Vol. 37,No.6 ANTIMICROBIALAGENTSANDCHEMOTHERAPY,June 1993, p. 1324-1328
0066-4804/93/061324-05$02.00/0
Copyright © 1993, American Society forMicrobiology
High
Specificity
of
cphA-Encoded Metallo-,-Lactamase
from
Aeromonas hydrophila AE036 for
Carbapenems and Its
Contribution to
13-Lactam
Resistance
BERNARDEITASEGATORE,1 ORIETIAMASSIDDA,2t GIUSEPPE
SATrA,3
DOMENICO SETACCI,' ANDGIANFRANCOAMICOSANTEl*Departmentof BiomedicalSciencesand Technologiesandof Biometrics, FacultyofMedicine, Universityof L'Aquila, Localita Collemaggio, I-67100 L'Aquila, DepartmentofMicrobiology, University of Siena, Siena,2
andDepartmentof Microbiology, Cattolica University of Rome, Rome,3Italy Received 7 December1992/Accepted 23 March 1993
TheAeromonashydrophila AE036 chromosome contains acphAgeneencodinga
metallo-1-lactamase
highly
activeagainstcarbapenem antibiotics. This enzymewasinduced in strain AE036tothesame extentbyboth benzylpenicillin andimipenem.When thecphAgenewasinserted intoplasmidpACYC184,usedtotransform Escherichia coli DH5a, the MICs of imipenem, meropenem, and penem HRE664 for recombinant clone DHSa(pAA2OR), expressing the Aeromonasmetallo-13-lactamase,
were significantly increased, but those of penicillins andcephalosporinswere not.When themetallo-13-lactamase
purifiedfrom E.coliDH5a(pAA20R) wasassayed with several13-lactam
substrates,ithydrolyzedcarbapenemsbutnotpenicillinsorcephalosporinsefficiently.
These resultsdemonstrate that thismetallo-13-lactamase
possesses anunusualspectrumofactivity
compared with all the other class B enzymes identifiedsofar,beingactiveonpenemsandcarbapenems
only.
Thisenzyme maythus contributetothedevelopment ofresistancetopenemsandcarbapenemsbutnotother-lactams.
Limited diffusionof
3-lactam
antibioticsthroughtheouter membrane of gram-negative bacteria and the presence of3-lactamases
in theperiplasm
are two of the mainreasonsfor the failure oftreatment for infections caused by these microorganisms (4, 10, 13, 19). In addition, morethan one
13-lactamase
typemay beproduced
by
theseorganisms
after antibiotic induction, so it may be difficult to assess the precise role of each enzyme in1-lactam
resistance. In particular,amonggram-negative rods,strains ofAeromonas spp. have been reported to produce up to three different 13-lactamases(16).
Some authors used thecatalytic
proper-ties of a
13-lactamase
with respect to anumber of 3-lactamcompounds to correlate the theoretically predictable MICs andtheexperimentallyobserved MICs (6, 13, 17).
In a previous paper, Iaconis and Sanders (7) reported somepropertiesoftwoinducible
1-lactamases
produced by Aeromonas spp.; in particular, Aeromonas hydrophila AER19M produces two enzymes, called Al and A2. The formerwasdescribedas acephalosporinasewithapIof 7.0 andamolecularmassof 42,500Da, whereasA2(pI 8.0)was described as a,-lactamase highlyactive against penicillins and carbapenems but sensitiveto EDTA. Recently, amet-allo-p-lactamase
encodedbyA. hydrophilaAE036cphAwas cloned in Escherichia coli DH5ot, and the gene was se-quenced by Massidda et al. (11). In the present work, we evaluated the contribution of this enzymeto the resistance patternshowedbyE. coliDH5a(pAA20R),
which was used to express the metallo-,-lactamase toeliminate the hydro-lytic contribution of the other Aeromonasj3-lactamase
present in the original strain. The properties of the
cphA-*Correspondingauthor.
tPresent address: InstituteofMicrobiology, University of An-cona,Ancona, Italy.
encoded enzyme were alsocompared with those of a similar enzyme (A2) previously characterized by other authors(7).
