Due to Enterobacter aerogenes Resistant to Imipenem

JOURNAL OF CLIMCAL MICROBIOLOGY, Jan. 1993, p. 123-127 0095-1137/93/010123-05$02.00/0

Vol. 31, No. 1

Copyright ©) 1993, American Society for Microbiology

Clinical and Bacteriological Study of Nosocomial Infections Due to Enterobacter aerogenes Resistant to Imipenem C. DE CHAMPS,1" C. HENQUELL,2 D. GUELON,3 D. SIROT,2 N. GAZUY,4 AND J. SIROT2 Service d'Hygiene Hospitali&re' and Service de Bact6riologie-Virologie,2 Faculte de Medecine, 28, Place Henri Dunant, 63001 Clermont-Ferrand Cedex, and De6partement d'Anesthesie-Reanimation3 and Service de Nephrologie,4 H6pital G. Montpied, 63000 Clermont-Ferrand Cedex, France Received 18 May 1992/Accepted 13 October 1992

Enterobacter aerogenes strains resistant to imipenem were isolated in 10 patients, 7 of whom had received imipenem-cilastatin. The strains were differentiated by biotype, antibiotype, and plasmid content. All of the strains overproduced a chromosomal cephalosporinase and lost a major outer membrane protein with a size of about 40 kDa. In 5 of the 10 patients, E. aerogenes strains resistant to extended-spectrum cephalosporin were isolated during the same stay. In three patients, the similarity between the imipenem-susceptible and -resistant strains suggests the occurrence of mutation and reversion in vivo. The combination imipenem-cilastatin has been critically important for use with multiresistant strains of Enterobacter spp., but its use increases the risk of selection of imipenem-resistant strains. Imipenem was first used in Clermont-Ferrand hospitals in 1986. For 4 years, we observed no imipenem-resistant (IPM-R) strains among strains of Enterobacteriaceae. The Enterobacter species isolated in many infections were often resistant to all antibiotics with the exception of imipenem. Since 1990, resistance to imipenem has been observed in Enterobacter aerogenes, partly because of a lack of porin (4, 10). Thirteen strains of E. aerogenes resistant to imipenem were isolated in 1991 in our hospital. We collected clinical and bacteriological data to try to explain the occurrence of this recent resistance.

MATERIALS AND METHODS Clinical data. In 1991, E. aerogenes strains resistant to imipenem were isolated in 10 patients in Clermont-Ferrand hospitals (2,329 beds). Their charts were reviewed for this study by an intensive care unit (ICU) consultant. The severity of illness on admission was assessed by the simplified acute physiologic score (14). The antibiotic treatment of each patient from admission onward was recorded. Microbiological study. Twenty-three strains of E. aerogenes (13 IPM-R and 10 imipenem susceptible [IPM-S]) from these 10 patients were isolated. The biotypes were determined with the Api 20 E and Api 50 CH systems (bioMerieux, Marcy l'Etoile, France). The results of the Api 50 CH were analyzed by aggregation according to the mean distance (unweighted pair group method with arithmetic average [18a]), on the basis of the Gower coefficient of similarity (7), by J. M. Boeufgras (bioMerieux). (i) Antibiotic susceptibility testing. The susceptibility of strains was determined by the disk diffusion method on Mueller-Hinton agar. Strains were classified as susceptible (S), intermediate, and resistant (R) according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (1). The MICs of carbapenems (imipenem, meropenem) were determined in Mueller-Hinton broth by a microdilution technique (Autodiluter II; Dynatech Laboratories, Inc.). Inocula of 105 to 106 CFU/ml were distributed with a multipoint *

