes among P. aeruginosa isolates were O6 (30.8%), O11 (16.1%), O1 (13.4%), O3 (8.5%) and O12 (6.5%). From 1993 the number of imipenem-resistant P. aeruginosa strains increased, and a high incidence of imipenem-resistant strains was observed in 2000–2001. Table 3 shows the distribution of imipenem-resistant P. aeruginosa strains among the most frequently encountered serotypes. The highest number of imipenem-resistant strains was found to be in the isolates of serotypes O6 and O11. 3.4. Distribution of imipenem-resistant P. aeruginosa strains among hospital wards The highest number of P. aeruginosa isolates was collected from children hospitalized in the intensive care unit, general surgery and urology wards. The analysis of imipenem-resistant strains from the intensive care unit isolated in 1993–2002 showed different resistance patterns (Table 4). The most frequently encountered were P. aerug-
inosa strains characterized by imipenem-resistance and susceptibility to other antibiotics. The highest number of strains in this group was of serotype O11 and they were characterized by resistance to imipenem (MIC90 = 16 mg/L) and intermediate resistance to meropenem (MIC90 = 8 mg/L). Imipenem-resistant P. aeruginosa strains of serotype O11 isolated in 2000 and 2001 were clonally related, as shown by the PFGE results (Fig. 2). In 2001, the imipenem-resistant P. aeruginosa strains consisted of 35% of all the isolates from the general surgery ward. The analysis of resistance patterns of imipenem-resistant isolates showed that the most frequently encountered strains were resistant to imipenem only (Table 5). They were all of serotype O6, characterized by the MIC values 16 mg/L for imipenem and 4 mg/L for meropenem. Genotyping of the imipenem-resistant P. aeruginosa strains of serotype O6 by the PFGE method showed that strains isolated in 2001 and 2002 were clonally related and had nearly identical restriction patterns, whereas strains from 2000 had different restriction patterns (Fig. 3). 4. Discussion The hospital’s antibiotic-giving policy plays a key role in the increase and spread of antibiotic resistant strains. Some
Table 4 Antibiotic resistance of Pseudomonas aeruginosa isolates obtained from children hospitalized in the intensive care unit in 1993–2002 Year
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Total
No. of isolates
24 35 37 31 31 21 30 32 50 42 333
No. (%) antibiotic resistant isolates A
B
A+B
IPM
A + IPM
B + IPM
A + B + IPM
Total
1 (4.2) 6 (17.1) 2 (5.4) 1 (3.2) 2 (6.4) 2 (9.5) 2 (6.7) 3 (9.4) 5 (10) 1 (2.4) 25 (7.5)
0 1 (2.9) 7 (18.9) 1 (3.2) 1 (3.2) 1 (4.8) 2 (6.7) 0 1 (2) 6 (14.3) 20 (6)
2 (8.3) 5 (14.3) 10 (27.0) 2 (6.4) 4 (12.9) 2 (9.5) 3 (10) 2 (6.2) 0 2 (4.8) 32 (9.6)
1 (4.2) 1 (2.9) 2 (5.4) 4 (12.9) 1 (3.2) 4 (19) 4 (13.3) 8 (25) 18 (36) 4 (9.8) 47 (14.1)
0 0 1 (2.7) 2 (6.4) 2 (6.4) 0 1 (3.3) 2 (6.2) 2 (4) 0 10 (3)
1 (4.2) 0 0 0 4 (12.9) 3 (14.3) 3 (10) 3 (9.4) 1 (2.0) 4 (9.8) 19 (5.7)
2 (8.3) 0 1 (2.7) 1 (3.2) 1 (3.2) 3 (14.3) 3 (10) 2 (6.2) 1 (2) 3 (7.1) 17 (5.1)
7 (29.2) 13 (37.1) 23 (62.2) 11 (35.5) 15 (48.4) 15 (71.4) 18 (60.0) 20 (62.5) 28 (56.0) 20 (47.6) 170 (51.1)
A, aminoglycosides; B, beta-lactams; IPM, imipenem.
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Table 5 Antibiotic resistance of Pseudomonas aeruginosa isolates obtained from children hospitalized in the general surgery ward in 1993–2002 Year
1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 Total
No. of isolates
17 21 22 22 28 31 33 34 40 40 288
No. (%) antibiotic resistant isolates A
B
A+B
IPM
A + IPM
B + IPM
A + B + IPM
Total
1 (5.9) 5 (23.8) 2 (9.1) 3 (13.6) 2 (7.1) 3 (9.7) 1 (3.0) 0 0 1 (2.5) 18 (6.3)
1 (5.9) 1 (4.8) 1 (4.5) 0 1 (3.6) 4 (12.9) 2 (6.1) 2 (5.9) 2 (5.0) 1 (2.5) 15 (5.2)
0 0 1 (4.5) 3 (13.6) 4 (14.3) 1 (3.2) 4 (12.1) 0 0 0 13 (4.5)
0 0 0 1 (4.5) 1 (3.6) 1 (3.2) 2 (6.1) 6 (17.6) 14 (35.0) 10 (25.0) 35 (12.1)
0 0 1 (4.5) 0 2 (7.1) 0 2 (6.1) 3 (8.8) 1 (2.5) 1 (2.5) 10 (3.5)
0 0 0 0 3 (10.7) 1 (3.2) 1 (3.0) 1 (2.9) 0 0 6 (2.1)
1 (5.9) 0 2 (9.1) 1 (4.5) 0 1 (3.2) 3 (9.1) 8 (23.5) 4 (10.0) 3 (7.5) 23 (8.0)
3 (17.6) 6 (28.6) 7 (31.8) 8 (36.4) 13 (46.4) 11 (35.5) 15 (45.5) 20 (58.8) 21 (52.5) 16 (40.0) 120 (41.7)
A, aminoglycosides; B, beta-lactams; IPM, imipenem.