MATERIALS AND METHODS
Bacterial strains. The strains ofA.hydrophila (AE036) and E.coli[DH5a andDH5at(pAA2OR)]used herewerethe same asthose usedin a previous work (11). PlasmidpAA20R has a 2.0-kb EcoRI Aeromonas fragment containing the cphA geneand inserted intopACYC184, which doesnothave an ampicillinresistancemarker for selection. The chromosomal AmpC 3-lactamase in E. coli DH5aL and E. coli DH5a (pAA2OR) was undetectable.
Inductionkinetics forA.hydrophila AE036 3-lactamases.A. hydrophilaAE036wasgrownaerobically overnightin brain heart infusion broth with shaking (180 rpm) at 37°C, the culturewasdiluted 10-fold withfreshmedium, and aliquots of 100 ml were reincubated. After 2 h, the inducer (ben-zylpenicillin or imipenem) was added, and the resulting ,B-lactamase
production
was tested at different times(see
Fig. 1). Inbrief, the culture was centrifugedat 10,000 xg, resuspendedin5 ml of25mMphosphate buffer (pH 7.4),and disrupted byultrasonictreatmentwithaLabLine ultrasonic disintegrator. Thesuspensionwasclarifiedat 105,000xgin a Beckman L8 70 ultracentrifuge; the supernatantwas dia-lyzed for 4 h against 25 mM cacodylate buffer (pH 7.4) containing 50 ,M ZnCl2 and used to determine activity against imipenem and nitrocefin with or without 20 mM EDTA.Theprotein contentsweredeterminedby the proce-dure of Bradford (3) with bovine serum albumin as the standard.MIC determinations. MICs for eitherE. coliDH5aorE. coli DH5a(pAA2OR) producing the metallo-,B-lactamase were determined by the double-dilution technique with Mueller-Hinton broth andabacterial inoculum of 5 x 104or 5 x 108 CFU/ml, respectively, as previouslydescribed (2). 1324
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A. HYDROPHILA cphA-ENCODED METALLO-3-LACTAMASE 1325 All the drugs tested in these experiments were kindly
do-nated by the following manufacturers: Merck Sharp & Dohme, Rome, Italy (imipenem); ICI, Cheshire, England (meropenem); Hoechst, Milan, Italy (cefotaxime and HRE664); Squibb, Princeton, N.J. (aztreonam); and Smith-Kline-Beecham, Betchworth, England (carbenicillin). Ben-zylpenicillin and cephaloridin were supplied by Sigma, St. Louis, Mo. Nitrocefin was purchased from Unipath S.p.A., Milan, Italy.
Enzymepurification.The
metallo-3-lactamase
waspurified asfollows. E. coli DH5a(pAA20R)containing the cphA gene was grown overnight in brain heart infusion broth at 37°C with shaking as reported above. Bacterial cells were col-lected bycentrifugation and disrupted by ultrasonic treat-ment, and the clear lysates were loaded onto a Sephadex G-75column equilibrated with 50 mM cacodylate buffer (pH 7.4) containing 50 ,uM ZnCl2. The active fractions were assayedwith imipenem as the substrate and pooled. Subse-quently, 25 ml of this pool was diluted 1:1 with double-distilled water and loaded into aRotoforapparatus (Bio-Rad, Richmond, Calif.). The sample was mixed with 2% Ampho-line togenerate a pH gradient ranging from 3 to 10. Separa-tion was carried out at 4°C for 4 h at 1,500 V and aconstant power of 12 W. The metallo-,-lactamase was found in the fractions with pHs ranging from 7.3 to 8.2. Fractions with pHsranging from 7.8 to 8.2 were used to determine kinetic parameters. This enzyme preparation contained 138 U of specific activity per mg when