Postantibiotic Sub-MIC Effects of Vancomycin, Roxithromycin, Sparfloxacin, and Amikacin

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1992, p /92/ $02.00/0 Copyright X) 1992, Americn Society for Microiology Vol. 36, No. 9 Postntiiotic Su-MIC Effects of Vncomycin, Roxithromycin, Sprfloxcin, nd Amikcin INGA ODENHOLT-TORNQVIST,* ELISABETH LOWDIN, AND OTTO CARS Deprtment of Infectious Diseses, Uppsl University, Uppsl, Sweden Received 14 Jnury 1992/Accepted 15 June 1992 The su-mic effects (SMEs) nd the postntiiotic su-mic effects (PA SMEs) of vncomycin, roxithromycin, nd sprfloxcin for Streptococcus pyogenes nd Streptococcus pneumonie nd of mikcin for Escherichi coli nd Pseudomons eruginos were investigted. A postntiiotic effect ws induced y exposing strins to 10x the MIC of the ntiiotic for 2 h in vitro. After the induction, the exposed cultures were wshed to eliminte the ntiiotics. Unexposed controls were treted similrly. Therefter, the exposed cultures (PA SME) nd the controls (SME) were exposed to different suinhiitory concentrtions (0.1, 0.2, nd 0.3 x the MIC) of the sme drug nd growth curves for period of 24 h were compred. In generl, the PA SMEs were much more pronounced thn the SMEs. However, for mikcin nd E. coli the SME of 0.2 nd 0.3 x the MIC lso hd n initil ctericidl effect. The longest PA SMEs were demonstrted for the comintions with the most pronounced killing during the induction nd for the comintions which exhiited the longest PAEs. In the erly 1940s, the dosge schedules devised for penicillin therpy were sed on the ssumption tht drug concentrtions must e mintined ove the MIC. This ssumption ws sed on previous experiments with the sulfonmides (1, 26). However, few yers fter the introduction of penicillin, it ws shown oth in niml experiments nd in clinicl studies tht discontinuous penicillin therpy ws s effective s continuous one, even if the concentrtions in serum hd fllen under the MIC (4, 26, 28). At tht time, Egle et l. showed, oth in vitro nd in thigh infection model in mice, tht there ws lg phse efore regrowth of cteri fter first chllenge of penicillin (5, 6). This concept roused new interest in the 1970s nd ws nmed the postntiiotic effect (PAE) (3, 14, 33). The PAE hs een extensively studied oth in vitro nd in vivo nd hs een cited s one explntion for the success of intermittent dosing regimens (3, 32). However, in most ntiioticcterium comintions the sum of the time tht the drug concentrtion is ove the MIC nd the PAE does not cover the whole dosing intervl. Also, in the in vivo sitution, suprinhiitory concentrtion of drug will lwys e followed y suinhiitory concentrtions (su-mics). We hve shown erlier tht suinhiitory ntiiotic concentrtions my hve different effects on cteri exposed previously to suprinhiitory ntiiotic concentrtions (postntiiotic su-mic effect [PA SME]), compred with the effects on cteri not previously exposed to ntiiotics (su-mic effect [SME]) (18, 20). In most of the investigted comintions with P-lctm ntiiotics, we found pronounced difference in time etween the PA SME nd the SME, with two exceptions. In the comintions with no PAE, neither PA SME nor n SME ws found, nd in the comintion of imipenem with Pseudomons eruginos, oth long PA SME nd n SME were seen (20). The im of this study ws to investigte the occurrence of PA SMEs nd SMEs for compounds other thn,b-lctm ntiiotics, such s vncomycin, roxithromycin, sprfloxcin, nd mikcin. * Corresponding uthor (This mteril ws presented in prt t the 31st Interscience Conference on Antimicroil Agents nd Chemotherpy, Chicgo, Ill., 29 Septemer to 2 Octoer 1991.) MATERIALS AND METHODS Cultures. The strins used in this study were s follows: Streptococcus pyogenes, group A, M12, NCTC P 1800, Streptococcus pneumonie ATCC 6306, P. eruginos ATCC 27853, nd Escherichi coli ATCC The grmpositive strins were grown in Todd-Hewitt roth, nd the grm-negtive strins were grown in Mueller-Hinton roth supplemented with 