JAC Advance Access originally published online on January 28, 2003
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Journal of Antimicrobial Chemotherapy (2003) 51, 565-574
© 2003 The British Society for Antimicrobial Chemotherapy
Relationship between ß-lactamase production, outer membrane protein and penicillin-binding protein profiles on the activity of carbapenems against clinical isolates of Acinetobacter baumannii
1 Department of Microbiology, University of Seville, Apdo. 914, 41009 Seville; 2 University Hospital Virgen Macarena, Seville; 3 Center of Molecular Biology ‘Severo Ochoa’, Autonomous University of Madrid, Madrid, Spain
Received 30 July 2002, returned 25 September 2002, revised 14 November 2002; accepted 20 November 2002
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Abstract |
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Twenty blood isolates of Acinetobacter baumannii were studied, representing eight pulsed-field gel electrophoresis patterns and all different antimicrobial susceptibility patterns observed during 1995–97 at the University Hospital Virgen Macarena, Seville, Spain. The MIC90s (mg/L) of imipenem and meropenem decreased from 16 to 0.5 and from 8 to 4, respectively, in the presence of BRL 42715 (BRL) but not clavulanic acid. Hydrolysing activity (nmol/min/mg) of bacterial supernatants against cefaloridine ranged from 8.8 to 552.3 for A. baumannii type I (imipenem MICs
2), which expressed only a ß-lactamase of pI 
9, and from 12.3 to 1543.5 for A. baumannii type II (imipenem MICs 4), which expressed a ß-lactamase of pI 9 and two others of pI 6.3 and 7. The hydrolysing activities of A. baumannii type II against imipenem, meropenem and oxacillin were higher than those observed for A. baumannii type I. Ten outer membrane protein (OMP) profiles (A. baumannii types I and II) were visualized on 10% SDS–PAGE gels with 6 M urea, whereas only five OMP profiles (A. baumannii types I and II) were differentiated in 12% SDS–PAGE gels. Five A. baumannii with OMP profile type B, characterized by the absence of a 22.5 kDa OMP, were resistant to meropenem and/or imipenem. Twelve penicillin-binding protein (PBP) patterns were observed. PBP patterns of A. baumannii type II were characterized by the absence of a 73.2 kDa band (PBP 2). We concluded that production of ß-lactamases of pI 6.3 and 7.0 and reduced expression of PBP 2 are the most frequently observed mechanisms of resistance to carbapenems. In some isolates, loss of a 22.5 kDa OMP is also related to resistance to carbapenems. Keywords: Acinetobacter baumannii, carbapenems, ß-lactamases, outer membrane proteins, penicillin-binding proteins
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Antimicrobial therapy is limited against infections caused by strains of Acinetobacter baumannii resistant to carbapenems.1,2 The mechanisms underlying resistance to carbapenems in A. baumannii are still poorly understood, but they would be expected to be similar to those described in other Gram-negative bacteria (GNB): production of carbapenem-hydrolysing ß-lactamases (carbapenemases),3–13 decreased outer membrane permeability caused by the loss or reduced expression of porins,14–18 overexpression of multidrug efflux pumps19,20 and alterations in penicillin-binding proteins (PBPs).21–23 A combination of several mechanisms may be present in the same microorganism, as has also been observed in other GNB.16,19,24–26
The aim of this study is to evaluate the role of ß-lactamase production, outer membrane proteins (OMPs) and PBPs on the activity of carbapenems against clinical isolates of A. baumannii.
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Materials and methods |
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Bacterial strains
Twenty non-consecutive A. baumannii isolates obtained from blood (January 1995 to December 1997) at the Department of Microbiology, University Hospital Virgen Macarena, Seville, Spain were studied. Isolates represented all the different antimicrobial resistance patterns within the same or different DNA genetic profiles, as defined by pulsed-field gel electrophoresis (PFGE) (see below). Reference strains used in this study are listed in Table 1.
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Identification and typing methods
Preliminary identification and susceptibility to antimicrobials were determined using the Neg-Combo 6I type panels and the Walk-Away system (Dade-Behring, Sacramento, CA, USA). Definitive identification and biotyping were carried out according to the biochemical scheme described by Bouvet & Grimont.29
Genetic typing of genomic DNA digested with SmaI (Boehringer-Mannheim, Madrid, Spain) was carried out by PFGE as described by Allardet-Servent et al.30 PFGE patterns were compared using the recommendations of Tenover et al.31
Antimicrobial susceptibility testing
A microdilution assay was used according to the NCCLS guidelines.32 Ampicillin, piperacillin, cefoxitin, cefaloridine, cefotaxime, gentamicin, tobramycin, amikacin, tetracycline and ciprofloxacin were from Sigma (Madrid, Spain); ticarcillin, clavulanic acid and BRL 42715 (BRL) were from SmithKline Beecham (Madrid, Spain); meropenem was from Zeneca Farma (Madrid, Spain); ceftazidime was from Glaxo–Wellcome (Madrid, Spain); cefepime and aztreonam were from Bristol-Myers Squibb (Madrid, Spain); imipenem was from Merck Sharp & Dohme (Madrid, Spain); and sulbactam was from Pfizer (Madrid, Spain).
