Comparative activity of several antimicrobial agents against nosocomial Gram-negative rods isolated across Canada

Department of Microbiology, Mount Sinai and Princess Margaret Hospitals, University of Toronto, Toronto, Ontario The Canadian Antimicrobial Resistance Study Group comprises: Anne-Marie Bourgault MD FRCPC, Hopital St-Luc, Montreal, Quebec; James Brunton MD FRCPC, The Toronto Hospital, Toronto, Ontario; Kevin Forward MD FRCPC, Victoria General Hospital, Halifax, Nova Scotia; Daryl Hoban PHD, Health Sciences Centre, Winnipeg, Manitoba; Jean R Joly MD FRCPC, St Sacrement Hospital, Ste-Foy, Quebec; Tom J Louie MD FRCPC, Calgary General Hospital, Calgary, Alberta; Vivian Loo MD FRCPC, Royal Victoria Hospital, Montreal, Quebec; Anne Phillips MD FRCPC, The Toronto Hospital, Toronto, Ontario; Robert Rennie PHD, University of Alberta Hospitals, Edmonton, Alberta; and John A Smith MD FRCPC, Vancouver General Hospital, Vancouver, British Columbia Correspondence and reprints: Dr DE Low, Department of Microbiology, Mount Sinai Hospital, 600 University Avenue, Room 1487, Toronto, Ontario M5G 1X5. Telephone (416) 586 4435, Fax (416) 586 8746, e-mail low@mshri.on.ca Received for publication August 4, 1994. Accepted October 13, 1994 SR SCRIVER, CANA DIAN ANTI MI CRO BIAL RESIS TANCE STUDY GROUP, DE LOW. Com para tive ac tiv ity of sev eral an ti mi cro bial agents against no so comial Gramnegative rods iso lated across Can ada. Can J In fect Dis 1995;6(2):7682. In 1992, a sur veil lance study was per formed in Can ada to de ter mine the sus cep ti bil ity of no so comial Gramnegative rods to sev eral wide spec trum an ti mi cro bi als. Con secu tive iso lates from 10 in sti tu tions, as well as ad di tional strains of se lected spe cies of En tero bac te riaceae that are known to pos sess the Bush group 1 betalactamase, were tested for sus cep ti bil ity to 12 an ti mi cro bi als. Thirdgeneration cepha lo sporin re sis tance was found to be as high as 29% in En tero bac ter cloa cae that pos sesses the Bush group 1 betalactamase and less than 4% in those iso lates not pos sess ing this en zyme. Ce fepime equalled or ex ceeded the ac tiv ity of the thirdgeneration cepha lo sporins against the spe cies of En tero bac te riaceae that dem on strated re sis tance to the thirdgeneration cepha lo sporins.

