Lactamase-Producing and Carbapenemase-Producing Enterobacter

JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2008, p. 1037–1044 0095-1137/08/$08.00⫹0 doi:10.1128/JCM.00197-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 3

Successive Emergence of Extended-Spectrum ␤-Lactamase-Producing and Carbapenemase-Producing Enterobacter aerogenes Isolates in a University Hospital䌤 M. Biendo,1* B. Canarelli,1 D. Thomas,1,2 F. Rousseau,1 F. Hamdad,1 C. Adjide,1 G. Laurans,1,2 and F. Eb1,2 Service de Bacte´riologie et d’Hygie`ne CHU NORD, Place Victor Pauchet, 80054 Amiens, Cedex 1 France,1 and Laboratoire de Bacte´riologie, Faculte´ de Me´decine, 80036 Amiens, Cedex France2 Received 26 January 2007/Returned for modification 3 March 2007/Accepted 15 January 2008

Sixty-two clinical isolates of Enterobacter aerogenes resistant to expanded-spectrum cephalosporins were collected between July 2003 and May 2005. Among these isolates, 23 (37.1%) were imipenem (IPM) susceptible, and 39 (62.9%) were IPM insusceptible, of which 89.7% (35/39) were resistant and 10.3% (4/39) were intermediate. Isolate genotypes were compared by pulsed-field gel electrophoresis. Of 62 isolates, 48 belonged to epidemic pulsotype A (77.4%). This pulsotype included 37.5% and 58.4% of ␤-lactam phenotypes b and a, respectively. Nine isolates (14.5%) belonged to pulsotype E, which included 22.3% and 77.7% of phenotypes b and a, respectively. The ␤-lactamases with pIs of 5.4, 6.5, 8.2, and 8.2 corresponded to extended-spectrum ␤-lactamases (ESBLs) TEM-20, TEM-24, SHV-5, and SHV-12, respectively. Of 39 IPM-insusceptible E. aerogenes isolates, 26 (66.6%) were determined to be metallo-␤-lactamase producers, by using a phenotypic method. Of these isolates, 24 harbored a blaIMP-1 gene encoding a protein with a pI of >9.5, and two carried the blaVIM-2 gene encoding a protein with a pI of 5.3, corresponding to ␤-lactamases IMP-1 and VIM-2, respectively. The remaining 13 (33.4%) isolates were negative for the blaIMP-1 and blaVIM-2 genes but showed an alteration of their outer membrane proteins (OMPs). Ten of these isolates produced the two possible OMPs (32 and 42 kDa), with IPM MICs between 8 and 32 ␮g/ml, and three others produced only a 32-kDa OMP with IPM MICs >32 ␮g/ml. This work demonstrates that, in addition to resistance to expanded-spectrum cephalosporins, IPM resistance can occur in ESBL-producing E. aerogenes isolates by carbapenemase production or by the loss of porin in the outer membrane. (A.U.H.) in 2002. To determine whether the strains were epidemiologically related, all of the strains were characterized by antibiotic resistance phenotyping and pulsed-field gel electrophoresis (PFGE). In addition, ␤-lactamases were characterized by the determination of their isoelectric points and by gene sequencing, and outer membrane protein (OMP) analysis was performed. (This study was presented in part at the 16th European Congress of Clinical Microbiology and Infectious Diseases [5]).

Enterobacter aerogenes has recently emerged as an important hospital pathogen (13). The prevalence of this bacterial species has increased considerably since the introduction of extendedspectrum cephalosporins into clinical practice (18). Various resistance mechanisms have been described in this species, such as extended-spectrum ␤-lactamases (ESBLs), plasmidmediated production (21), and hyperproduction of the Bush group 1 chromosomally mediated cephalosporinases (32). Although these enzymes are specifically characteristic of some Enterobacter spp., the appearance of similar plasmid-mediated ␤-lactamases in Klebsiella pneumoniae and Escherichia coli raises concerns about the spread of resistance (21, 32). For both ESBL-producing and AmpC-producing isolates, carbapenems are the only ␤-lactam agents active against both resistance mechanisms. Data from previous studies have shown that both ESBL and AmpC ␤-lactamase producers had reduced susceptibility to imipenem (IPM) due to either carbapenemhydrolyzing enzymes (carbapenemase) (30), decreased membrane permeability due to loss of porin in the outer membrane (14, 39), or active efflux (27). The IPM resistance of ESBL-producing E. aerogenes strains was observed for the first time at Amiens University Hospital

MATERIALS AND METHODS Patient and bacterial strain data. From July 2003 to May 2005, 62 clinical isolates of E. aerogenes resistant to expanded-spectrum cephalosporins were identified from four A.U.H. wards. Among these isolates, 23 (37.1%) were IPM susceptible (IPM-S), and 39 (62.9%) were IPM insusceptible (IPM-Ins). These isolates were collected from a group of 22 adult patients, consisting of 12 men (54.5%) and 10 women (45.5%) with a mean age of 67.1 years (range, 34 to 90 years). These patients had been hospitalized for periods of 7 to 181 days (median, 64.5 days). The time to acquisition of E. aerogenes colonization or infection ranged from 7 to 132 days (median, 46.7 days). The temporal occurrence of colonization in these patients showed the presence of one or more colonized and then infected patients on the wards at the time of colonization acquisition by a new patient and a constant rate of transmission throughout the study period. Seven patients were admitted to the hepatology and gastroenterology ward, seven were admitted to the polyvalent intensive care unit (ICU), seven were admitted to the general and visceral surgery ward, and one was admitted to the respiratory ICU. Eight patients were admitted for digestive adenocarcinoma, seven for acute pancreatitis, five for multiple trauma, one for prostate adenocarcinoma, and one for posttraumatic leg necrosis. Eleven patients had received IPM plus an expanded-spectrum cephalosporin prior to the isolation of the organisms, and 11 patients had not received IPM prior to the isolation of the

* Corresponding author. Mailing address: Laboratoire de Bacte´riologie-Hygie`ne, CHU Nord—Place Victor Pauchet—80054 Amiens, Cedex 1, France. Phone: 03 22 66 84 30. Fax: 03 22 66 84 98. E-mail: [email protected]. 䌤 Published ahead of print on 30 January 2008. 1037

1038

BIENDO ET AL.

