Journal of Antimicrobial Chemotherapy (2003) 51, 565–574DOI: 10.1093/jac/dkg097Advance Access publication 28 January 2003

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© 2003 The British Society for Antimicrobial Chemotherapy

Relationship between β-lactamase production, outer membrane protein and penicillin-binding protein profiles on the activity of carbapenems

against clinical isolates of Acinetobacter baumannii

Felipe Fernández-Cuenca1*, Luis Martínez-Martínez1,2, Mª Carmen Conejo1, Juan A. Ayala3,

Evelio J. Perea1,2 and Alvaro Pascual1,2

1Department of Microbiology, University of Seville, Apdo. 914, 41009 Seville; 2University Hospital Virgen Macarena, Seville; 3Center 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

Twenty blood isolates of Acinetobacter baumannii were studied, representing eight pulsed-fieldgel electrophoresis patterns and all different antimicrobial susceptibility patterns observedduring 1995–97 at the University Hospital Virgen Macarena, Seville, Spain. The MIC90s (mg/L) ofimipenem and meropenem decreased from 16 to 0.5 and from 8 to 4, respectively, in the presenceof BRL 42715 (BRL) but not clavulanic acid. Hydrolysing activity (nmol/min/mg) of bacterialsupernatants against cefaloridine ranged from 8.8 to 552.3 for A. baumannii type I (imipenemMICs ≤ 2), which expressed only a β-lactamase of pI ≥ 9, and from 12.3 to 1543.5 for A. baumanniitype II (imipenem MICs ≥ 4), which expressed a β-lactamase of pI ≥ 9 and two others of pI 6.3 and7. The hydrolysing activities of A. baumannii type II against imipenem, meropenem and oxacillinwere higher than those observed for A. baumannii type I. Ten outer membrane protein (OMP) pro-files (A. baumannii types I and II) were visualized on 10% SDS–PAGE gels with 6 M urea, whereasonly 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, wereresistant to meropenem and/or imipenem. Twelve penicillin-binding protein (PBP) patterns wereobserved. PBP patterns of A. baumannii type II were characterized by the absence of a 73.2 kDaband (PBP 2). We concluded that production of β-lactamases of pI 6.3 and 7.0 and reducedexpression of PBP 2 are the most frequently observed mechanisms of resistance to carba-penems. In some isolates, loss of a 22.5 kDa OMP is also related to resistance to carbapenems.

Keywords: Acinetobacter baumannii, carbapenems, β-lactamases, outer membrane proteins, penicillin-binding proteins

Introduction

Antimicrobial therapy is limited against infections caused bystrains of Acinetobacter baumannii resistant to carbapenems.1,2

The mechanisms underlying resistance to carbapenems inA. baumannii are still poorly understood, but they would beexpected to be similar to those described in other Gram-nega-tive bacteria (GNB): production of carbapenem-hydrolysingβ-lactamases (carbapenemases),3–13 decreased outer mem-brane 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 inthe same microorganism, as has also been observed in otherGNB.16,19,24–26

The aim of this study is to evaluate the role of β-lactamaseproduction, outer membrane proteins (OMPs) and PBPs onthe activity of carbapenems against clinical isolates ofA. baumannii.

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*Corresponding author. Tel: +34-95-455-2862; Fax: +34-95-437-7413; E-mail: felipefc@supercable.es

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Materials and methods

Bacterial strains

Twenty non-consecutive A. baumannii isolates obtained fromblood (January 1995 to December 1997) at the Department ofMicrobiology, University Hospital Virgen Macarena, Seville,Spain were studied. Isolates represented all the different anti-microbial resistance patterns within the same or differentDNA genetic profiles, as defined by pulsed-field gel electro-phoresis (PFGE) (see below). Reference strains used in thisstudy are listed in Table 1.