T HE IN TRO DUC TION OF THIRD-GENERATION CEPHALO- sporins has im proved our abil ity to treat se ri ous in fections caused by Gram-negative patho gens.How ever, increased use of these agents has been fol lowed by the emer gence of re sis tance to them.This re sis tance can be medi ated by di min ished outer mem brane per me abil ity, pro duction of the chro mo somally me di ated in duci ble beta-lactamase, ac qui si tion of plas mid me di ated betalactamases or com bi na tions of these mecha nisms (1)(2)(3).Initially, ex tended spec trum cephalo sporins were be lieved to be sta ble to both chro mo so mal and plas mid me di ated en zymes.How ever, since 1985, out breaks and spo radic cases of in fections due to ceftazidime-resistant or gan isms with plasmidmediated ex tended spec trum beta-lactamases have been described in Europe, the United States and else where (4)(5)(6)(7)(8).Spo radic mu ta tions lead ing to sta ble derepression of the chro mo somally me di ated in duci ble beta-la ct amase also result in re sis tance to third-generation cepha lo sporins (1,(9)(10)(11)(12).These Bush group 1 (BGI) enzymes are gen er ally iden ti fied in spe cies of En tero bacter, Citro bac ter, Ser ra tia, Provi dencia, Mor gan ella, indole-positive Pro teus and Pseu do mo nas ae rugi nosa.
Ce fepime is an ex tended spec trum cepha lo sporin that is con sid ered to be a 'fourth -generation' cepha lo sporin.This group of cepha lo sporins has a slightly dif fer ent penicillinbinding pro tein target-binding profile, lower af fin ity for chromo somally en coded beta-la ct amases, par ticu larly BGI, and a higher rate of pene tra tion through outer mem brane porins of Gram-negative bac te ria com pared with other cepha lo sporins.These dif fer ences en hance the spec trum of bac te ri cidal ac tivity of ce fepime rela tive to all avail able cepha lo sporins (13).Other stud ies have dem on strated that Gram-negative isolates that are re sis tant to one or more third-generation cephalo sporins are sus cep ti ble to ce f epime (14,15).In a re cent Ca na dian sur vey, it was de ter mined that 23% of iso lates of En tero bac ter cloa cae were cef tazidime re sis tant (16).
Ta zo bac tam is a re cently stud ied peni cil lanic acid sul fone that in hib its a wide va ri ety of lac ta mases (17)(18)(19)(20)(21).The com bina tion of ta zo bac tam with peni cil lins and broad spec trum cepha lo sporins has dem on strated broad an ti mi cro bial ac tiv ity (19,20).
The pur pose of this study was to de ter mine the in vi tro activ ity of ce fepime and piper acil lin/ta zo bac tam, two new an timi cro bial agents, in ad di tion to sev eral other an ti mi cro bi als against a se lected group of no so comial Gram-negative isolates from sev eral cen tres across Can ada.