J. CLIN. MICROBIOL.

TABLE 1. Oligonucleotides used as primers for PCR amplification of ␤-lactams and aminoglycosides resistance genes Gene

Amplicon length (bp)a

Primer

Sequence (5⬘ 3 3⬘)

ESBL blaTEM blaTEM blaSHV blaSHV

A B A B

TTA-GAC-GTC-AGG-TGG-CAC-TT GGA-CCG-GAG-TTA-CCA-ATG-CT TCG-GCC-TTC-ACT-CAA-GGA-TG ATG-CCG-CCG-CCA-GTC-ATA-TC

972

MBL blaIMP-1 blaIMP-1 blaVIM-2 blaVIM-2

A B A B

GAA-GGC-GTT-TAT-GTT-CAT-AC GTA-AGT-TTC-AAG-AGT-GAT-GC GTT-TGG-TCG-CAT-ATC-GCA-AC AAT-GCG-CAG-CAC-CAG-GAT-AG

587

A B

TAT-GAG-TGG-CTA-AAT-CGA-T CCC-GCT-TTC-TCG-TAG-CA

E F

GCC-TGA-CGA-TGC-GTG-GA GAC-TTG-ACC-TGA-ATG-TTT-GG

A B

GCA-GGG-TGT-GGA-ATA-CG ACA-GAC-CGA-GAA-GGC-TTA-TG

aac(6⬘)-Ib Integron 5⬘-CS 3⬘-CS intL3 a b

785

382 395 1,000 2,000 760

Positionb

Reference or accession no.

90–105 1062–1042 103–121 988–970

41

1241 1808 821–881 1206–1186

33

494 872

35

1190–1206 1341–1327

M73819 M73819

200 940

35

41

33

Values represent the expected size of the amplification. Range represents the nucleotide position of the primer in the sequence.

organisms, but these patients were treated with cefepime (FEP) or cefpirome (CPO) plus gentamicin (G). The clinical isolates collected from these patients were identified as E. aerogenes by using an ID32 system (Bio-Rad, Marne-la-Coquette, France) according to the manufacturer’s instructions. Table 2 shows the sources of these isolates. Phenotypic detection of ESBL-, AmpC-, and MBL-producing isolates. Isolates were screened for ESBL production by using a double-disk synergy test (DDST). Mueller-Hinton medium was inoculated, and disks containing the standard 30-␮g quantities of cefotaxime (CTX), FEP, CPO, ceftazidime (CAZ), and aztreonam (ATM) were placed 30 mm (center-to-center) from an amoxicillinclavulanic acid (20 and 10 ␮g, respectively) disk. After disks were incubated, an enhanced zone of inhibition between any one of the ␤-lactam disks and the amoxicillin-clavulanic acid disk was interpreted as presumptive evidence of the presence of an ESBL. The same isolates were also screened for AmpC production by using a disk potentiation test on Mueller-Hinton agar containing, as the inhibitor, cloxacillin (250 ␮g/ml) (A.E.S. Chemunex, Bruz, France), according to the manufacturer’s instructions. After the disks were incubated, the absence of the inhibition diameter from around the amoxicillin-clavulanic acid zone and the potentiation of the zone of inhibition around CTX, FEP, CPO, CAZ, and ATM indicated the possible presence of AmpC ␤-lactamase. Metallo-␤-lactamase (MBL) production was detected by using the MBL Etest strips (AB Biodisk, Solna, Sweden) with the IPM-Ins E. aerogenes isolates, according to the manufacturer’s instructions. Antimicrobial susceptibility. MICs were determined for all isolates studied by using an Etest method (AB Biodisk, Solna, Sweden). The MICs were determined for the following antibiotics: cefoxitin (FOX), CTX, CAZ, FEP, CPO, ATM, IPM, G, tobramycin (T), netilmicin (N), amikacin (A), trimethoprim-sulfamethoxazole (SXT), and ofloxacin (OFX). Escherichia coli ATCC 25922 was used as the MIC reference strain. The MIC results were interpreted according to the Comite´ de l’Antibiogramme de la Socie´te´ Française de Microbiologie guidelines (12). Plasmid and chromosomal DNA preparation, Southern blotting, and hybridization. Plasmid and chromosomal DNA extraction from the clinical isolates was performed using a Qiagen plasmid kit (Qiagen, Courtaboeuf, France) and a GNOME kit (Qbiogene, France) according to the manufacturers’ instructions. Plasmid and chromosomal DNAs were detected by 0.8% agarose gel electrophoresis. The sizes of the plasmids were determined by comparison with the plasmid standards pUC8 (2.678 kb), pBR322 (4.362 kb), RP4 (54 kb), and pIP173 (125.800 kb) (from Collection Institut Pasteur [CIP] strains). Plasmid and genomic DNAs were digested with EcoRI, and the digested fragments were analyzed by electrophoresis using a 1-kb ladder (Promega, Madison, WI) as a DNA size marker. The restriction fragments were transferred from an agarose gel to a nylon membrane (Amersham Hybond-N⫹; GE Healthcare