Identification and typing methods

Preliminary identification and susceptibility to antimicro-bials were determined using the Neg-Combo 6I type panelsand the Walk-Away system (Dade-Behring, Sacramento,CA, USA). Definitive identification and biotyping werecarried out according to the biochemical scheme described byBouvet & Grimont.29

Genetic typing of genomic DNA digested with SmaI(Boehringer-Mannheim, Madrid, Spain) was carried out byPFGE as described by Allardet-Servent et al.30 PFGE patternswere compared using the recommendations of Tenover et al.31

Antimicrobial susceptibility testing

A microdilution assay was used according to the NCCLSguidelines.32 Ampicillin, piperacillin, cefoxitin, cefaloridine,cefotaxime, gentamicin, tobramycin, amikacin, tetracyclineand ciprofloxacin were from Sigma (Madrid, Spain); ticar-cillin, clavulanic acid and BRL 42715 (BRL) were fromSmithKline Beecham (Madrid, Spain); meropenem was fromZeneca Farma (Madrid, Spain); ceftazidime was from Glaxo–Wellcome (Madrid, Spain); cefepime and aztreonam werefrom Bristol-Myers Squibb (Madrid, Spain); imipenem wasfrom Merck Sharp & Dohme (Madrid, Spain); and sulbactamwas from Pfizer (Madrid, Spain).

The MICs of imipenem and meropenem were also deter-mined in the presence of inhibitors of serine β-lactamasesclavulanic acid (2 mg/L) and BRL (4 mg/L).

Isoelectric focusing of β-lactamases

Crude extracts of sonicated cells were concentrated by fil-tration (Pall Filtron 10K; Northborough, MA, USA) and sub-jected to isoelectric focusing (IEF), using the PhastSystemapparatus (Pharmacia Biotech, Uppsala, Sweden).

Bands of β-lactamases were visualized with 500 mg/Lnitrocefin (Oxoid, Madrid, Spain). The isoelectric point (pI)of β-lactamases and their inhibition profiles were determinedas described previously.33

Hydrolysing activity of β-lactamases

The hydrolysing activity of β-lactamases (concentrated crudeextracts of sonicated cells) against 0.1 mM cefaloridine(Sigma), 1 mM oxacillin (Sigma) and 0.1 mM imipenem andmeropenem was determined by UV spectrophotometry (DU640; Beckman, Fullerton, CA, USA).

One unit of hydrolytic activity (U) against cefaloridine,oxacillin, imipenem or meropenem was defined as the amountof enzyme that hydrolyses 1 µmol of the respective β-lactamper minute.

Inactivation of ticarcillin, ceftazidime, imipenem, mero-penem and oxacillin was tested by the cloverleaf34 and doubledisc35 methods.

Analysis of OMP profiles

OMP profiles were studied by SDS–PAGE, using both 12%polyacrylamide gels and 10% polyacrylamide gels with 6 Murea, as described previously.36 Proteins were stained withCoomassie Blue R-250 (Sigma). A commercial kit (SDS–PAGE Standards, low-range; Bio-Rad) was used for molecu-lar weight standards.

Table 1. Reference strains used in this study

Strain Study Source or reference

Acinetobacter baumannii 19606 reference pattern of OMP profiles ATCCEscherichia coli 25922 susceptibility testing ATCCEscherichia coli JM 109 (pPTN 103) microbiological assay of β-lactamases

(positive control)27

Escherichia coli W3110 reference pattern of PBP profiles and molecular weight standards of PBPs

28

Micrococcus spp. HUS-97 microbiological assay of β-lactamases (indicator strain)

this study

Pseudomonas aeruginosa 27853 susceptibility testing ATCCStaphylococcus aureus 8325 molecular size marker of DNA for PFGE NCTC

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Labelling and detection of PBPs

A conjugate of iodine-125 (Bolton and Hunter reagent;Pharmacia Biotech, Barcelona, Spain) and ampicillin was usedfor labelling PBPs.37,38 Binding assays of PBPs were carriedout according to the method of Spratt.39 Samples (∼50 µg)were fractionated on 12% SDS–PAGE, using the SE 200Mighty Small II system (Hoefer, Amersham Pharmacia Bio-tech).