imipenem was used as the substrate and showed a purification factor of about 600 and ayield relativetothe total amount ofmetallo-13-lactamaseof 19%. One unitwas defined as 1 ,umol of imipenem hydro-lyzedper minby 0.01 mg of protein at 30°C.Mrandisoelectricpointdeterminations. Polyacrylamidegel electrophoresis was done as described by Laemmli (8). Proteins (standards and metallo-13-lactamase) were stained with Coomassieblue R-250. Isoelectric focusing(IEF)was performed with clarified cell extracts or pure
metallo-13-lactamase aspreviouslyreported (14). Nitrocefinwas used as the chromogenic substrate to reveal the f-lactamase activity in the focused gel as previously described (12). When necessary,1-lactamase
activity was detected by the iodometric technique with imipenem as the substrate as previously described (11).Treatment with EDTA and pCMB. For evaluation of the effect ofEDTA on 3-lactamase activity, experiments were performed at 30°C by use of50 mM cacodylate buffer (pH 7.4)with orwithout 50 ,uMZnCl2 andwith 100and 200 ,uM imipenem, respectively, as the substrate. For thispurpose, weusedeitherpurifiedenzyme(50nM)orcrudeextractsof A. hydrophila AE036 (100 ,ul) obtained after induction with benzylpenicillin. The interaction was studied byincubating the enzymesamples with20 mM EDTA anddeterminingthe residualactivity at differentincubationtimes: 0, 5, 15,30,60, and90 min.
Samples ofpurifiedenzyme (50nM)were also incubated with 0.5mMp-hydroxymercuribenzoate (pCMB)for 10min in 50 mMcacodylate buffer(pH 7.4)with 50p,M ZnCl2,and theresidual activity was measured in thepresence of 100 and 200,uMimipenem.
Kinetic parameter determinations. The activity of the purified
metallo-p-lactamase
was monitored spectrophoto-metrically by use of a Perkin-Elmer lambda-2 instrument and 50 mMcacodylatebuffer(pH7.4)
containing50,uMZnCl2
at 30°C. Under these conditions, thehydrolysis
of each sub-stratewithout theenzymewaslower than 1%, althoughthe hydrolysiswas notnegligiblewhen Tris bufferwasused, as1606 E
140
-E 120 -E .g° 10-86
b 0 40 4 0 26 w a-(1 0 4 6 12 1626
24 28 TIME(hours)FIG. 1. Time course of metallo-,-lactamase production in A. hydrophila AE036. Symbols: *, control (withoutan inducer);
5,
imipenem(5 ,ug/ml)astheinducer;A,benzylpenicillin(100,g/ml) asthe inducer. Theactivitywasmonitoredatvarioustimeswith100 and 200pLM
imipenem asthe substrate. Forexperimental details, seethetext.we previously reported (15). Hydrolysis rates were deter-mined with
imipenem
(50
to200p,M;
X = 299nm),
cephalo-ridine(80
to 240 FM; X = 260nm),benzylpenicillin (250
to1,000 p,M;
X= 230nm),
meropenem(100 to 300p,M;
A= 299nm),
HRE664(10to100pM;
A=265nm),aztreonam(100to 1,000 pM;X=318nm),
carbenicillin(200
to800pM;
A=235nm),
cefotaxime(30 to120 pM; X = 260nm), and nitrocefin(50
to 150 p,M; X = 482nm).
In thepresence
ofhigher
substrate
concentrations, 0.2-cm-path-length
quartz cu-vettes were used.Catalytic
rate constant(kcat)
and Km values were obtained by applying the integrated Hanesequation
tothe initialratesofhydrolysis. Inseparateexper-iments,we alsodetermined the
enzymatic
activityincrude extracts ofA.hydrophila
AE036 to obtaininformation
on basalactivity
oractivity
after induction withimipenem,
cephaloridine,
benzylpenicillin,
andnitrocefin(see
Table2).