Bio-Sciences, Orsay, France) by the Southern method (4). A digoxigenin DNA labeling and detection kit (Roche Diagnostics, Mannheim, Germany) was used for labeling the probes blaTEM, blaSHV, blaIMP-1, and blaVIM-2 and for hybridization. Isoelectric focusing (IEF). Sonicated extracts were prepared from cultures in brain heart infusion, by ultrasonic disintegration, as described previously (29). ␤-Lactamases with known isoelectric points (pI) (TEM-1, 5.4; TEM-24, 6.5; SHV-1, 7.6; SHV-4, 7.8; IMP-1, ⬎9.5; and VIM-2, 5.3) were focused in parallel with the extracts. ␤-Lactamase activity was detected directly on the gel by nitrocefin (Oxoid Ltd., Basingstoke, United Kingdom) (29). PCR for the detection of TEM and SHV genes. PCR was carried out with a final volume of 50 ␮l containing 25 ␮l Go Taq Green Master Mix (Promega, Madison, WI), 20 ␮M (each) primer, and 2 ␮l of plasmid DNA preparation. The cycling conditions were 2 min at 95°C, followed by 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and then 72°C for 5 min. PCR for the detection of MBL (IMP-1 and VIM-2) genes. PCR was carried out with 1 ␮l of plasmid DNA. The reaction conditions consisted of predenaturation at 94°C for 2 min and then 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, and then 72°C for 5 min. PCR mapping of the integrons (integron PCR), the IntL3 integrase gene (integrase gene PCR), and the aminoglycoside resistance gene. PCR amplification of the detection of class 1 integrons was performed with the primers 5⬘-CS and 3⬘-CS, using DNA as the template. Combinations of the 5⬘-CS primer and the blaIMP-1 or blaVIM-2 gene and the 3⬘-CS primer and the blaIMP-1 or the blaVIM-2 gene were used for the determination of the genetic content of class 1 integrons. For the detection of the intL3 and aac(6⬘)-Ib genes, PCR was performed with specific oligonucleotide primers. The annealing step was carried out at 57°C and 55°C for the intL3 and aac(6⬘)-Ib genes, respectively. The primer sizes of the expected amplification products are listed in Table 1. Amplification reactions were carried out on a thermal Cycler 2400 instrument (Applied Biosystems, Foster City, CA). The PCR products and the base pairs (bp) DNA Smart ladder (Eurogentec, Belgium) were analyzed by 1% agarose gel electrophoresis with Tris-EDTA as the running buffer. The gels were stained with ethidium bromide, and a single observed band was photographed on a UV light transilluminator. Sequencing of the blaTEM, blaSHV, blaIMP-1, blaVIM-2, aac(6ⴕ)-Ib, and intL3 gene-specific PCR products. PCR products containing these genes were purified with a QIAquick PCR purification kit (Qiagen, France) and subjected to direct sequencing on an ABI PRISM 377 automated sequencer (Applied Biosystems, Foster City, CA). The identities of the genes encoding TEM, SHV, IMP-1, VIM-2, the aac(6⬘)-Ib gene, and the IntL3 enzymes were resolved by sequencing the blaTEM, blaSHV, blaIMP-1, blaVIM-2, aac(6⬘)-Ib, and intL3 gene-specific PCR products with the same primers. The comparison of the determined nucleotide

VOL. 46, 2008

ESBL- AND MBL-PRODUCING E. AEROGENES ISOLATES

sequences with the sequence databases was performed with an updated version of FASTA and BLAST-2 software. Multiple sequence alignments were performed with a CLUSTAL program. OMP analysis. The OMPs were isolated as sodium lauryl sarcosinate (1.7%) (Sigma-Aldrich, Germany), insoluble material from cell envelopes obtained by the sonication of bacteria grown in brain heart infusion broth (16). The electrophoretic analysis of OMPs was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as described elsewhere (23). Western blotting analysis of SDS-PAGE-separated OMPs was carried out with the buffers and conditions described by Towbin et al. (38). Western blotting analysis of proteins separated by SDS-PAGE was carried out by transfer to Immobilon membranes (Millipore, Saint-Quentin Yvelines, France). Membranes were then incubated sequentially with 5% skim milk– phosphate-buffered saline (PBS), anti-OmpK36 serum, alkaline phosphataselabeled goat anti-rabbit immunoglobulin G (IgG) (Sigma-Aldrich, Saint-Quentin, France), and 5-bromo-4-chloro-3-indolylphosphate disodium–nitroblue tetrazolium (BCIP-NBT) (7). Incubations were carried out for 1 h, and washing steps with 0.05% Tween 20-PBS were included after each incubation step. Polyclonal antibodies directed against the E. coli porins were recognized by the E. aerogenes porins, as reported previously (28). Purification of the OmpK36 protein from selected strains and antiserum was performed according to previously published procedures (1). Epidemiological typing. PFGE was performed as described previously (19), using SpeI (3 U/␮l) as the restriction enzyme. Digested whole-cell DNA was separated on a 1% agarose gel (pulsed field certified; Bio-Rad, France) with a contour-clamped homogeneous electric field system (CHEF-DRII system; GenePath, Bio-Rad, France). The gel was stained with ethidium bromide and visualized with a Gel Doc 2000 system (Bio-Rad, France). A lambda ladder (Bio-Rad, France) was used as the molecular weight marker, and E. coli O157:H7 (BioRad, France) and E. aerogenes 1846 and 1847 (CIP strains) were used as reference strains. Patterns were analyzed by visual comparison of the printed digital gel images and were interpreted as described previously (37). A percentage of similarity between each pulsotype was computed by Molecular Analyst Fingerprinting software (Bio-Rad, France), and Simpson’s index of diversity (36) was used to analyze the PFGE results.