The MICs of imipenem and meropenem were also determined in the presence of inhibitors of serine ß-lactamases clavulanic acid (2 mg/L) and BRL (4 mg/L).
Isoelectric focusing of ß-lactamases
Crude extracts of sonicated cells were concentrated by filtration (Pall Filtron 10K; Northborough, MA, USA) and subjected to isoelectric focusing (IEF), using the PhastSystem apparatus (Pharmacia Biotech, Uppsala, Sweden).
Bands of ß-lactamases were visualized with 500 mg/L nitrocefin (Oxoid, Madrid, Spain). The isoelectric point (pI) of ß-lactamases and their inhibition profiles were determined as described previously.33
Hydrolysing activity of ß-lactamases
The hydrolysing activity of ß-lactamases (concentrated crude extracts of sonicated cells) against 0.1 mM cefaloridine (Sigma), 1 mM oxacillin (Sigma) and 0.1 mM imipenem and meropenem was determined by UV spectrophotometry (DU 640; Beckman, Fullerton, CA, USA).
One unit of hydrolytic activity (U) against cefaloridine, oxacillin, imipenem or meropenem was defined as the amount of enzyme that hydrolyses 1 µmol of the respective ß-lactam per minute.
Inactivation of ticarcillin, ceftazidime, imipenem, meropenem and oxacillin was tested by the cloverleaf34 and double disc35 methods.
Analysis of OMP profiles
OMP profiles were studied by SDS–PAGE, using both 12% polyacrylamide gels and 10% polyacrylamide gels with 6 M urea, as described previously.36 Proteins were stained with Coomassie Blue R-250 (Sigma). A commercial kit (SDS–PAGE Standards, low-range; Bio-Rad) was used for molecular weight standards.
Labelling and detection of PBPs
A conjugate of iodine-125 (Bolton and Hunter reagent; Pharmacia Biotech, Barcelona, Spain) and ampicillin was used for labelling PBPs.37,38 Binding assays of PBPs were carried out according to the method of Spratt.39 Samples (
50 µg) were fractionated on 12% SDS–PAGE, using the SE 200 Mighty Small II system (Hoefer, Amersham Pharmacia Biotech).
Bands of PBPs were visualized by autoradiography (AGFA CURIX films, Madrid, Spain). Radioactivity of the 125I-labelled PBPs was quantified using the CYCLON Storage Phosphor System (Packard, La Jolla, CA, USA). Arbitrary units of radioactivity (AURs) were determined by calculating the height of the peaks of radioactivity. AURs were expressed as relative units (URLs) [with reference to that of PBP 5 (100% intensity) of every isolate], because the band corresponding to this PBP was homogeneously labelled in all the isolates evaluated.
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Results |
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Typing methods
The 20 isolates were identified as A. baumannii. Five biotypes (2, 6, 9, 11 and 18) and eight PFGE patterns (A–H) were recognized (Table 2).
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Antimicrobial susceptibility testing
Ampicillin, piperacillin, ticarcillin, cefaloridine, cefoxitin and cefotaxime were the ß-lactams with the lowest antimicrobial activity against the A. baumannii evaluated (MIC90s 512 mg/L) (Table 2). Cefepime (MIC90s 4 mg/L) was four times more active than ceftazidime (MIC90s 16 mg/L).
Carbapenem MICs (mg/L) ranged from 0.06 to 32 (imipenem) and from 0.5 to 16 (meropenem). Two types of isolate were defined with respect to the MICs of imipenem: type I (MIC range 0.06–2 mg/L) and type II (MIC range 4– 32 mg/L).
Sulbactam was the ß-lactamase inhibitor tested with the highest intrinsic activity: MIC range 1–32 mg/L. MICs of clavulanic acid and BRL were 64 mg/L for all isolates. The MIC90s of carbapenems were unaffected (meropenem) or decreased twice (imipenem) in the presence of clavulanic acid. The MIC90s of imipenem were reduced four times (type I isolates) and 128 times (type II isolates) in the presence of BRL (Table 3). In contrast, the MIC90s of meropenem were not reduced (type I isolates) or decreased four times (type II isolates) when this carbapenem was combined with BRL.