'~900 E 800o 700-6 800-E 500-400 300-< 200 U_ 100 w E---.- ... ... a. 0 @ 0 4 8 12 18 20 24 28 TIME(hours)
FIG. 2. Timecourseofnitrocefin-hydrolyzing ,B-lactamase pro-duction in A. hydrophilaAE036. Symbols: *,control
(without
aninducer);
0,
imipenem(5pg/ml)astheinducer;A,
benzylpenicillin
(100p,g/ml)astheinducer. Theactivitywasmonitoredasreported
inFig. 1with 80and100pM nitrocefinasthe substrate. VOL.37, 1993
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ANTIMICROB. AGENTS CHEMOTHER.
TABLE 2. Basal activity and activity after induction of
P-lactamases
in crude extracts ofA. hydrophila AE036Activity (nmolmin-' mg-'of protein) Antibiotic Control+ Aft Afterinductiona
Control 20mM
nduction'
+20 mM EDTA EDTA Imipenem 1.1 1.0 63.6 2.8 Cephaloridine 0.5 0.01 10.45 6.7 Benzylpenicillin 0.01 0.01 44.7 42.4 Nitrocefin 228 228 4,420 4,420a The inducerwas100,ugofbenzylpenicillinperml.
o ---[----m ==== =l
0 15 30 45 60 75 90 105
TIME(min)
FIG. 3. TimecourseofEDTA inhibition of metallo-3-lactamase activity determined byuseofpurifiedenzymeobtained from E. coli DH5a and crudeextracts obtained afterinduction from A. hydro-phila AE036. Symbols, *, crude extractsafterinduction; [l,
puri-fiedenzyme. The activitywas monitored in bothcaseswith
imi-penem(100 and200FLM)asthe substrate.Forexperimental details,
seethetext.
RESULTS
P-Lactamase
induction. The basalactivityofP-lactamases
in A. hydrophila AE036 was found to be negligible with imipenem but easily detectable with nitrocefin. Moreover,asreported in Fig. 1 and 2, both imipenem andbenzylpenicillin actedasgood 1-lactamaseinducers in the tested strain. After
induction,twoenzymes,with pIs of 6.6 and 7.0,wereeasily
detectable with nitrocefin; athird enzyme,with apI of 8.0
and active against imipenem but with a poor affinity for nitrocefin,wasidentified by IEFanalysis using imipenemas
the substrate (datanot shown)or by a spectrophotometric
assay. Enzymeproduction reached a maximum during the
first 2 h ofbacterialgrowth after induction. Wewereunable
todetectanymetallo-1-lactamaseactivity with nitrocefin by
use ofIEF analysis of the crude extracts and the purified
enzyme, inkeeping with the previousreportby Massiddaet al. (11); the isoelectric pointwasdetermined with 20,ug ofa
pure enzyme preparation run onthe same gel. By sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, we
foundan
M,
of about 28 kDa for thepure enzyme and, byIEFanalysis,we foundanisoelectric point of 8.0 + 0.1, in agreementwith thedata reported elsewhere for thisenzyme
(5, 11).
Interaction with
non-1-lactam
compounds. Both crude extractsand thepure enzyme werestrongly inhibited whenincubatedwith 20 mM EDTA.The loss of activity reacheda
maximum after 30 min oftreatment, asreported in Fig. 3.
Thesmall differences in the inhibition data reported for the
pure enzyme and crude extracts are probably due to the different amountsof metallo-1-lactamase in thesamples.
The interaction withpCMB revealed a significant
inhibi-tion of thepure
metallo-o-lactamase
after10min ofincuba-tion(17% residual activity). The A2protein produced byA. hydrophila AER19M was inhibited to a lesser extent, al-though these datawereobtained underdifferent
experimen-tal conditions, asreported in Table 1.
Substrate
hydrolysis
determinations. We used imipenem, cephaloridine, benzylpenicillin, and nitrocefin in kineticas-says to evaluate the contribution to 3-lactam hydrolysis of the P-lactamases, whether constitutively produced or in-ducedinA.hydrophilaAE036.Imipenemisreportedtobea
verygood substrate for metallo-,B-lactamases, whereas
ben-zylpenicillin and cephaloridine are reported to be good substrates for serine ,B-lactamases of classes A and C, respectively.