RESULTS Antimicrobial resistance. One hundred percent of the strains were resistant to FOX (MIC, ⬎256 ␮g/ml), CAZ (MIC, ⬎256 ␮g/ml), SXT (MIC, ⬎32 ␮g/ml⬎), and OFX (MIC, ⬎32 ␮g/ml); 96.7% were resistant to T (MIC, 12 to 48 ␮g/ml) and N (MIC, 16 to 96 ␮g/ml); 93.6% were resistant to A (MIC, 32 to 64 ␮g/ml) and ATM (MIC, 48 to ⬎256 ␮g/ml); 56.4% were resistant to IPM (MIC, 12 to ⬎32 ␮g/ml); 29.1% were resistant to CTX (MIC, ⬎32 ␮g/ml); and 22.5% were resistant to FEP and CPO (MICs, ⬎32 ␮g/ml). Of the strains tested, 74.1% were intermediate to FEP (MIC, 8 to 32 ␮g/ml) and CPO (MIC, 12 to 32 ␮g/ml), 70.9% were intermediate to CTX (MIC, 8 to 32 ␮g/ml), 6.4% were intermediate to IPM (MIC, 8 ␮g/ml), 6.4% were intermediate to ATM (MIC, 24 to 32 ␮g/ml), and 3.2% were intermediate to A (MIC, 12 to 16 ␮g/ml). Four phenotypes were defined on the basis of their resistance to ␤-lactams: phenotype a (Foxr Ctxr⫹i Fepr⫹i Cpor⫹i Atmr⫹i Ipmr⫹i Cazr, where “r ⫹ i” means insusceptible) included 61.2% (38/62) of the strains; phenotype b (Foxr Ctxr⫹i Fepr⫹i Cpor⫹i Atmr⫹i Cazr) included 35.4% (22/62) of the strains; phenotype c (Foxr Ctxi Atmi Cazr, where “i” means intermediate) included 1.7% (1/62) of the strains; and phenotype d (Foxr Ctxr Atmr Ipmr Cazr) consisted of 1.7% (1/62) of the strains. Two aminoglycoside resistance phenotypes were also identified: phenotype e (Tr Nr Ar⫹i) included 91.9% (57/62) of the strains, and phenotype f (Gr Tr Nr) corresponded to 4.8% (3/62) of the strains.

1039

ESBL-like phenotypes and MBL production. The DDST detected the presence of ESBLs in 60 of the 62 isolates studied. They were intermediate or resistant to CTX (MIC, 8 to 32 ␮g/ml), FEP (MIC, 8 to ⬎32 ␮g/ml), CPO (MIC, 12 to 32 ␮g/ml), and ATM (MIC, 24 to ⬎256 ␮g/ml) and resistant to CAZ (MIC, ⬎256 ␮g/ml). Clavulanic acid inhibited the enzymes which hydrolyzed these substrates and subsequently enhanced the zones of inhibition of these substrates. Two of 62 (3.2%) isolates were DDST negative. They were sensitive to FEP and CPO and produced ␤-lactamases which were inhibited by cloxacillin, presumably indicating AmpC ␤-lactamase production. Twenty-six of the 39 IPM-Ins isolates were MBL positive by Etest, and the 13 remaining strains were MBL nonproducers. Plasmid and chromosomal DNA encoding ␤-lactamases. All plasmid and chromosomal DNA were obtained from E. aerogenes isolates. These isolates had one or more plasmids with the following molecular sizes: 3.5, 6, 10, and 20 kb. Six- to twenty-kilobase plasmids were present in all ESBL-producing E. aerogenes isolates. The plasmid DNA obtained was recognized by blaTEM-, blaSHV-, and blaIMP-specific primers used in this experiment, but not by blaVIM-2. Repeated attempts to detect the presence of plasmids capable of hybridizing with the blaVIM-2 probe failed. Meanwhile, the fragments of the EcoRI-digested genomic DNA showed hybridization, suggesting a chromosomal location. On the other hand, repeated attempts to detect blaTEM, blaSHV, and blaIMP-1 on plasmids were successful, confirming the specificity of the PCR assay. Phenotypic characterization of ␤-lactamases: the problem of multiplicity within the phenotype. The MICs of the ␤-lactam drugs and the results of IEF analysis of the 62 isolates studied suggested ESBL expression in 60 isolates and MBL expression in 39 isolates. Two isolates (Table 2, no. 61 and 62) were ESBL and MBL nonproducers but were suspected producers of highlevel chromosomally mediated AmpC ␤-lactamase. Five ␤-lactamase bands with pI values of 5.4 (5 isolates), 6.5 (39 isolates), 8.2 (55 isolates), ⬎9.5 (24 of 39 IPM-Ins isolates), and 5.3 (2 of 39 IPM-Ins isolates) were identified (Table 2). These results suggested the presence of ESBL and MBL, for which the frequent association made it difficult to interpret resistance phenotypes. PCR analysis was used initially to confirm and/or identify the ␤-lactamases observed by IEF. PCR using specific primers identified the presence of TEM (52/62 isolates), SHV (58/62 isolates), IMP-1 (24/39 IPM-Ins isolates), and VIM-2 genes (2/39 IPM-Ins isolates). The specific PCR products encoding enzymes with pIs of 5.4 (n ⫽ 5), 6.5 (n ⫽ 10), ⬎9.5 (n ⫽ 10), and 5.3 (n ⫽ 2) were selected for sequencing (one or two strains were selected from those isolated at the beginning of the epidemic and one or two strains isolated at the end of the epidemic, and the others were selected at random during the epidemic). All specific PCR products encoding SHV, with a pI of 8.2, were sequenced because SHV-5 and SHV-12 have the same pI values. Identification and nucleotide sequencing of the blaTEM, blaSHV, blaIMP, and blaVIM genes. The nucleotide sequences of the E. aerogenes blaTEM gene were compared to the known sequences of blaTEM-1, accession no. AF309824, and blaTEM-24, accession no. X65253 (10) (98 to 100% identity). Two muta-