Fig. 2. PFGE typing of the imipenem-resistant P. aeruginosa strains isolated from children hospitalized in the intensive care unit. Lines: , marker; 1–13, strains from 2001 (serotype O11); 14–16, strains from 2000 (serotype O11); 17–19, strains from 2001 (serotype O6).
Fig. 3. PFGE typing of the imipenem-resistant P. aeruginosa strains isolated from children hospitalized in the general surgery ward. Lines: , marker; 1–2, strains (serotype O6) from 2000; 3–14, strains (serotype O6) from 2001; 15–18, strains (serotype O6) from 2002.
antimicrobials, such as imipenem, are more susceptible to the development of resistance during treatment, leading to failure in therapy [15]. Imipenem therapy in patients with P. aeruginosa infections caused an increase in imipenem resistance as well as resistance to piperacillin with tazobactam and ceftazidime [16]. The results of a 3-year study (1997–2000) on the usage of beta-lactams and P. aeruginosa resistance indicated that periods of intensive imipenem consumption correlated with periods of increased P. aeruginosa resistance to imipenem, ceftazidime and piperacillin with tazobactam [17]. The epidemiological situation of our hospital cannot be compared with other paediatric hospitals in Poland. Our specificity is connected with the treatment of children from the whole country with serious diseases, often with defects of the immunology system, and often colonized or infected with hospital bacterial strains as a result of earlier hospitalization in other hospitals. Furthermore these patients had been treated many times with a broad spectrum of different antimicrobials, which results in the only therapeutic option for our hospital to be the use of carbapenems. The susceptibility of Gram-negative bacterial strains isolated from clinical specimens of children hospitalized in the intensive care unit of our hospital from 1997 to 2000 was studied [18]. Extended spectrum beta-lactamases and AmpC beta-lactamase producers among Enterobacteriaceae isolates from this intensive care unit continued to be a serious therapeutic problem, although the carbapenems were highly active against these phenotypes. This epidemiological situation was the cause of a high consumption of carbapenems, both imipenem and meropenem, in our hospital. In fact, there was a four-fold increase in the period 1993–2002. In 2003, the usage of carbapenems increased further and was five-fold higher in comparison to 1993. Therefore, P. aeruginosa strains resistant to imipenem and susceptible to other antibiotics appeared in our hospital as a direct consequence of this high usage of carbapenems. The percentage of imipenem-resistant P. aeruginosa strains increased from 4.3% in 1993 to 26.4% in 2001. Results obtained in the SENTRY program for the period 1997–1999 indicated a decrease in carbapenem susceptibility of P. aerug-
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inosa isolates from three regions: in Canada for imipenem, in South America for meropenem and in Europe for both drugs [3]. Imipenem had probably been a selective agent, because in the majority of countries meropenem was just introduced to therapy or its usage was limited at this time. The analysis of data from the time-period 1992–2000 obtained in different programs of antibiotic susceptibility studies showed that resistance of P. aeruginosa to imipenem in Europe varied between 21 and 31%, but in Greece reached 64% [19]. On a molecular level the imipenem resistance of the majority of these selected P. aeruginosa strains was connected with mutation in the oprD gene. In our studies, the analysis of P. aeruginosa strains of the most frequently encountered serotypes showed that the number of strains resistant to imipenem and simultaneously susceptible to other antibiotics reached a peak in our hospital in 2000–2001. Meropenem was characterized by higher activity than imipenem and about 10% of imipenem-resistant strains were susceptible to meropenem. The clonally related, imipenem-resistant P. aeruginosa strains of serotype O11, from the intensive care unit, showed only intermediate resistance to meropenem. The clonally related imipenem-resistant P. aeruginosa strains of serotype O6, from the general surgery ward, were meropenem-susceptible. The mechanism of imipenem-resistance in some of the P. aeruginosa strains was connected with the efflux system (unpublished data). Furthermore, among the imipenem-resistant P. aeruginosa isolates were found 11 multi-resistant strains which produced metallo-beta-lactamase VIM-4 [20]. The first strain appeared in 1998 and P. aeruginosa strains harbouring this metallobeta-lactamase have become endemic in our hospital since then.
Acknowledgment The excellent technical assistance of Teresa Guzik and Małgorzata Walus is gratefully appreciated.
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