Bands of PBPs were visualized by autoradiography(AGFA CURIX films, Madrid, Spain). Radioactivity of the125I-labelled PBPs was quantified using the CYCLON StoragePhosphor System (Packard, La Jolla, CA, USA). Arbitraryunits of radioactivity (AURs) were determined by calculatingthe height of the peaks of radioactivity. AURs were expressedas relative units (URLs) [with reference to that of PBP 5(100% intensity) of every isolate], because the band corres-ponding to this PBP was homogeneously labelled in all theisolates evaluated.

Results

Typing methods

The 20 isolates were identified as A. baumannii. Five biotypes(2, 6, 9, 11 and 18) and eight PFGE patterns (A–H) wererecognized (Table 2).

Antimicrobial susceptibility testing

Ampicillin, piperacillin, ticarcillin, cefaloridine, cefoxitinand cefotaxime were the β-lactams with the lowest anti-microbial 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 ofisolate 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).

Table 2. Relationship among biotypes and PFGE patterns, and activity of 13 antimicrobial agents against type I (imipenem MICs 0.06–2 mg/L) and type II (imipenem MICs 4–32 mg/L) clinical isolates of A. baumannii

IPM, imipenem; MEM, meropenem; TIC, ticarcillin; CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam; FEP, cefepime; SUL, sulbactam; GEN, gentamicin;AMK, amikacin; TOB, tobramycin; TET, tetracycline; CIP, ciprofloxacin. ND, not determined.

PFGE pattern

MICs (mg/L)

Isolate Biotype IPM MEM TIC CTX CAZ ATM FEP SUL GEN AMK TOB TET CIP

Type IHUS 167 11 H 0.06 0.5 16 8 8 32 0.5 1 0.03 0.5 0.03 2 ≤0.06HUS 296 18 C 0.125 0.5 16 16 8 32 2 2 16 32 8 128 64HUS 31 9A E 0.25 0.5 16 64 16 64 32 1 >512 64 64 64 64HUS 108 9A E 0.25 2 32 64 32 64 32 4 >512 64 64 64 128HUS 158 2 A 0.5 2 128 256 64 256 32 2 512 64 2 256 64HUS 451a 2 A 1 2 128 512 128 512 32 4 >512 256 8 256 512HUS 431 9B G 1 4 >512 >512 >512 >512 32 4 16 0.5 8 4 8HUS 331 2 A 2 2 64 256 64 256 32 4 >512 128 4 128 32HUS 58 2 A 2 2 64 256 64 256 32 2 512 4 4 256 32ATCC 19606 ND ND 0.25 0.5 16 16 8 32 8 1 8 8 4 4 0.5

Type IIHUS 576 6 D 4 4 >512 256 64 32 16 32 >512 64 128 32 64HUS 457 18 C 4 4 >512 32 16 64 4 8 128 16 16 8 32HUS 531 6 C 8 8 >512 256 64 32 16 32 >512 128 256 32 64HUS 211 9B F 16 8 >512 512 64 128 16 16 8 64 8 4 8HUS 538 9B G 16 8 >512 64 32 128 8 32 64 256 16 4 8HUS 753 9B F 16 16 >512 >512 >512 >512 128 16 16 64 8 4 16HUS 48 6 B 16 8 >512 256 64 32 8 32 >512 64 128 32 64HUS 253 6 B 16 8 >512 512 >512 128 128 32 >512 512 512 256 512HUS 451b 6 B 16 8 >512 256 64 32 16 32 >512 8 256 32 64HUS 349 6 B 32 8 >512 256 64 32 16 4 >512 32 512 4 64HUS 373 9B G 32 16 >512 >512 >512 >512 64 32 16 64 8 4 16

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Table 3. Effect of BRL 42715 (BRL; 4 mg/L) on the activity of imipenem (IPM) and meropenem (MEM), β-lactamase distribution (isoelectric point) and hydrolysing activity of type I and type II A. baumannii against 0.1 mM cefaloridine (CFL), 0.1 mM imipenem (IPM), 0.1 mM meropenem (MEM) and 1 mM oxacillin (OXA)

CL, cloverleaf method; DD, double disc method.aND, hydrolysis not detected.bResults of hydrolysis expressed qualitatively: difficult to read (IND), not detectable (0), minimal (1), moderate (2) and high (3).