3

2

10 11 12 13

14

5

6

23 24

25

8

9

30

11

31

29

Bronchial aspirate Bronchial aspirate

Anorectal swab

Bronchial aspirate Bronchial aspirate Deep pus Femoral catheter

Bile Blood

Blood Anorectal swab Peritoneal fluid Anorectal swab Stool

Bronchial aspirate Bone biopsy Anorectal swab Anorectal swab

Stool Deep pus Deep pus Anorectal swab

Polyvalent ICU

Hepatology and gastroenterology

General and visceral surgery








Polyvalent ICU

Ward

Clinical features

Deceased

Improved

Deceased

Deceased

Deceased

Deceased

Deceased

Deceased

Deceased

Deceased

Improved

Patient outcomeb

R

S

R

R S

S

S

S R

R S R R R

R S R

S

S R S S

R

R

R R

R

S

R

S R

Relevant resistance characteristic (IPM-S)c

TEM-24

TEM-20

TEM-24

TEM-20 TEM-20

TEM-24

TEM-24

TEM-24 TEM-24

6.5

5.4

6.5

5.4 5.4

6.5

6.5

6.5 6.5

6.5 6.5 6.5 6.5 6.5

6.5

TEM-24 TEM-24 TEM-24 TEM-24 TEM-24 TEM-24

6.5

6.5

6.5 6.5 6.5 6.5

6.5

6.5

6.5 6.5

6.5

6.5

6.5

6.5 6.5

pI value

TEM-24

TEM-24

TEM-24 TEM-24 TEM-24 TEM-24

TEM-24

TEM-24

TEM-24 TEM-24

TEM-24

TEM-24

TEM-24

TEM-24 TEM-24

TEM

SHV-12

SHV-12

SHV-12

SHV-12

SHV-12 SHV-12

SHV-12 SHV-12 SHV-12 SHV-12 SHV-12

SHV-12 SHV-12 SHV-12

SHV-12

SHV-12 SHV-12 SHV-12 SHV-12

SHV-12

SHV-12

SHV-12 SHV-12

SHV-12

SHV-12

SHV-12

SHV-12 SHV-12

SHV

8.2

8.2

8.2

8.2

8.2 8.2

8.2 8.2 8.2 8.2 8.2

8.2 8.2 8.2

8.2

8.2 8.2 8.2 8.2

8.2

8.2

8.2 8.2

8.2

8.2

8.2

8.2 8.2

pI value IMP

⫺ ⫹ ⫺ ⫺ ⫺

⫹ ⫺ ⫺ ⫺ ⫺ ⫺

⬎9.5

⫺ ⫹ ⫺ ⫺

⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺

⬎9.5 ⬎9.5 ⬎9.5 IMP-1 IMP-1 IMP-1

IMP-1

IMP-1

IMP-1

IMP-1

⫹

⫹

⫺

⫺ ⬎9.5

⫹

⫹

⬎9.5

⫹ ⫺

⫺ ⫺

⫹

⬎9.5

⬎9.5

⫹ ⫹ ⫹

⫹

⬎9.5 IMP-1

⫹

⫹ IMP-1

⫹

⫹

⬎9.5

⬎9.5

⫹

⫹

⬎9.5

⫹ ⫹

⫹ ⫹

⬎9.5 ⬎9.5

⫹

⫹

⫺

⫺ ⬎9.5

⫹

⫹

⬎9.5

⫹

aac(6⬘) -Ib

⫹

intL3

⬎9.5

pI value

Gene detected

⫹

VIM

OMP patterne

⬎9.5 IMP-1

IMP-1

IMP-1

IMP-1

IMP-1 IMP-1

IMP-1

IMP-1

IMP-1

␤-Lactamase gene productsd

A1

A

A

A A

A

A

A A

A A A A A

A A A

A

A B A A

A

A

A A

A

A

A

A A

PFGE type

BIENDO ET AL.

10

27 28

26

18 19 20 21 22

7

15 16 17

9

Bronchial aspirate

Urine Bronchial aspirate Anorectal swab

6 7

8

Blood

Vaginal exudate Stool

Stool Decubitus ulcer

Source of isolation

5

4

3

1 2

1

4

Isolate no.

Patient no.

TABLE 2. Characteristics of the 62 IPM-S or IPM-Ins ESBL-producing E. aerogenes isolates

1040 J. CLIN. MICROBIOL.

36 37

38 39

40 41

42 43 44

45 58

46

47 48 49 50

14

15

16

17

18

19

20

Polyvalent ICU



Polyvalent ICU

Polyvalent ICU


Polyvalent ICU

Polyvalent ICU



Respiratory ICU

Respiratory ICU

Improved

Deceased

Improved

Deceased

Improved

Improved

Improved

Improved

Improved

Deceased

R S

S

S R

R R R R

S R R

I I R S

R

R R

R S S

S R

R S

I R

S R

S

I

TEM-24

TEM-24

TEM-24

TEM-24 TEM-24 TEM-24

TEM-24 TEM-24

TEM-24 TEM-24

TEM-20 TEM-20

TEM-24

TEM-24

6.5

6.5

6.5

6.5 6.5 6.5

6.5 6.5

6.5 6.5

5.4 5.4

6.5

6.5

SHV-12 SHV-12




SHV-12

SHV-5 SHV-5


SHV-5 SHV-5

SHV-12 SHV-12

SHV-12 SHV-12

SHV-12

SHV-12

8.2 8.2

8.2 8.2 8.2 8.2

8.2 8.2 8.2

8.2 8.2 8.2 8.2

8.2

8.2 8.2

8.2 8.2 8.2

8.2 8.2

8.2 8.2

8.2 8.2

8.2

8.2

IMP-1

IMP-1

IMP-1 IMP-1

IMP-1

IMP-1

VIM-2 VIM-2

⫹

⬎9.5

⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺

OmpK32⫹ OmpK32⫹ OmpK32⫹ OmpK32⫹ OmpK42⫹ OmpK32⫹ OmpK42⫾ OmpK32⫹ OmpK42⫾ OmpK32⫹ OmpK32⫹ OmpK32⫹ OmpK32⫹ OmpK32⫹

OmpK42⫾ OmpK42⫾ OmpK42⫾ OmpK42⫾

OmpK42⫹ OmpK32⫹ OmpK42⫺ OmpK32⫹ OmpK42⫹ OmpK32⫹ OmpK42⫺ OmpK32⫹ OmpK45⫹ OmpK42⫹ OmpK38⫹ OmpK32⫹