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MICs of gentamicin, amikacin, tobramycin and tetracycline showed a biotype-dependent variation. All isolates were resistant to ciprofloxacin (MIC range 8–512 mg/L), except HUS 167 (MIC
0.06 mg/L). Isoelectric focusing of ß-lactamases
A band of pI 9 inhibited by cloxacillin but not clavulanic acid or EDTA was observed in all the isolates (Table 3). Two bands of pIs 7.0 and 6.3, respectively, which were weakly inhibited by clavulanic acid but not by either cloxacillin or EDTA, were also expressed in type II, but not in type I isolates. The band of pI 5.8 (inhibited by EDTA) was detected in six type II isolates. Finally, a band of pI 5.4 (inhibited only by clavulanic acid) was observed in seven type II isolates.
Hydrolysis of cefaloridine, carbapenems and oxacillin
Hydrolysis of cefaloridine (mU/mg of protein) ranged from 18.8 to 552.2 (type I isolates) and from 12.3 to 1543.5 (type II isolates) (Table 3). The hydrolysing activities of type II isolates against carbapenems and oxacillin were higher than those observed in type I isolates (Table 3). For type I isolates these values (mU/mg) ranged between not detectable and 0.11 (imipenem), not detectable and <0.05 (meropenem), and not detectable and 120.1 (oxacillin), whereas those for type II isolates ranged between 0.41 and 1.5 (imipenem), 0.18 and 0.88 (meropenem), and 126.9 and 506.5 (oxacillin).
Hydrolysis of ticarcillin and imipenem was detected in all isolates using the cloverleaf method, whereas hydrolysis of oxacillin, meropenem and ceftazidime was only detected in 18, 16 and 11 isolates, respectively (Table 3). Using the double disc method, hydrolysis was detected in 18 (ticarcillin and oxacillin), 17 (imipenem), 10 (meropenem) and seven (ceftazidime) isolates. The agreement obtained between the cloverleaf method and the double disc method was 90% (ticarcillin, oxacillin), 85% (imipenem), 80% (ceftazidime) and 70% (meropenem).
OMP profiles
Ten OMP patterns were observed in 10% SDS–PAGE gels with 6 M urea, whereas only five OMP patterns were observed in 12% SDS–PAGE gels without urea (Figures 1 and 2; Table 4). The most representative profile A1 (n = 6) observed in 10% SDS–PAGE gels with 6 M urea showed six bands of 48.3 (Omp 1), 41.0 (Omp 2a), 33.0 (Omp 3a), 26.5 (Omp 4a), 24.0 (Omp 5a) and 23.7 kDa (Omp 5b), and one band of variable electrophoretic mobility (40–45 kDa; Omp 2b). In 12% SDS–PAGE gels, the most frequently observed profiles were A1* (n = 6), A2* (n = 6) and B* (n = 5). Seven major bands with relative mobilities of 43.3 (Omp 1a*), 34.8 (Omp 2*), 32.3 (Omp 3b*), 30.2 (Omp 4a*), 25.8 kDa (Omp 5*), 23.7 (Omp 6*) and 22.5 kDa (Omp 7*) were present in OMP profile A1*. Profile A2* differed from profile A1* in the absence of Omp 5*. Profile B* (n = 5) was characterized by the presence of four bands similar to Omp 1a*, Omp 2*, Omp 3b* and Omp 6*, the presence of one band of 31.4 kDa (Omp 3c*), instead of Omp 3b*, and by the absence of Omp 7*.
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PBP profiles
Twelve highly complex patterns of PBPs were observed. Five bands of 93 (PBP 1a), 64 (PBP 3), 49 (PBP 4), 47 (PBP 4b) and 38 kDa (PBP 5) were expressed in type I and II isolates, whereas the other bands showed a variable distribution. The PBP pattern of type II isolates differed from those of type I isolates by the absence of one band of 73 kDa, corresponding to PBP 2 (Figure 3).
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For type I isolates, the percentage of radioactivity (URLs) of the 125I-labelled PBPs ranged between 425.8 and 2397.2% (PBP 1a), 110.1 and 477.2% (PBP 1c), 109.6 and 503.1% (PBP 2), 450.1 and 3352.2% (PBP 3), and 93.9 and 153.7% (PBP 4), except for HUS 431, which showed the lowest URLs of type I isolates (Table 5). For type II isolates, however, these values were lower than those for type I isolates: 53.5–307.9% (PBP 1a), 43.8–113.8% (PBP 1c), 168.9–556.6% [(PBP 3), except for HUS 457 (803.75%)] and 68.6–116.3% (PBP 4/4b).
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Discussion |
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The A. baumannii isolates we studied showed susceptibility or moderate resistance to imipenem and meropenem (MICs of both carbapenems were
32 mg/L). The synergic effect of BRL on the activity of imipenem and, to a lesser extent, meropenem indicates that production of serine ß-lactamases is involved in the resistance to carbapenems.