inoculator (MIC 2000; Dynatech). Imipenem was provided by Merck Sharp and Dohme, and meropenem was provided by ICI Pharmaceutical Inc. (ii) P-Lactamase preparation and 13-lactamase assays. Sonic extracts were prepared from cultures in Trypticase soy broth by ultrasonic disintegration as previously described (12). They were studied by electrofocusing (LKB 2117 Multiphor apparatus). The enzyme activities were located in the gels with chromogenic cephalosporin CM 32150. P-Lactamase activity was determined spectrophotometrically in sodium phosphate buffer (10 mM, pH 7) at 30°C with a double-beam spectrophotometer (model PU 880; Pye Unicam, Philips). One unit of 1-lactamase activity was defined as the amount of enzyme hydrolyzing 1 ,umol of cephalothin per min at 30°C. (iii) Agarose gel electrophoresis of large plasmids. Plasmid DNA of clinical strains was prepared by the procedure described by Kado and Liu (11), and the crude extract was used directly for electrophoresis, which was performed in agarose gels (0.7%, wt/vol) for 90 min at 50 V. The size of the plasmids was determined by using pcFF04 (85 kb) and pcFF14 (180 kb) as standards. (iv) Preparation and electrophoresis of OMPs. Cell membranes were prepared from exponential-phase cultures. Outer membranes were obtained by using 0.3% (wt/vol) N-lauroylsarcosine (Sigma, St. Louis, Mo.) and by centrifugation. The crude outer membrane proteins (OMPs) (pellets) were then collected as described by Gutmann et al. (9). Protein concentrations were determined according to Lowry's method. OMP preparations (10 ,ug per lane) were applied to sodium dodecyl sulfate-polyacrylamide (12%) gel electrophoresis (SDS-PAGE) gels, stained with Coomassie blue, and destained with H20-methanol-acetic acid. When several strains were obtained in the same patient, IPM-R strains were compared with the IPM-S strains. RESULTS Clinical data. The 10 patients were hospitalized in three ICUs before the isolation of IPM-R strains, 8 of them in the same unit, at different periods in 1991. The simplified acute physiologic score mean was 14 (range, 11 to 18). Patient

Corresponding author. 123

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TABLE 1. Clinical and bacteriological data Length Patient

ofdsta (days)"