Asreported in Table 2, the basal activitywaslowagainst
eitherimipenemorcephaloridine, although benzylpenicillin was a surprisingly poor substrate. After induction, the
activity of crudeextracts increased for all thecompounds, particularly for benzylpenicillin (4,400-fold increase). Incu-bation with 20 mM EDTA indicated that benzylpenicillin
was nota substrate for the metallo-1-lactamase and that it
wasprobably hydrolyzed by the otherenzymes induced in strainAE036. To confirmthesefindings,weusedapurified
preparation of the metallo-1-lactamase to determine the kinetic parameters for the set of 1-lactams tested, as
re-portedinTable3. These data confirmed the resultsobtained with the crude preparations of the induced enzyme. Ben-zylpenicillinwas avery poorsubstrate, and kcat values for
nitrocefin andcephaloridine werenegligible compared with
those for imipenem; penem HRE664 showed the highest
TABLE 1. Molecularparametersof the metallo-,B-lactamasefrom A. hydrophila AE036 (cphA gene) in comparison withthose of theA2proteinproduced byA. hydrophila AER19M
Result for:
Molecular parameter
AE036metallo-o-lactamase A2proteina
Molecular mass 28,000 31,500
Isoelectricpoint 8.0 8.0
Inhibitionby EDTA + +
SensitivitytopCMB High (residual activity, <20%) Low(residual activity, 83%)
Nitrocefin hydrolysis Low High
Aztreonamaffinity Very low (>1 mM) Intermediate(50FtM)
aThedatafor theA.
hydrophila
AER19M enzyme were obtained underdifferent
experimental conditions (0.1 M phosphate buffer, pH 7.0, nitrocephin as thesubstrate). I-0 -J cc LL 0 1O 1326 SEGATORE ET AL. 1 on November 18, 2017 by guest http://aac.asm.org/ Downloaded from
A. HYDROPHILA cphA-ENCODED METALLO-j3-LACTAMASE 1327 TABLE 3. Kineticparametersof themetallo-t-lactamase from A.hydrophila AE036(cph,A gene)in comparison
with thoseof the A2proteinproduced by A.hydrophilaAER19M
AE036metallo-,B-lactamase A2protein'
Antibiotic
k,,,t
(s-) Km(>±M) (lk/K
Relativekcat
RelativeVm Km (>M)kcat(s-' K. (pM) (106) (h-1s-1) (% (%) ~tM Nitrocefin 0.4 96 0.004 100 100 30.6 Benzylpenicillin 3 630 0.0047 750 170 124 Carbenicillin 10 500 0.02 2,500 NDb ND Cephaloridine 0.3 200 0.0015 75 ND ND Cefotaxime 0.1 34 0.0029 25 0.5 15 Aztreonam ND >1000 ND ND 3.2 53.3 HRE664 2.5 1.9 1.32 6,250 ND ND Imipenem 140 86 1.6 35,000 68.5 30 Meropenem 53 250 0.21 13,250 ND ND
aThe data forthe A.hydrophilaAER19M enzymewere obtained under different experimentalconditions (0.1 Mphosphate buffer,pH 7.0,nitrocefinasthe substrate).
bND, notdetermined.
affinity for the
metallo-p-enzyme,
while aztreonam was poorlyrecognized.Invitro
susceptibility.