Hydrolysis

MICs (mg/L) microbiological assayb

IPM + BRL

MEM + BRL

spectrophotometry (mU/mg)a IPM MEM OXA

Isolates IPM MEM pI β-lactamases CFL IPM MEM OXA CL DD CL DD CL DD

Type IHUS 167 0.06 0.06 0.5 0.5 ≥9 18.8 ND ND ND 1 0 0 0 IND 0HUS 296 0.125 0.06 0.5 0.5 ≥9 22.5 ND ND ND 1 0 0 0 IND 0HUS 31 0.25 0.25 0.5 0.5 ≥9 81.8 0.09 ND 59.4 1 1 0 0 1 1HUS 108 0.25 0.25 2 1 ≥9 64.8 ND ND 120.1 1 1 0 0 1 2HUS 158 0.5 0.5 2 2 ≥9 285.5 0.11 <0.05 100.9 1 1–2 1 IND 1 2HUS 451a 1 0.5 2 2 ≥9 506.3 0.07 <0.05 49.4 1 1 1 0 1 2HUS 431 1 0.25 4 4 ≥9 192.2 <0.05 <0.05 28.9 1 IND 1 IND 2 2HUS 331 2 0.5 2 2 ≥9 518.5 0.10 <0.05 82.3 2 1 1 0 2 1HUS 58 2 0.5 2 2 ≥9 552.2 0.07 ND 88.2 1–2 2 1 0 1–2 2

Type IIHUS 576 4 0.125 4 0.5 ≥9; 7; 6.3; 5.8; 5.4 660.1 1.1 0.37 440.5 2 2 1 IND 2 2HUS 457 4 0.125 4 1 ≥9; 7; 6.3; 5.4 99.6 0.6 0.26 174.9 3 2 2 1 2 2HUS 531 8 0.25 8 0.5 ≥9; 7; 6.3; 5.8; 5.4 914.1 1.5 0.41 126.9 2 2 1 1 3 2HUS 211 16 0.25 8 2 ≥9; 7; 6.3; 5.4 369.8 0.41 0.27 235.8 3 3 2 1 3 3HUS 538 16 0.125 8 4 ≥9; 7; 6.3 12.3 0.55 0.18 185.8 2 1 1–2 1 3 1HUS 753 16 0.25 16 4 ≥9; 7; 6.3 42.3 0.98 0.23 506.5 3 2 2 1 3 2HUS 48 16 0.25 8 0.5 ≥9; 7; 6.3; 5.8; 5.4 1308.5 0.78 0.45 283.5 2 2 2 1 2–3 3HUS 253 16 1 8 4 ≥9; 7; 6.3; 5.8; 5.4 182.4 1.3 0.22 362.6 2 2 2 1 2–3 3HUS 451b 16 0.25 8 0.5 ≥9; 7; 6.3; 5.8; 5.4 1543.5 1.2 0.88 408.7 2–3 2 1 2 2–3 3HUS 349 32 0.25 8 1 ≥9; 7; 6.3; 5.8 1412.7 0.6 0.63 362.9 2 2 1 2 2–3 3HUS 373 32 0.25 16 8 ≥9; 7; 6.3 41.1 0.95 0.24 373.5 3 2 2 1 3 2

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Sulbactam was the β-lactamase inhibitor tested with thehighest intrinsic activity: MIC range 1–32 mg/L. MICs ofclavulanic acid and BRL were ≥64 mg/L for all isolates. TheMIC90s of carbapenems were unaffected (meropenem) ordecreased twice (imipenem) in the presence of clavulanicacid. The MIC90s of imipenem were reduced four times (type Iisolates) and 128 times (type II isolates) in the presence ofBRL (Table 3). In contrast, the MIC90s of meropenem werenot reduced (type I isolates) or decreased four times (type IIisolates) when this carbapenem was combined with BRL.