Antibiotic" treatment 8 days

before strain

E. aerogenes strain (classification)'

Site or method of isolation (diam by disk diffusion, mm)

~~~isolation

MICd

(mg/liter)

1

13

P-CAZ-OFX

la (IPM-R)

C.V. catheter (16)

32

2

28 47

None None

2a (IPM-R) 2b (IPM-R)

C.V. catheter (18) Urine (18)

16 16

3

35 68 97

IPM-TEC IPM None

3a (IPM-S) 3b (IPM-R) 3c (IPM-S)

Eschar (30) Eschar (16) Bronchoalveolar specimen (29)

0.5 32 1

4

8 29 29

P-AN-CAZ-MTR IPM-OFX-TEC IPM-OFX-TEC

4a (IPM-S) 4b (IPM-S) 4c (IPM-R)

Bronchoalveolar specimen (24) Arterial catheter (24) Blood culture (17)

2 4 16

5

12 28 31 39 47

P-MTR IPM-AN IPM IPM IPM

5a (IPM-S) Sb (IPM-S) Sc (IPM-S) Sd (IPM-R) Se (IPM-S)

Incisional wound (25) Blood culture (23) Incisional wound (24) Incisional wound (17) Incisional wound (23)

2 4 2 16 4

6

15 18 50 64

AMC-OFX IPM-GN None PIP-TEC

6a (IPM-R) 6b (IPM-R) 6c (IPM-R) 6d (IPM-S)

Arterial catheter (18) Urine (18) Retroperitoneal abcess (17) C.V. catheter (24)

16 16 16 2

7

17

PIP-AN-IPM-CIP

7a (IPM-R)

Bronchoalveolar (16)

16

8

17

IPM-TEC-FA

8a (IPM-R)

Blood culture (20)

9

23 46

TCC-CIP IPM-AN

9a (IPM-S) 9b (IPM-R)

Incisional wound (27) Incisional wound (16)

2 16

10

14

IPM-AN-TEC

10a (IPM-R)

Bronchoalveolar specimen (16)

16

8

aLength of stay before isolation of E. aerogenes strains. b p, penicillin G; CAZ, ceftazidime (ESB); OFX, ofloxacin; IPM, imipenem plus cilastatin; TEC, teicoplanin; AN, amikacin; MTR, metronidazol; GN, gentamicin; PIP, piperacillin; CIP, ciprofloxacin; FA, fusidic acid; TCC, ticarcillin plus clavulanic acid. C Susceptibility determined by disk diffusion with IPM. d Determined by a microdilution technique in Mueller-Hinton broth.

profiles and antecedent antibiotic therapy are summarized in Table 1. Patients 1, 2, and 6 did not receive imipenem-cilastatin before isolation of IPM-R E. aerogenes strains. Patient 1 was treated for pneumonia with penicillin and then with ceftazidime and ofloxacin. After 13 days of treatment, one IPM-R strain, la, was isolated from the central venous (C.V.)

catheter, which was removed. The fever decreased. Patient 2 was admitted to the ICU for renal failure. Nineteen days after the end of a peroperative prophylactic treatment with cefamandole, an IPM-R E. aerogenes strain (2a) was isolated from the C.V. catheter. After removal of the catheter, the patient improved after treatment with the combination imipenem-cilastatin (750 mg/day) for 9 days and amikacin for 3 days. Nine days after the end of treatment, an IPM-R strain (2b) appeared in urine, which was followed by septic shock. The patient improved with the combination of imipenemcilastatin and amikacin. Patient 6 had been receiving amoxicillin-clavulanic acid and ofloxacin before IPM-R E. aerogenes organisms were isolated from urine (strain 6b) and the arterial catheter (strain 6a). Another IPM-R strain (6c) was isolated from a retroperitoneal abscess 8 days after the end of a first 14-day course of imipenem-cilastatin (6 g/day) treatment, and one IPM-S strain (6d) was isolated 14 days

later. Seven patients (patients 3 to 5 and 7 to 10) received imipenem-cilastatin before the occurrence of an IPM-R strain. Patient 3 had been receiving imipenem-cilastatin and teicoplanin when an IPM-S E. aerogenes strain (3a) was isolated from an eschar. Thirty-three days later, during a second course of imipenem treatment, an IPM-R strain was isolated from the eschar (3b). One month later, when the patient had no antibiotic treatment, an IPM-S strain (3c) was isolated from a protected bronchoalveolar specimen. Patient 4 was hospitalized after thoracic and abdominal trauma and right hepatectomy. After 21 days of imipenem-cilastatin, teicoplanin, and ofloxacin treatment, an IPM-R E. aerogenes strain (4c) and a Xanthomonas maltophilia strain were isolated from a blood culture. Simultaneously, an IPM-S strain (4b) was isolated from an arterial catheter. The fever did not decrease until 20 g of aztreonam, 12 g of moxalactam, and 1.6 g of amikacin were given per day, as determined by the bactericidal levels of the serum. Patient 5 was treated with penicillin and metronidazole for a wound infection when an IPM-S E. aerogenes strain was isolated from the incisional wound. Septicemia due to Pseudomonas aeruginosa and Escherichia coli occurred. Treatment with imipenem and amikacin was started. Under treatment with

VOL. 31, 1993