MA TE RI ALS AND METH ODS
Bac te rial iso lates: Ap proxi mately 100 con secu tive non du plicate no so comial iso lates of com mon Gram-negative bac te rial patho gens were col lected from 10 cen tres across Can ada.Par tici pat ing cen tres were asked to sub mit an ad di tional 50 iso lates of spe cies known to har bour BGI beta-lactamases, spe cifi cally: E cloa cae, En tero bac ter spe cies, Citro bac ter fre un dii, Mor gan ella mor ganii, Pro teus vul garis, Provi den cia spe cies, Ser ra tia marc es cens and P ae rugi nosa.Iso lates were iden ti fied us ing stan dard meth od olo gies (22) and fro zen at -70°C in phos phate buff ered glyc erol.All iso lates were subcul tured twice be fore sus cep ti bil ity test ing.Sus cep ti bil ity test ing: Broth mi cro di lu tion was per formed ac cord ing to Na tional Com mit tee for Clini cal Labo ra tory Standards guide lines (23).Mi cro di lu tion pan els were pre pared by dis pen sa tion of cation-supplemented Mueller-Hinton broth con tain ing twofold-concentration in cre ments of an ti mi cro bial agents in 100 µL ali quots into plas tic 96-well trays.In ocu lum sus pen sions equal to a 0.5 McFar land stan dard were fur ther di luted and added to the mi cro di lu tion trays to achieve a fi nal in ocu lum of 5x10 5 col ony form ing units (CFU)/mL.Col ony counts were per formed to con firm the fi nal in ocu lum.Fol lowing in ocu la tion, mi cro di lu tion trays were in cu bated at 35°C in am bi ent air for 16 to 20 h.Af ter in cu ba tion, the mini mum inhibi tory con cen tra tion (MIC) was de fined as the low est con centra tion of an ti mi cro bial agent with no visi ble evi dence of growth.Ce fepime (Bristol-Myers Squibb, Con necti cut), piper acil lin (Lederle Labo ra to ries, New Jer sey), ta zo bac tam (Lederle Labo ra to ries), ce fo taxime (Hoechst-Roussel Phar ma ceu ti cals Inc, New Jer sey), cef tazidime (Glaxo, North Caro lina), cef tri axone (Hoffman-La Roche, Inc, New Jer sey), ti car cil lin (Beecham Labo ra to ries, Ten nes see), cla vu la nate (Beecham Labo ra to ries) and imipe nem (Merck Sharp and Dohme, New Jer sey) were ob tained from their re spec tive manu fac tur ers.Gen ta mi cin, to bra mycin, cipro floxa cin and trimethoprimsulfamethoxazole pow ders were ob tained from Sigma (Sigma Chemi cal Co, Mis souri).Ta zo bac tam was com bined with piper acil lin at a fixed con cen tra tion of 4.0 g/mL.Cla vulanic acid was com bined with ti car cil lin in a ra tio of ti car cil lin to cla vu la nate of 2:1.
Escheri chia coli ATCC 25922 and P ae rugi nosa ATCC 27853 were used as con trol strains for broth mi cro di lu tion test ing.
Third-generation cepha lo sporin re sis tance was rare in spe cies where re sis tance would be char ac ter ized by the presence of plasmid-mediated ex tended spec trum betalactamases, spe cifi cally E coli and K pneu mo niae.Rates of re sis tance to cef tazidime in these iso lates were 1.1% and 2.9%, re spec tively.How ever, in iso lates with BGI betalactamases, rates of re sis tance to the third-generation cephalo sporins were sig nifi cant.E cloa cae dem on strated the highest lev els of re sis tance to cef tazidime (28.9%).Iso lates re sis tant to cef tazidime also dem on strated cross-resistance to other third-generation cepha lo sporins.The most ac tive cepha lo sporin tested against all iso lates was ce fepime.Iso lates of E coli, K pneu mo niae and P mirabilis were more sus cep ti ble to piper acil lin/ta zo bac tam (MIC 90 32.0 g/mL) than those with type 1 cepha lo spori nases.Of the iso lates of Entero bac te riaceae and P ae rugi nosa re sis tant to piper acil lin alone, 290 (16.6%) and 34 (14.8%),re spec tively, be came sen si tive when piper acil lin was com bined with ta zo bac tam.
For all iso lates tested, the most ac tive agents were the ami no gly cosides, imipe nem and cipro floxa cin.Cipro floxa cin re sis tance was less than 2.0% in all iso lates of En tero bac teriaceae with the no ta ble ex cep tion of En tero bac ter spe cies, in which 5.1% of iso lates had MICs to cipro floxa cin of greater than 4.0 g/mL.
Imipe nem ex hib ited ex cel lent in vi tro ac tiv ity.Re sis tance was de tected only in P ae rugi nosa (23.3%) and En tero bac ter spe cies (2.3%).The high est in ci dence of ami no gly co side resis tance was found in iso lates of S marc es cens, in which 26.2% and 22.1% of iso lates were gen ta mi cin-and tobramycin-resistant, re spec tively.In all other spe cies tested, in clud ing P ae rugi nosa, ami no gly co side re sis tance was less than 10%.To bra my cin was much more ef fec tive against isolates of P ae rugi nosa than gen ta mi cin, be cause only 2.7% were re sis tant to this ami no gly co side whereas 15.1% of isolates were re sis tant to gen ta mi cin.