OmpK42⫾ OmpK42⫾ OmpK42⫾ OmpK42⫹

⫹ ⫹

⫹ ⫹

5.3 5.3

⫺

⫺

⫺ ⫺

⫹ ⫹ ⫹ ⫹

⫺ ⫹ ⫹

⫹ ⫹ ⫹ ⫺

⫹

⫹ ⫺ ⫺

⫹ ⫺ ⫺

⫺ ⫹

⫹

OmpK42⫺ OmpK32⫹ OmpK42⫹ OmpK32⫹ OmpK42⫹ OmpK32⫹

⫺

⬎9.5

⫹ ⫹

⫺ ⫹

⫹ ⫹

⬎9.5 ⬎9.5

⫹

⫹

OmpK42⫹ OmpK32⫹ OmpK42⫾ OmpK32⫹

⫺

⫹

⬎9.5

⬎9.5

A3

A

C D

A1 A2 A1 A1

E E A2

F A E A

A

A A

A A A

A A

C A

A A

A A

A

A

c

b

Isolates 61 and 62 were derepressed cephalosporinase producers. The outcome was favorable with respect to the E. aerogenes infections. S, susceptible; I, intermediate; R, resistant. d The ESBL and MBL genes shown in bold type were sequenced. Some additional ESBLs and MBLs, shown in regular type, were identified by phenotypic, biochemical, and PCR tests, but the genes were not sequenced. e OmpK⫹, presence of Omp band; OmpK⫺, absence of Omp band; OmpK⫾, presence of weak Omp band.

a

61a

22

Anorectal swab Anorectal swab

Anorectal swab Decubitus ulcer Blood Pancreatic biopsy Urine Peritoneal fluid Pancreatic biopsy Deep pus Peritoneal fluid Wound exudate Blood

Intravascular catheter

Deep pus Bronchial aspirate

Anorectal swab Anorectal swab Jugular exudate

Anorectal swab Tracheal aspirate

Deep pus Anorectal swab

Urine Urine

Anorectal swab Bile

Bronchial aspirate Anorectal swab

Tracheal aspirate Blood 62a HB101 Strain

59 60

21

54 55 56 57

51 52 53

34 35

33

32

13

12

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BIENDO ET AL.

tions, T747C and G914A, resulted in two amino acid substitutions, M182T and G238S, in the TEM-20 ␤-lactamase with a pI of 5.4 (2), and five additional amino acid substitutions were observed, as previously described (9), for TEM-24 with a pI of 6.5. The SHV gene sequences were compared with those of blaSHV-5, accession no. AY386368, and blaSHV-12, accession no. AY008838. The blaSHV gene, which exhibited a 99 to 100% nucleotide homology, corresponded to the homologies observed for blaSHV-5 (6) and blaSHV-12 (22). Sequences for ESBL SHV enzymes, with a pI of 8.2, had a G-to-A mutation in the bla gene, resulting in amino acid substitutions G236S and E237K found in SHV-5 ␤-lactamase in four isolates. Two additional amino acid substitutions, G238S and E240K, were observed for SHV-12 ␤-lactamase in 54 isolates. The nucleotide sequences of the blaIMP-1 and blaVIM-2 genes from our E. aerogenes strains were identical to those known for genes encoding IMP-1 (100% homology with sequences for Serratia marcescens, accession no. S71932) (31) and VIM-2 (99% homology with sequences for Pseudomonas aeruginosa, accession no. AF191564 and AY242984) (34), respectively. These enzymes with pIs of ⬎9.5 and 5.3 corresponded to IMP-1 and VIM-2 ␤-lactamases, respectively. OMP characterization. Thirteen IPM-Ins and MBL-nonproducing isolates as well as 7 IPM-S and MBL-nonproducing isolates were characterized by SDS-PAGE. IPM-Ins and IPM-S strains demonstrated only two major proteins, of about 32 and 42 kDa. In contrast, E. coli HB101 produced four possible OMPs, of 45, 42, 38, and 32 kDa (Table 2). Three of thirteen IPM-Ins strains for which MICs of IPM were more than 32 ␮g/ml did not express the 42-kDa OMP but produced only the 32-kDa OMP, whereas the remaining 10 strains for which MICs of IMP were between 8 ␮g/ml and 32 ␮g/ml expressed both 32- and 42-kDa OMPs with a weak band of 42-kDa porin. Some minor nonspecific reactions were observed for the Western blot with endogenous E. coli porins, presumably because of the high degree of homology between enterobacterial porins. Integrons and integrase PCR results. The use of the 5⬘-CS and 3⬘-CS primers allowed the detection of class 1 integrons and the sizes of any inserted gene cassette. The frequency of integron carriage was 100% (39/39) of the E. aerogenes IPMIns isolates. The inserted gene cassette sizes that were detected were 1 and 2 kb. The integrase gene PCR showed a frequency of 79.4% (31/39) of IntL3-positive IPM-Ins isolates. The intL3 gene was found in 79.1% (19/24) of isolates of IMP-1 producers, and both of the VIM-2-producer isolates were intL3 positive. The PCR mapping technique revealed that 94.8% (37/39) of IPM-Ins isolates carried the aac(6⬘)-Ib genes. Of these, 24 isolates were associated with blaIPM-1, and two were associated with blaVIM-2. The sequences of intL3 and aac-(6⬘)-Ib products were 100% identical to previously published sequences (accession no. AF416297 [11] and accession no. 231133 [37a], respectively). Sequence analysis of both the 1-kb and the 2-kb integrons revealed a structure with at least two gene cassettes, containing blaIMP-1 and aac-(6⬘)-Ib. Molecular epidemiology. Six SpeI patterns, named A to F, were found among the 62 E. aerogenes isolates. The major epidemic pattern A comprised 87% (54/62) of isolates genetically related to subtypes A1 (1 isolate), A2 (2 isolates), A3 (1