MICs of gentamicin, amikacin, tobramycin and tetra-cycline showed a biotype-dependent variation. All isolateswere resistant to ciprofloxacin (MIC range 8–512 mg/L),except HUS 167 (MIC ≤ 0.06 mg/L).

Isoelectric focusing of β-lactamases

A band of pI ≥ 9 inhibited by cloxacillin but not clavulanicacid or EDTA was observed in all the isolates (Table 3). Twobands of pIs 7.0 and 6.3, respectively, which were weaklyinhibited by clavulanic acid but not by either cloxacillin orEDTA, were also expressed in type II, but not in type Iisolates. The band of pI 5.8 (inhibited by EDTA) was detected

in six type II isolates. Finally, a band of pI 5.4 (inhibited onlyby clavulanic acid) was observed in seven type II isolates.

Hydrolysis of cefaloridine, carbapenems and oxacillin

Hydrolysis of cefaloridine (mU/mg of protein) ranged from18.8 to 552.2 (type I isolates) and from 12.3 to 1543.5 (type IIisolates) (Table 3). The hydrolysing activities of type IIisolates against carbapenems and oxacillin were higher thanthose observed in type I isolates (Table 3). For type I isolatesthese values (mU/mg) ranged between not detectable and0.11 (imipenem), not detectable and <0.05 (meropenem), andnot detectable and 120.1 (oxacillin), whereas those for type IIisolates ranged between 0.41 and 1.5 (imipenem), 0.18 and0.88 (meropenem), and 126.9 and 506.5 (oxacillin).

Hydrolysis of ticarcillin and imipenem was detected in allisolates using the cloverleaf method, whereas hydrolysis ofoxacillin, meropenem and ceftazidime was only detected in18, 16 and 11 isolates, respectively (Table 3). Using thedouble disc method, hydrolysis was detected in 18 (ticarcillinand oxacillin), 17 (imipenem), 10 (meropenem) and seven(ceftazidime) isolates. The agreement obtained between thecloverleaf method and the double disc method was 90%

Table 4. OMP and PBP profiles of type I and type II isolates of A. baumannii

ND, not determined.

OMP profile

Isolate Biotype PFGE pattern 10% SDS–PAGE–6 M urea 12% SDS–PAGE PBP profile

HUS 158 2 A A1 A2* A1HUS 331 2 A A1 A2* A1HUS 451a 2 A G A2* A2HUS 58 2 A G A2* A1

HUS 48 6 B A1 A1* HHUS 349 6 B A1 A1* JHUS 253 6 B B1 A1* IHUS 451b 6 B A1 A1* JHUS 531 6 C B1 A1* KHUS 576 6 D B2 A1* G1

HUS 31 9 E A2 A2* C1HUS 108 9 E A1 A2* C1HUS 211 9 F C1 B* B2HUS 753 9 F C2 B* B2HUS 431 9 G C1 B* B1HUS 373 9 G C2 B* KHUS 538 9 G C2 B* B2ATCC 19606 ND ND H E* ND

HUS 167 11 H D D* D

HUS 296 18 C E C* EHUS 457 18 C F C* G1

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(ticarcillin, oxacillin), 85% (imipenem), 80% (ceftazidime)and 70% (meropenem).

OMP profiles

Ten OMP patterns were observed in 10% SDS–PAGE gelswith 6 M urea, whereas only five OMP patterns wereobserved in 12% SDS–PAGE gels without urea (Figures 1and 2; Table 4). The most representative profile A1 (n = 6)observed in 10% SDS–PAGE gels with 6 M urea showed sixbands 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; Omp2b). In 12% SDS–PAGE gels, the most frequently observedprofiles were A1* (n = 6), A2* (n = 6) and B* (n = 5). Sevenmajor bands with relative mobilities of 43.3 (Omp 1a*), 34.8(Omp 2*), 32.3 (Omp 3b*), 30.2 (Omp 4a*), 25.8 kDa (Omp5*), 23.7 (Omp 6*) and 22.5 kDa (Omp 7*) were present inOMP profile A1*. Profile A2* differed from profile A1* inthe absence of Omp 5*. Profile B* (n = 5) was characterizedby 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*.