E. coli DH5a(pAA2OR) containing thecphA gene wasused toevaluate the contribution of the metallo-3-lactamaseto 3-lactam resistancefor selected com-pounds. AsTable 4 shows, thisenzyme apparently confers resistance only tocarbapenems (imipenem and meropenem) orpenem HRE664. Asreported byMassidda et al.(11), the MICs ofcarbapenemsforA. hydrophila AE036 were similar when the same inoculum was used. Benzylpenicillin and cephaloridine, easily hydrolyzed by most3-lactamases,
werescarcelyinfluencedunder ourexperimental conditions.DISCUSSION
Metallo-p-lactamases
represent a growing threat in resis-tancephenomenabecause of their broadspectrum ofactivity against ,B-lactam substrates, includingthe so-called "stable compounds"(9).The recentappearance of a plasmid-medi-ated enzymebelongingtoclass Bisnowcausing problemsin the treatmentof infectious diseasesmediatedbyorganisms able to express these class B enzymes (18). Recently, Massidda et al.(11)
reported the sequencing of a gene encoding an A. hydrophilametallo-1-lactamase
showing stronghydrolyticactivity against imipenemandmeropenem. The reported amino acid sequence of the protein showed partial homology to that of themetallo-1-lactamases
pro-duced by Bacillus cereus and Bacteroides fragilis. The authors also concluded thatthe enzymeproducedinAero-TABLE 4. MICs determined forE. coliDHSawithorwithout themetallo-p-lactamasegene
MICforE.coliDH5aat theindicatedCFU/ml:
Antibiotic
Without
thecphA With thecphAgene gene 104 108 104 108 Imipenem 0.25 0.5 0.5 128 Meropenem 0.0078 0.5 0.06 64 HRE664 0.5 1 16 128 Aztreonam 0.125 128 0.125 128 Benzylpenicillin 32 64 32 128 Carbenicillin 2 64 4 128 Cefotaxime 0.015 2 0.03 4 Cephaloridine 1 16 2 16
monasspp.islesscloselyrelated to theseenzymes, suggest-ing a new variant among class B
j-lactamases.
Recently, Bakken et al.(1) andIaconisandSanders(7) publishedsome interestingpapersconcerningAeromonas sp.,-lactam resis-tance causedby twoinducible enzymes subsequently puri-fied and characterized. Theseenzymes, called Al and A2, appear to be a cephalosporinase and a penicillin- and car-bapenem-hydrolyzingP-lactamase,
respectively (7). More-over, the results reported inthis study show thatA. hydro-phila AE036possesses two inducible enzymes, as revealed by IEF analysis, and that its crude extracts hydrolyze carbapenems. Benzylpenicillin and imipenem were good inducers of bothj-lactamases,
confirming the ability of imipenem to cause induction in Aeromonas spp. (9). The substrate profile of the purifiedmetallo-p-lactamase
also proved tobeinteresting. Carbapenemsandpenem HRE664 werefound to begood substrates, whereas benzylpenicillin and cephalosporins were poorly hydrolyzed. Wild-typeA. hydrophila AE036 was able to hydrolyze benzylpenicillin andcephaloridine bymeansof asecond serineenzyme, most probably a cephalosporinase with a pI of 7.0. When we consider thekineticparameters of themetallo-1-lactamase,
we observe a correlation between the lack of resistance under anyconditionsand the lackofhydrolysisof the tested compound. As reported in Table 4, strainDH5a(pAA20R)
wasalso susceptibletocarbapenemsandHRE664 when we used an inoculum of 104CFU/ml,
but the effect of the metallo-,B-lactamase reallybecameimportantat108CFU/ml.
Finally, the purified enzyme was compared with the A2 enzyme,previouslydescribedbyothers(7).
Asindicated in Tables 1and3, therearesomedifferencesbetween thetwo proteins,butprobablyonlysequencingof thegeneencoding the A2enzyme canhelpus todecidewhether thereare two distinctP-lactamases
in Aeromonas spp. Inconclusion,
in this paper we describe ametallo-p-lactamase
that is pro-ducedbyA.hydrophila
and thathydrolyzesbenzilpenicillin
and
cephalosporins
at a low level and mostprobably
doesnotconferresistancetothesedrugs.Itwill be very
important
for future studiestodeterminewhich factorsareinvolved in thenarrowsubstratespecificity
of thisprotein.
ACKNOWLEDGMENTS
Thisworkwaspartially supportedby fundsfrom theConsiglio Nazionale delle Ricerche and Ministero dell'Universita e della RicercaScientifica.
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ANTIMICROB. AGENTS CHEMOTHER. We thank P.U.Varaldo,MorenoGalleni, and Antonio Felici for
critical reviews ofthe manuscript and Arduino Oratore for interest inthis study.
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