J. CLIN. MICROBIOL.

isolate), and A4 (1 isolate). The ␤-lactam phenotype a was present in 59.2% (32/54) of the isolates, phenotype b was present in 37% (20/54) of the isolates, and phenotypes c and d were present in 1.9% (1/54) of pulsotype A. Profiles B, C, D, E, and F corresponded to sporadic cases (only one isolate, except for C and E, which had two and three isolates, respectively) (Table 2). Pulsotype A was identified from patients on all wards included in this study. At the 73% similarity breakpoint, the patterns were categorized into five clusters (I to V). Cluster II contained 87% of the isolates. The percentage of similarity between isolates of this cluster reached at least 80% and belonged to the PFGE pattern A. For the whole set of isolates, the index of diversity was 0.10, indicating their homogeneity and their clonality. DISCUSSION Enterobacter aerogenes is a common agent of hospital-acquired infection. Isolates generally exhibit high resistance to broad-spectrum antibiotics, with the exception of IPM. The emergence of extended-spectrum cephalosporin- and carbapenem-resistant strains has been documented (3). IPM was first used in our institution in 1987. For 15 years, we did not observed any IPM-Ins Enterobacteriaceae strains. Since 2002, resistance to IPM has been observed with E. aerogenes isolated from four wards at A.U.H. The occurrence of these multiresistant bacteria is a crucial problem, as no antibiotic options are available. In every case, the survival of patients is seriously impaired. Throughout the epidemic, we observed the coexistence of IPM-S and IPM-Ins ESBL-producing strains in the same patients (16 of 22 studied patients) over a period of time. Among these patients, 11 had stool and anorectal screening samples that were positive for E. aerogenes ESBL IPM-S strains. Our data suggest that most hospitalized patients included in this study harbored E. aerogenes strains within their endogenous digestive flora and that they were initially colonized before their admission to the hospital. In these previously colonized patients, the substitution of the original strain with a multiresistant strain generally occurs during hospitalization. This can be explained either by the selection of resistant strains after broad-spectrum antibiotic therapy or cross-transmission from critically ill, heavily colonized patients who require prolonged and intensive nursing and medical care. Most of our patients had malignant diseases. They had undergone digestive surgery and had received IPM therapy or an expanded-spectrum ␤-lactam plus G. This suggests that malignancy, surgery, and selective pressure from antibiotics constitute a risk factor for the acquisition of ␤-lactamase-producing isolates. E. aerogenes infections are difficult to treat because of their intrinsic resistance, and IPM has generally been used successfully. In the case of treating IPM-Ins strains, we proposed the combination of sulbactam-ampicillin plus FEP but without much success. The use of FEP as an alternative therapy is highly controversial (17). Thirteen of twenty-two patients (59%) in this study died during the study period from septic shock or from their underlying disease. De Gheldre et al. (15) reported a crude mortality rate of 38% in their series. Our study suggests the spread of a prevalent clone through

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ESBL- AND MBL-PRODUCING E. AEROGENES ISOLATES

four wards at our hospital, which is in agreement with data in the literature (8). This prevalent clone had plasmids carrying genes coding for ESBLs (TEM-24 and SHV-12) and MBL (IMP-1). At the same time, nine isolates of the prevalent clone may have acquired additional plasmid ␤-lactamases, SHV-5 in four cases and TEM-20 in five cases. Some ESBL-producing E. aerogenes isolates present sporadic pulsotypes and produce the same enzymes (TEM-24, SHV-12, and IMP-1). This might be explained by an epidemic of plasmids and/or transposons. It should be noted that two VIM-2-producing Ipmr strains were isolated from samples obtained from one patient at the end of the epidemic. This may suggest the risk of emergence of other VIM-2 producers, but this was not the case in the present study, probably because of the drastic epidemic control measures implemented that ensured eradication of these strains. The combination analysis of blaIMP-1 and blaVIM-2 with the 5⬘-CS and 3⬘-CS primers for the detection of class 1 integrons indicated that blaIMP-1 and blaVIM-2 are part of a class 1 integron. Based on the sizes of the amplified fragments, the structure of the integron of E. aerogenes was identical to that found in Pseudomonas aeruginosa (25, 40). The fact that the aac(6⬘)-Ib and the intL3 genes were amplified from blaIMP-1- and blaVIM-2-positive E. aerogenes strains implies that the aac(6⬘)-Ib and intL3 genes are well conserved among these blaIMP- and blaVIM-positive strains. Interestingly, most isolates that expressed either IMP-1 or VIM-2 ␤-lactamases were resistant to most aminoglycosides, thus limiting the choice of drugs active against multiresistant E. aerogenes. The aac(6⬘)-Ib-encoded enzyme, a 6⬘-Nacetylaminoglycoside acetyltransferase type I [AAC(6⬘)-I], was characterized by one amino acid substitution (L119S). This substitution was responsible for the susceptibility of G and the resistance of T, N, and A (24). Increased MICs for carbapenems were observed with thirteen ESBL-producing E. aerogenes strains. Screening for carbapenemases was negative for these strains. The OMP characterization results showed that there were five patients (patients 16, 17, 20, 21, and 22) (Table 2) infected with both IPM-S and IPM-resistant isolates. We did not quantify the level of production of the ␤-lactamases; however, the differences in susceptibility appeared to be the loss or reduction of the 42-kDa porin. Decreased susceptibility to carbapenems is uncommon in ESBL-producing E. aerogenes isolates. It has been demonstrated that IPM resistance in enterobacteria due to a decrease in OMP requires the expression of the AmpC chromosomal ␤-lactamase or a class A ESBL (26). The emergence of E. aerogenes strains with a decreased susceptibility to IPM is of concern. Arpin et al. reported that 4.6% of their E. aerogenes strains were resistant to IPM (3). Moreover, Malleá et al. (27) described clinical E. aerogenes strains presenting a complex resistance strategy associating ␤-lactamase production, impermeability, and active efflux. In our study, the efflux mechanism cannot be excluded but was not specifically investigated. In conclusion, this study demonstrated the spread of a multidrug-resistant E. aerogenes clone particularly well adapted to the hospital environment. Strict infection control measures against such isolates should be implemented to prevent their further dissemination throughout the hospital. The careful antibiotic usage policy in hospital wards and the need for close