DIS CUS SION
The rapid ini tia tion of ef fec tive an ti mi cro bial ther apy for the  (24).Treat ment is of ten em piri cal and based pri mar ily on knowl edge of the an ti mi crobial sus cep ti bil ity of the most com mon bac te ria en coun tered.
It is there fore cru cial to con duct broadly based sur veil lance stud ies to gen er ate sus cep ti bil ity data that can help pre dict sus cep ti bil ity of a spe cies to an an ti mi cro bial or group of an timi cro bi als.How ever, it is also nec es sary to know or highly sus pect what spe cies are re spon si ble for the dis ease syndrome in a given pa tient popu la tion.
In light of the in creas ing im por tance of En tero bac ter species as im por tant no so comial patho gens, changes in the beta-lactam sus cep ti bil ity pro file of these iso lates are of great con cern.Sand ers (12) found that, al though all iso lates of E cloa cae will pro duce a BGI beta-lactamase on in duc tion, 28.9% of these iso lates have be come con sti tu tive pro duc ers.
Ce fepime, a fourth-generation cepha lo sporin, is struc turally simi lar to ce fo taxime, cef tri ax one and cefti z ox ime.However, a qua ter nary nitrogen-containing sub sti tu tion on the posi tively charged third car bon and the nega tive charge on po si tion four of the cepha lo sporin nu cleus gives ce fepime zwit te ri onic prop er ties that al low ce fepime to pene trate very rap idly through the outer mem brane of Gram-negative bac teria.As well, ce fepime, like other fourth-generation cepha losporins, is sta ble against BGI beta-lactamases (13).
For all iso lates, the most ac tive cepha lo sporin tested was ce fepime.Among En tero bac te riaceae, more than 98% of all iso lates were sus cep ti ble to this agent.The ac tiv ity of cefepime equalled or ex ceeded that of third-generation cepha losporins.Iso lates dem on strat ing re sis tance to third-generation cepha lo sporins re mained sus cep ti ble to ce fepime.Ma jor differ ences in ac tiv ity be tween ce fepime and the thirdgeneration cepha lo sporins were found in iso lates with BGI beta-lactam ases, ie, E cloa cae, En tero bac ter ae ro genes, C fre un dii, M mor ganii and S marc es cens.Against P ae ruginosa, ce fepime and cef tazidime were more ac tive than ce fotaxime, but these dif fer ences are likely the re sult of dif fer ent per me abili ties for these agents (25) as op posed to betalactamase sta bil ity.
For all iso lates tested, the ac tiv ity of piper acil lin was highly po ten ti ated when com bined with the beta-lactamase in hibi tor ta zo bac tam.The beta-lactam/beta-lactamase in hibitor com bi na tion of piper acil lin/ta zo bac tam was more ef fec tive than ti car cil lin/cla vu la nate against iso lates pos sess ing BGI beta-lactamases, iso lates found in this study and oth ers to be in creas ingly re sis tant to tra di tional third-generation thera peutic agents.
An ti mi cro bial re sis tance among iso lates of K pneu mo niae and E coli oc curred in fre quently.Re sis tance to thirdgeneration cepha lo sporins was rare, which con trasts sharply with re ports from Europe and spe cific ar eas of the United States, where the preva lence of re sis tance is high with the occur rence of no so comial out breaks (7,8).

CON CLU SIONS
This study dem on strated that third-generation cepha losporin re sis tance is a clini cally rele vant prob lem in iso lates known to pos sess BGI beta-lactamases.Al though crossresistance to other third-generation cepha lo sporins was seen, ce fepime and nonbeta-lactam agents re tained their activ ity.Plasmid-mediated ex tended spec trum beta-lactamase was not a sig nifi cant find ing.Tra di tional nonbeta-lactam agents were found to have re tained their ac tiv ity against noso comial Gram-negative rods.As well, ami no gly cosides are still highly ac tive against the ma jor ity of iso lates tested, with the no ta ble ex cep tion of S marc es cens.

TA BLE 1 Com para tive ac tiv ity of 12 an ti mi cro bi als against Gram-negative clini cal iso lates from 10 medi cal cen tres
CAN J INFECT DIS VOL 6 NO 2 MARCH/APRIL 1995 *Mini mum in hibi tory con cen tra tion (MIC) for 50 and 90% of iso lates tested.TMP/SMX Trimethoprim-sulfamethoxazole TA BLE 1 (con tin ued) *Mini mum in hibi tory con cen tra tion (MIC) for 50 and 90% of iso lates tested.†Includes 68 iso lates of En tero bac ter ae ro genes, 27 iso lates of En tero bac ter ag glom er ans and 39 En tero bac ter spe cies.TMP/SMX Trimethoprim-sulfamethoxazole

TA BLE 1 (con tin ued)
Mini mum in hibi tory con cen tra tion (MIC) for 50 and 90% of iso lates tested.TMP/SMX Trimethoprim-sulfamethoxazole CAN J INFECT DIS VOL 6 NO 2 MARCH/ APRIL 1995 man age ment of in fec tions caused by Gram-negative ba cilli can sig nifi cantly im prove out come *