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High specificity of cphA-encoded metallo-β-lactamase from Aeromonas hydrophila AEO36 for carbapenems and its contribution to β-lactam resistance

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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 Aeromonas

metallo-13-lactamase,

were significantly increased, but those of penicillins andcephalosporinswere not.When the

metallo-13-lactamase

purifiedfrom E.coliDH5a(pAA20R) wasassayed with several

13-lactam

substrates,ithydrolyzedcarbapenemsbutnotpenicillinsorcephalosporins

efficiently.

These resultsdemonstrate that this

metallo-13-lactamase

possesses anunusualspectrumof

activity

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 of

3-lactamases

in the

periplasm

are two of the mainreasons

for the failure oftreatment for infections caused by these microorganisms (4, 10, 13, 19). In addition, morethan one

13-lactamase

typemay be

produced

by

these

organisms

after antibiotic induction, so it may be difficult to assess the precise role of each enzyme in

1-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 the

catalytic

proper-ties of a

13-lactamase

with respect to anumber of 3-lactam

compounds 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, a

met-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. coli

DH5a(pAA20R),

which was used to express the metallo-,-lactamase toeliminate the hydro-lytic contribution of the other Aeromonas

j3-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(pH

7.4)

containing50,uM

ZnCl2

at 30°C. Under these conditions, the

hydrolysis

of each sub-stratewithout theenzymewaslower than 1%, althoughthe hydrolysiswas notnegligiblewhen Tris bufferwasused, as

1606 E

140

-E 120 -E .g° 10-

86

b 0 40 4 0 26 w a-(1 0 4 6 12 16

26

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 200

pLM

imipenem asthe substrate. Forexperimental details, seethetext.

we previously reported (15). Hydrolysis rates were deter-mined with

imipenem

(50

to200

p,M;

X = 299

nm),

cephalo-ridine

(80

to 240 FM; X = 260nm),

benzylpenicillin (250

to

1,000 p,M;

X= 230

nm),

meropenem(100 to 300

p,M;

A= 299

nm),

HRE664(10to100

pM;

A=265nm),aztreonam(100to 1,000 pM;X=318

nm),

carbenicillin

(200

to800

pM;

A=235

nm),

cefotaxime(30 to120 pM; X = 260nm), and nitrocefin

(50

to 150 p,M; X = 482

nm).

In the

presence

of

higher

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 Hanes

equation

tothe initialratesofhydrolysis. Inseparate

exper-iments,we alsodetermined the

enzymatic

activityincrude extracts ofA.

hydrophila

AE036 to obtain

information

on basal

activity

or

activity

after induction with

imipenem,

cephaloridine,

benzylpenicillin,

andnitrocefin

(see

Table

2).

'~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

an

inducer);

0,

imipenem(5pg/ml)astheinducer;

A,

benzylpenicillin

(100p,g/ml)astheinducer. Theactivitywasmonitoredas

reported

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 AE036

Activity (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 4,420

a 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 basalactivityof

P-lactamases

in A. hydrophila AE036 was found to be negligible with imipenem but easily detectable with nitrocefin. Moreover,as

reported 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, by

IEFanalysis,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 when

incubatedwith 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 of

incuba-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 kinetic

as-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 under

different

experimental conditions (0.1 M phosphate buffer, pH 7.0, nitrocephin as the

substrate). I-0 -J cc LL 0 1O 1326 SEGATORE ET AL. 1 on November 18, 2017 by guest http://aac.asm.org/ Downloaded from

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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

Relative

kcat

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 most

3-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. hydrophila

metallo-1-lactamase

showing stronghydrolyticactivity against imipenemandmeropenem. The reported amino acid sequence of the protein showed partial homology to that of the

metallo-1-lactamases

pro-duced by Bacillus cereus and Bacteroides fragilis. The authors also concluded thatthe enzymeproducedin

Aero-TABLE 4. MICs determined forE. coliDHSawithorwithout themetallo-p-lactamasegene

MICforE.coliDH5aat theindicatedCFU/ml:

Antibiotic

Without

thecphA With thecphA

gene 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-hydrolyzing

P-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 both

j-lactamases,

confirming the ability of imipenem to cause induction in Aeromonas spp. (9). The substrate profile of the purified

metallo-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 the

metallo-1-lactamase,

we observe a correlation between the lack of resistance under anyconditionsand the lackofhydrolysisof the tested compound. As reported in Table 4, strain

DH5a(pAA20R)

wasalso susceptibletocarbapenemsandHRE664 when we used an inoculum of 104

CFU/ml,

but the effect of the metallo-,B-lactamase reallybecameimportantat108

CFU/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 distinct

P-lactamases

in Aeromonas spp. In

conclusion,

in this paper we describe a

metallo-p-lactamase

that is pro-ducedbyA.

hydrophila

and thathydrolyzes

benzilpenicillin

and

cephalosporins

at a low level and most

probably

does

notconferresistancetothesedrugs.Itwill be very

important

for future studiestodeterminewhich factorsareinvolved in thenarrowsubstrate

specificity

of this

protein.

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.