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bacteriologic monitoring of the local ecology seem to be the most important measures to stop this development. ACKNOWLEDGMENTS We thank Patrice Courvalin of the Centre National de Reference des Antibiotiques, Institut Pasteur, Paris, for kindly providing us with the E. aerogenes Ipmr control strains used in this study. REFERENCES 1. Alberti, S., G. Marque´s, S. Camprubi, S. Merino, J. M. Tomas, F. Vivanco, and V. J. Benedi. 1993. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins. Infect. Immun. 61:852–860. 2. Arlet, G., S. Goussard, P. Courvalin, and A. Philippon. 1999. Sequences of the genes for the TEM-20, TEM-21, TEM-22, and TEM-29 extended-spectrum ␤-lactamases. Antimicrob. Agents Chemother. 43:969–971. 3. Arpin, C., C. Coze, A. M. Rogues, J. P. Gachie, C. Bebear, and C. Quentin. 1996. Epidemiological study of an outbreak due to multidrug-resistant Enterobacter aerogenes in a medical intensive care unit. J. Clin. Microbiol. 34:2163–2169. 4. Biendo, M., G. Laurans, J. F. Lefebvre, F. Daoudi, and F. Eb. 1999. Epidemiological study of an Acinetobacter baumannii outbreak by using a combination of antibiotyping and ribotyping. J. Clin. Microbiol. 37:2170–2175. 5. Biendo, M., G. Laurans, B. Canarelli, D. Thomas, F. Hamdad-Daoudi, F. Rousseau, C. Adjide, and F. Eb. 2006. Emergence of Enterobacter aerogenes strains resistant to imipenem mediated by the coexistence of metallo-betalactamase production and outer membrane permeability, abstr. P1246, p. 112. Abstr. 16th European Congress of Clinical Microbiology and Infectious Diseases, Nice, France. 6. Billot-Kein, D., L. Gutmann, and E. Collatz. 1990. Nucleotide sequence of the SHV-5 ␤-lactamase gene of a Klebsiella pneumoniae plasmid. Antimicrob. Agents Chemother. 34:2439–2441. 7. Blake, M. S., K. H. Johnston, G. J. Russell-Jones, and E. C. Gotschlich. 1984. A rapid, sensitive method for detection of alkaline phosphate-conjugated anti-antibody on Western blots. Anal. Biochem. 136:175–179. 8. Bosi, C., A. Davin-Regli, C. Bornet, M. Malleá, J-M Page`s, and C. Bollet. 1999. Most Enterobacter aerogenes strains in France belong to a prevalent clone. J. Clin. Microbiol. 37:2165–2169. 9. Bush, K., and G. Jacoby. 1997. Nomenclature of TEM ␤-lactamases. J. Antimicrob. Chemother. 39:1–3. 10. Chanal, C., M. C. Poupart, D. Sirot, R. Labia, J. Sirot, and R. Cluzel. 1992. Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 ␤-lactamase genes. Antimicrob. Agents Chemother. 36:1817–1820. 11. Collis, C. M., M. J. Kim, S. R. Partridge, H. W. Stokes, and R. M. Hall. 2002. Characterization of the class 3 integron and the site-specific recombination system it determines. J. Bacteriol. 184:3017–3026. 12. Comite´ de l’Antibiogramme de la Socie´te´ Française de Microbiologie (CASFM). 2004. Concentrations, diame`tres, critiques et re`gle de lecture interpre´tative pour Enterobacteriaceae, p. 25–28. In Communique´ 2004. Socie´te´ Française de Microbiologie, Paris, France. 13. Davin-Regli, A., D. Monnet, P. Saux, C. Bosi, R. Charrel, A. Barthelemy, and C. Bonnet. 1996. Molecular epidemiology of Enterobacter aerogenes acquisition: one-year prospective study in two intensive care units. J. Clin. Microbiol. 34:1474–1480. 14. De Champs, C., C. Henquell, D. Guelon, D. Sirot, N. Gazuy, and J. Sirot. 1993. Clinical and bacteriological study of nosocomial infections due to Enterobacter aerogenes resistant to imipenem. J. Clin. Microbiol. 31:123–127. 15. De Gheldre, Y., N. Maes, F. Rost, R. De Ryck, P. Clevenbergh, J. L. Vincent, and M. J. Struclens. 1997. Molecular epidemiology of an outbreak of multidrug-resistant Enterobacter aerogenes infections and in vivo emergence of imipenem resistance. J. Clin. Microbiol. 35:152–160. 16. Filip, C., G. Fletcher, J. L. Wulf, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodiumlauryl sarcosinate. J. Bacteriol. 115:717–722. 17. Goethaert, K., M. Van Looveren, C. Lammens, H. Jansens, A. Baraniak, M. Gniadkowski, K. Van Herck, P. G. Jorens, H. E. Demey, M. Ieven, L. Bossaert, and H. Goossens. 2006. High-dose cefepime as an alternative treatment for infections caused by TEM-24 ESBL-producing Enterobacter aerogenes in severely-ill patients. Clin. Microbiol. Infect. 12:56–62. 18. Hubert, T. W., and J. S. Thomas. 1994. Detection of resistance due to inducible ␤-lactamase in Enterobacter aerogenes and Enterobacter cloacae. J. Clin. Microbiol. 32:2481–2486. 19. Jalaluddin, S., J.-M. Devaster, R. Scheen, M. Gerard, and J.-P. Butzler. 1998. Molecular epidemiological study of nosocomial Enterobacter aerogenes isolates in a Belgian hospital. J. Clin. Microbiol. 36:1846–1852. 20. Reference deleted. 21. Kaye, K. S., J. J. Engemann, H. S. Fraimow, and E. Abrutyn. 2004. Pathogens resistant to antimicrobial agents: epidemiology, molecular mechanisms, and clinical management. Infect. Dis. Clin. N. Am. 18:467–511.

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