imipenem-cilastatin, four strains were isolated-one IPM-S strain (5b) from blood culture and two IPM-S strains (5c and 5e) and one IPM-R strain (5d) from the incisional wounduntil it was cured with local treatment. Patient 7 was admitted for aspiration pneumonia. An IPM-R E. aerogenes strain (7a) was isolated from the protected bronchoalveolar specimen 48 h after the imipenem-cilastatin and ciprofloxacin treatments were stopped. The tracheal tube was removed, and no further E. aerogenes organisms were isolated. Patient 8 was admitted for septic shock and pneumonia. Treatment with imipenem-cilastatin, teicoplanin, and fusidic acid was started. Seventeen days later, patient 8 had septicemia due to IPM-R E. aerogenes 8a. Improvement was obtained after the patient was given an increased dosage of 6 g of imipenem-cilastatin per day in combination with 1 g of amikacin per day. Patient 9 was admitted for colectomy. An IPM-S E. aerogenes strain (9a) was isolated after 13 days of ticarcillin-clavulanic acid and ciprofloxacin treatment. Because of persistent fever, the treatment was replaced by imipenem-cilastatin and amikacin. Eleven days after the end of all antibiotic therapy, an IPM-R E. aerogenes strain (9b) was isolated. The strain was no longer isolated after local treatment. Patient 10 was admitted for pneumonia in the left lung after a right pneumonectomy. On the 10th day of treatment with imipenemcilastatin, amikacin, and teicoplanin, an IPM-R E. aerogenes strain (lOa) was isolated from the protected bronchoalveolar specimen. The patient's health improved, and the patient was extubated on the 16th day. Microbiologic results. The strains were characterized by the combination of biotype, resistance phenotype, and plasmid profile. (i) Biotype. With the Api 20 E gallery, only one strain (3b) did not use citrate. Five biotypes were obtained by the study of carbohydrate metabolism with Api 50 CH. The strains were differentiated on dulcitol, inulin, amidon, D-raffinose, 5-ketogluconate, and amygdalin. The two dulcitol-positive and D-raffinose-negative strains had 90.4% similarity to the other strains. The other 21 strains had 94% similarity to one another. (ii) Antibiotype. The IPM-R strains were resistant to extended-spectrum cephalosporins and to moxalactam. The MICs of imipenem ranged from 8 to 32 mg/liter. For each strain, except 9b, the MIC of imipenem was from two- to eightfold higher than that of meropenem. Among these strains, five phenotypes were differentiated on the basis of their susceptibility to aminoglycosides and trimethoprim. Nine strains were resistant to all antibiotics tested, with the exception of streptomycin, spectinomycin, and neomycin. Strain la was susceptible to gentamicin and was resistant to amikacin, tobramycin, and netilmicin (phenotype Akr Tmr Netr). The observation of this phenotype suggests there was production of an AAC(6')-I enzyme. Strain 2a was susceptible to amikacin and resistant to gentamicin, tobramycin, and netilmicin (phenotype Gnr Tmr Netr), suggesting the production of an AAC(3)-II enzyme. All strains, whether IPM-S or IPM-R, had a cephalosporinase of the same pl, 8.3. They all produced a transferable ,B-lactamase in addition to cephalosporinase as follows: TEM-1, pI 5.4 (patients 3 to 10); CAZ-6 (extended-spectrum p-lactamase [ESBI), pI 6.5 (strain la); and CTX-1 (ESB), pl 6.3 (strain 2b). One strain produced TEM-1 and CAZ-6 in addition to the cephalosporinase. Hydrolysis of cephalothin by the 1-lactamases was similar in IPM-S and IPM-R strains (10-4 to 10-3 mU/mg), 102_ to 103-fold higher than the activity of the wild-type strain of E. aerogenes, which had

IMIPENEM-RESISTANT ENTEROBACTER AEROGENES

1 97.4 66.2 45 40 j31

2

3 4

5

-Z

6

7

125

8

VW

21.5

144 4

FIG. 1. SDS-PAGE analysis of OMPs. Lanes 1, 2, and 3 (patient 3): IPM-R strain 3b, IPM-S strain 3c, and IPM-S strain 3a, respectively. Lanes 4 and 5 (patient 5): IPM-S strain Sb and IPM-R strain

Sd, respectively. Lanes 6 and 7 (patient 6): IPM-R strain 6c and IPM-S strain 6d, respectively. Lane 8 (patient 4): IPM-R strain 4c. Molecular mass in kilodaltons is shown on the left. Arrowhead, ca. 40-kDa protein. inducible cephalosporinase (2 x 10-6 mU/mg; MIC of imipenem, 0.03 mg/liter). (iii) Plasmid profile. Two different plasmid profiles were observed, with either one or two large plasmids. Of the IPM-R strains, 10 had a 180-kb plasmid, while 3 had 85- and 180-kb plasmids. The strains containing the 85-kb plasmid were resistant to amikacin and produced an ESB, characterized by its pI. On the basis of biotypes, resistance phenotypes, and plasmid profiles, only 3 of the 13 IPM-R strains were

similar.

(iv) OMPs. Three major OMPs, one about 40 kDa in size and a doublet of 35 and 36 kDa in size, were found in the wild-type strain of E. aerogenes and in the strains requiring MICs of