REFERENCES

1. Bakken, J. S., C.C. Sanders,R.B.Clark, and M. Hori. 1988.

P-Lactam

resistance inAeromonas spp. caused byinducible ,B-lactamases active

against penicillins,

cephalosporins,

and carbapenems.Antimicrob. AgentsChemother. 32:1314-1319. 2. Baron, E. J., and S. M. Finegold. 1990. Methods for testing

antimicrobial effectiveness, p. 171-194. In W. R. Bailey and Scott(ed.), Diagnostic microbiology, 8th ed. The C.V. Mosby Co.,St. Louis.

3. Bradford, M. M. 1976. Arapid and sensitive methodfor the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding.Anal. Biochem. 72:248-254. 4. Bush, K., and R. B. Sykes. 1984. Interaction of ,-lactam

antibioticswith,-lactamasesasa causeforresistance, p.1-31. In L.E. Bryan(ed.), Antimicrobial drug resistance. Academic Press, Inc., NewYork.

5. Felici, A., G.Amicosante, A. Oratore,R.Strom,P.Ledent,B. Joris,andJ.M.Frere.1993.Anoverview ofkinetic parameters ofclassB P-lactamases.Biochem. J. 291:151-155.

6. Frere, J.M.1989. Quantitativerelationship between sensitivity to ,B-lactam antibiotics and P-lactamase production in gram-negative bacteria. I. Steadystatetreatment. Biochem. Pharma-col. 38:1415-1426.

7.

Iaconiso

J. P.,andC. C. Sanders. 1990.Purificationand

charac-terizationof inducible

P-lactamases

inAeromonasspp. Antimi-crob. AgentsChemother.34:44-51.

8. Laemmli,U.K. 1970.Cleavage ofstructural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685.

9. Livermore, D. M. 1992. Carbapenemases. J. Antimicrob.

Chemother. 29:609-613.

10. Livermore, D. M. 1992. Interplay of impermeability and chro-mosomal 3-lactamase activity in imipenem-resistant Pseudo-monasaeruginosa.Antimicrob. Agents Chemother.36:2046-2048. 11. Massidda, O.,G. M. Rossolini, and G. Satta. 1991.The Aero-monashydrophila cphA gene: molecular heterogeneity among classBmetallo-1-lactamases.J. Bacteriol. 173:4611-4617. 12. Matthew, M.,A. M. Harris, M. J. Marshall, and G. W. Ross.

1975. The use of analyticalisoelectric focusing for detection and identification ofbeta-lactamases. J. Gen. Microbiol. 88:169-178. 13. Nikaido, H., and S. Normark. 1987. Sensitivity of E. coli to various ,-lactams is determined by the interplay of outer membranepermeability and degradation by periplasmic 3-lac-tamases: a quantitative predictive treatment. Mol. Microbiol. 1:29-36.

14. Oliva, B., M. C. Marinucci, G. Amicosante, A. Oratore, and P. M.Bennett.1987. Citrobacterdiversus ULA-27producestwo forms of a chromosomal P-lactamase. J. Antimicrob. Chemother. 20:23-35.

15. Salfi, V., G. Amicosante, and A. Oratore. 1978. Preliminary experimental dataoninhibition ofP-lactamaseIfromB. cereus byCu(II) andZn(II). Boll. Soc. Ital. Biol. Sper. 54:1298-1303. 16. Shannon,K., A. King,and I.Phillips. 1986. P-Lactamaseswith highactivityagainst imipenem and Sch 34343 fromAeromonas hydrophila.J.Antimicrob. Chemother. 17:45-50.

17. Waley, S. G. 1987.An explicit model for bacterial resistance: applicationto ,B-lactam antibiotics. Microbiol. Sci. 4:143-146. 18. Watanabe, M., S. Iyobe, M. Inoue, and S. Mitsuhashi. 1991.

Transferableimipenemresistance in Pseudomonasaenuginosa. Antimicrob.AgentsChemother. 35:147-151.

19. Werner, W.,C. C.Sanders,W.E.Sanders, andR.V.Goering. 1985. Role of P-lactamases and outer membrane proteins in multiple1-lactam resistance of Enterobacter cloacae. Antimi-crob.AgentsChemother. 27:455-459.

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Keywords: Sexual Rights, Sexual Health, Sexual Politics Source of Funding: Social Science and Humanities Research Council of Canada and the Ford Foundation Conflict of Interest

Consider now the same structural network, with a minor modification on one edge. The situation, depicted in Fig. 18 , makes the system non phase synchronizable.. Notice that when

Di questi otto punti sembrano resistere soltanto l’ultimo capitolo e il primo, legato ad un racconto, intitolato proprio “Mrs Dalloway in Bond Street” e pubblicato sul Dial nel

Francesco Berrardo per il travaglio di trabuchi sei […] et del travaglio d’altri trabuchi due […] caduno fatti d’escavatione alla palizzada appresso la portanova et altra

celebrated La Gazza Ladra and Il Barbiere di Siviglia whereas La Vestale and Giovanna d’Arco, by Viganò, represent two hits of dance music of the time. Rossini’s fandom was