PBP profiles

Twelve highly complex patterns of PBPs were observed. Fivebands 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. ThePBP pattern of type II isolates differed from those of type Iisolates by the absence of one band of 73 kDa, correspondingto PBP 2 (Figure 3).

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 URLsof type I isolates (Table 5). For type II isolates, however, thesevalues 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% (PBP4/4b).

Discussion

The A. baumannii isolates we studied showed susceptibilityor moderate resistance to imipenem and meropenem (MICs ofboth carbapenems were ≤32 mg/L). The synergic effect ofBRL on the activity of imipenem and, to a lesser extent, mero-penem indicates that production of serine β-lactamases isinvolved in the resistance to carbapenems.

A great diversity of β-lactamases was observed in mosttype II isolates. The band of pI ≥ 9 presumably correspondsto a non-inducible AmpC-type cephalosporinase, as Bou& Martínez-Beltrán40 have demonstrated in another study.Cephalosporinase activity, defined as hydrolysis of cefalorid-ine, did not significantly correlate with the MICs of imipenemand meropenem, suggesting that the expression of AmpCdoes 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 wasassociated with resistance to carbapenems, whereas theband of pI 5.4 (probably a TEM-type β-lactamase) wasunrelated.41,42

Figure 1. Outer membrane protein profiles A1 (lane 2), B1 (lane 3) andC1 (lane 4) observed in 10% SDS–PAGE gels with 6 M urea. Lane 1,molecular weight standards (kDa).

Figure 2. OMP profiles A2* (lane 2) and B* (lane 3) observed in 12%SDS–PAGE gels. Lane 1, molecular weight standards (kDa).

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The inhibition profile of bands of pI 6.3 and 7.0, and thehighest hydrolysing activity of type II isolates (expressingthese enzymes) against imipenem, meropenem and oxacillin,suggest that these β-lactamases are oxacillinases with a mod-erate hydrolysing activity against carbapenems. In contrast,the band of pI 5.8 was inhibited by EDTA and may be relatedto metallo-β-lactamases. Additional molecular and bio-chemical studies are in progress to determine the relationshipof these β-lactamases to those previously described in A. bau-mannii.3–13

Our isolates showed no decreased expression of the33–36 kDa OMP reported by Clark.18 Carbapenem resistancefor isolates with OMP profile B was related to the absence of a22.5 kDa OMP (Omp 7*). This observation was supported bythe results of BRL on the activity of imipenem and mero-penem: for type II isolates lacking Omp 7*, the BRLdecreased the MICs of imipenem and meropenem less thanfor type II isolates expressing this OMP. Bou et al.26 haveobserved isolates of A. baumannii resistant to carbapenemslacking a 22 kDa OMP, although its role in resistance to carba-penems has not been evaluated. The amino acid sequence of

this protein of 22 kDa and Omp 7*, and studies of proteolipo-somes reconstituted with these two proteins, will elucidatewhether they really are porins, and will help to determine theirrelationship to antimicrobial resistance.

A wide variation of PBP patterns was observed using the125I-ampicillin reagent. The absence (or reduced expression)of one band of PBP, which we named PBP 2, was related

Table 5. Quantification of the radioactive signal emitted by the 125I-ampicillin-labelled PBPs of type I and type II isolates of A. baumannii using the CYCLON system

Absolute units of radioactivity (AUR) were determined calculating for every band of PBP the height of the correspondent peaks of intensity. NA,not available; –, radioactivity not detected.aRelative units of radioactivity (URLs) were expressed as a percentage referred to the AUR of the PBP 5 of each isolate.