A great diversity of ß-lactamases was observed in most type II isolates. The band of pI 9 presumably corresponds to a non-inducible AmpC-type cephalosporinase, as Bou & Martínez-Beltrán40 have demonstrated in another study. Cephalosporinase activity, defined as hydrolysis of cefaloridine, did not significantly correlate with the MICs of imipenem and meropenem, suggesting that the expression of AmpC does not, per se, contribute to resistance to carbapenems.16,24,25
The expression of three bands of pI 6.3, 7.0 and 5.8 was associated with resistance to carbapenems, whereas the band of pI 5.4 (probably a TEM-type ß-lactamase) was unrelated.41,42
The inhibition profile of bands of pI 6.3 and 7.0, and the highest hydrolysing activity of type II isolates (expressing these enzymes) against imipenem, meropenem and oxacillin, suggest that these ß-lactamases are oxacillinases with a moderate hydrolysing activity against carbapenems. In contrast, the band of pI 5.8 was inhibited by EDTA and may be related to metallo-ß-lactamases. Additional molecular and biochemical studies are in progress to determine the relationship of these ß-lactamases to those previously described in A. baumannii.3–13
Our isolates showed no decreased expression of the 33–36 kDa OMP reported by Clark.18 Carbapenem resistance for isolates with OMP profile B was related to the absence of a 22.5 kDa OMP (Omp 7*). This observation was supported by the results of BRL on the activity of imipenem and meropenem: for type II isolates lacking Omp 7*, the BRL decreased the MICs of imipenem and meropenem less than for type II isolates expressing this OMP. Bou et al.26 have observed isolates of A. baumannii resistant to carbapenems lacking a 22 kDa OMP, although its role in resistance to carbapenems has not been evaluated. The amino acid sequence of this protein of 22 kDa and Omp 7*, and studies of proteoliposomes reconstituted with these two proteins, will elucidate whether they really are porins, and will help to determine their relationship to antimicrobial resistance.
A wide variation of PBP patterns was observed using the 125I-ampicillin reagent. The absence (or reduced expression) of one band of PBP, which we named PBP 2, was related to decreased susceptibility or resistance to carbapenems, in accordance with the results obtained by Neuwirth et al.21 in a clinical strain of Proteus mirabilis, for which resistance to carbapenems was due to the decreased affinity of PBP 2 (with similar mobility to the PBP 2 of our A. baumannii) to imipenem. Competition assays with imipenem and/or meropenem and 125I-ampicillin, or studies with imipenem and/or meropenem labelled with the Bolton and Hunter reagent, are necessary to find out the precise role of PBP 2 in the resistance of A. baumannii to carbapenem compounds.
Hyperproduction of the low molecular weight protein of 24 kDa (not saturable by imipenem) reported in the study by Gehrlen et al.23 was not observed in our isolates.
PBP bands of type II isolates showed lower radioactivity intensity (% URLs) than those of type I isolates. This may be due to hydrolysis of 125I-ampicillin by residual amounts of ß-lactamases (probably those of pI 6.3 and 7.0) not removed from membrane preparations after extensive and vigorous washing (three times of 1 min each in vortex). This problem could be resolved by using a potent inhibitor of ß-lactamases with a low affinity to PBPs, such as BRL 42715. Unfortunately, neither this compound nor any other with similar properties is available, which makes necessary the evaluation of other strategies of PBP analysis in A. baumannii.
Other possible mechanisms of resistance to carbapenems, such as the overproduction of efflux pumps19,20 or lipopolysaccharide alterations,43 could be expressed in some of our isolates for which the MICs of imipenem and meropenem were not completely related to the presence of any of the mechanisms evaluated in this study.
In conclusion, for the A. baumannii evaluated in our study, the mechanisms of resistance to carbapenems are multiple, with the production of oxacillinases and the absence of PBP 2 being most frequently observed. For some isolates, resistance to carbapenems is also related to the absence of an OMP of 22.5 kDa. Additional studies with isogenic mutants, which do or do not express these mechanisms individually and in combination with each other (double and triple mutants), must be developed to understand the precise role of these mechanisms in the resistance of A. baumannii to carbapenems.
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Acknowledgements |
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We thank P. Nordmann (Service de Bactériologie-Virologie, Hôpital de Bicêtre, France) for the gift of E. coli JM 109, E. Flores (Institute of Biochemistry of the Isla de la Cartuja of Seville, Spain) for excellent technical assistance in the quantification of PBPs, and E. Ramírez (University Hospital Vírgen Macarena of Seville, Spain) for her inestimable help in the PFGE study. We gratefully acknowledge the assistance of Janet Dawson in the preparation of the manuscript. We also thank the ‘Sociedad de Enfermedades Infeccciosas y Microbiología Clínica’ for their partial funding of the study of PBPs. This study was supported by grant 98/1027 from the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain.
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Footnotes |
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* Corresponding author. Tel: +34-95-455-2862; Fax: +34-95-437-7413; E-mail: felipefc{at}supercable.es
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