Percentage of relative units of radioactivity intensity (URLs) of 125I-ampicillin-labelled PBPsa

Isolates 1a 1c 2 3 4 5

Type IHUS 167 2064.3 (1b) 280.9 203.9 2021.9 153.7 100 HUS 296 920.3 166.9 292.7 2655.6 123.4 100HUS 31 2210 477.2 503.1 609.2 117.6 100HUS 108 2397.2 375.3 453.5 546.6 112.6 100HUS 158 2080 326.3 374.5 450.1 142.2 100HUS 451a 1442.4 292.2 305.2 3352.2 123.7 100HUS 431 100.5 44.3 79.4 199.4 56.4 100HUS 331 425.8 110.1 109.6 541.9 93.9 100HUS 58 1060 160.7 168.5 1761.5 115.2 100

Type IIHUS 576 155.8 43.8 – 207.8 96.4 100HUS 457 307.9 86.1 – 803.7 76.9 100HUS 531 NA NA NA NA NAHUS 211 226.7 113.8 – 368.5 102.2 100HUS 538 236.4 101 – 327.1 116.3 100HUS 753 295.2 51.3 – 556.6 98.1 100HUS 48 173.9 – – 366.5 92.7 100HUS 253 91.7 – – 168.9 83.6 100HUS 451b 76.3 67.7 – 324.7 89.6 100HUS 349 53.5 60.4 – 308.8 68.6 100HUS 373 203.1 – – 418.4 96.7 100

Figure 3. 12% SDS–PAGE pattern of 125I-ampicillin PBPs of E. coliW3110 (lane 1), A. baumannii HUS 457 (type II isolate; lane 2) andA. baumannii HUS 31 (type I isolate; lane 3).

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to decreased susceptibility or resistance to carbapenems, inaccordance with the results obtained by Neuwirth et al.21 in aclinical strain of Proteus mirabilis, for which resistance tocarbapenems was due to the decreased affinity of PBP 2(with similar mobility to the PBP 2 of our A. baumannii) toimipenem. Competition assays with imipenem and/or mero-penem and 125I-ampicillin, or studies with imipenem and/ormeropenem labelled with the Bolton and Hunter reagent, arenecessary to find out the precise role of PBP 2 in the resistanceof A. baumannii to carbapenem compounds.

Hyperproduction of the low molecular weight protein of24 kDa (not saturable by imipenem) reported in the study byGehrlen et al.23 was not observed in our isolates.

PBP bands of type II isolates showed lower radioactivityintensity (% URLs) than those of type I isolates. This may bedue to hydrolysis of 125I-ampicillin by residual amounts ofβ-lactamases (probably those of pI 6.3 and 7.0) not removedfrom membrane preparations after extensive and vigorouswashing (three times of 1 min each in vortex). This problemcould be resolved by using a potent inhibitor of β-lactamaseswith a low affinity to PBPs, such as BRL 42715. Unfortu-nately, neither this compound nor any other with similarproperties is available, which makes necessary the evaluationof other strategies of PBP analysis in A. baumannii.

Other possible mechanisms of resistance to carbapenems,such as the overproduction of efflux pumps19,20 or lipopoly-saccharide alterations,43 could be expressed in some of ourisolates for which the MICs of imipenem and meropenemwere not completely related to the presence of any of themechanisms 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 2being most frequently observed. For some isolates, resistanceto carbapenems is also related to the absence of an OMP of22.5 kDa. Additional studies with isogenic mutants, which door do not express these mechanisms individually and in com-bination with each other (double and triple mutants), must bedeveloped to understand the precise role of these mechanismsin the resistance of A. baumannii to carbapenems.

Acknowledgements

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 ofSeville, Spain) for excellent technical assistance in the quanti-fication of PBPs, and E. Ramírez (University Hospital VírgenMacarena of Seville, Spain) for her inestimable help in thePFGE study. We gratefully acknowledge the assistance ofJanet Dawson in the preparation of the manuscript. We alsothank the ‘Sociedad de Enfermedades Infeccciosas y Micro-biología Clínica’ for their partial funding of the study of

PBPs. This study was supported by grant 98/1027 from theFondo de Investigaciones Sanitarias, Ministerio de Sanidad yConsumo, Spain.

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