50 mg of C2" nd 25 mg of Mg2+ per liter. The grm-positive strins were cultured for 6 h t 37 C in 5% CO2 in ir, resulting in pproximtely 5 x 10' CFU/ml, nd the grm-negtive strins were cultured for 6 h t 37 C, resulting in pproximtely 109 CFU/ml. Antiiotics. The ntiiotics were otined s reference powders with known potencies from the following phrmceuticl compnies: vncomycin from Eli Lilly Sweden AB, Stockholm, Sweden; roxithromycin from Roussel Nordisk AB, Stockholm, Sweden; sprfloxcin from Rhone-Poulenc Rorer, Helsingorg, Sweden; nd mikcin from Bristol- Myers Squi, Bromm, Sweden. Dilutions were mde in distilled wter on the dy the experiments were performed. Determintion of MICs. MICs were determined in fluid medi y using twofold dilution with n inoculum of pproximtely 105 CFU of the test strin per ml nd were red fter 24 h. The MIC ws defined s the lowest concentrtion of the ntiiotic llowing no visile growth. Determintions of MICs were performed in triplicte on seprte occsions. Since the MIC of mikcin for E. coli ATCC ws higher thn tht reported y other uthors (see Results), the MIC for this comintion ws determined five times on different occsions. Induction of the postntiiotic phse nd determintion of the PAE. The following ntiiotic-cterium comintions were studied: vncomycin with S. pyogenes group A, M12, NCTC P 1800 nd with S. pneumonie ATCC 6306; roxithromycin with S. pyogenes group A, M12, NCTC P 1800 nd with S. pneumonie ATCC 6306; sprfloxcin with S. pyo-

2 VOL. 36, 1992 POSTANTIBIOTIC SUB-MIC EFFECTS 1853 TABLE 1. PAE, PA SME, nd SME for different ntiiotic-cterium comintions. Durtion (h) of effect' Antiiotic-cterium O.1x the MIC 0.2x the MIC 0.3x the MIC comintion PAE SME PA SME SME PA SME SME ' PA SME Vncomycin-S. pyogenes 3.5 ( ) 0 (0-0.1) 5.1 ( ) 0 (0-0.2) 6.8 ( ) >9.7-<22.7 <22.7 (9.4->22.7) M12, NCTC P 1800 (0.5->22.6) Vncomycin-S. pneumo- 3.0 ( ) 0 ( ) 8.8 ( ) 1.6 ( ) >8.3-< ( ) >21.3 (>21.3) nie ATCC 6306 (>7-<21.3) RoXithromycin-S. pyo- 5.0 ( ) 0.1 (0-0.3) 6.3 ( ) 0.1 (0-0.2) >9.3-< (0-0.5) >9.7-<22.7 genes M12, NCTC P (>9.3-<23) (>9.7-<23) 1800 RoXithromycin-S. pneu- >7.8-< (0) >7.8-< ( ) >7.8-< ( ) >7.8-<20.8 monie ATCC 6306 (6.0-<20.8) (8-<20.8) (>7.8-<21.3) (>7.8-<21.3) Sprfloxcin-S. pyogenes 1.9 ( ) 0.2 (0-0.4) 2.5 ( ) 0.6 ( ) 3.3 ( ) 1.4 ( ) 5.0 ( ) M12, NCTC P 1800 Sprfloxcin-S. pneumo- 2.5 ( ) 0.4 ( ) 3.1 ( ) 1.0 ( ) 3.8 ( ) 2.3 ( ) 6.0 ( ) nie ATCC 6306 Amikcin-E. coli ATCC 5.1 ( ) 0 (0-0.1) 5.5 ( ) >9.3-<22.3 >9.3-<22.3 >9.3-<22.3 >22.3 (>22.3) (>9.3->22.3) (> ) ( ) Amikcin-P. eruginos 1.4 ( ) 0 ( ) 1.7 ( ) 0.5 ( ) 2.2 ( ) 1.2 ( ) 3.3 ( ) ATCC Vlues were clculted y counting CFU nd re verges from three experiments. Vlues in prentheses re rnges from individul experiments. genes group A, M12, NCTC P 1800 nd with S. pneumonie ATCC 6306; mikcin with E. coli ATCC nd with P. eruginos ATCC Ech comintion ws studied three times on different occsions. After incution for 6 h, the test strins were diluted 101 to otin strting inoculum of 5 x 107 to 108 CFU/ml. Therefter, the strins in exponentil growth phse were exposed to 10 x the MIC of the ntiiotic for 2 h t 37 C. To eliminte the ntiiotics, the strins were wshed three times for 5 min ech time t 1,400 x g nd diluted into fresh medi. The control strins were treted similrly. To llow monitoring of growth fter the initil 2-h exposure, nd depending on the rte of killing during this period, the exposed strins were diluted either y fctor of 10-2 in fresh medium or not t ll (the comintions of roxithromycin with S. pneumonie, sprfloxcin with S. pneumonie, nd mikcin with E. coli were not diluted). In order to otin n inoculum s similr to the exposed cultures s possile, the controls were ll diluted The cultures with cteri in the postntiiotic phse nd the controls were therefter divided into four different tues. In order to determine the PAE, one tue of ech culture ws reincuted t 37 C for nother 22 h. Smples were withdrwn t 0 nd 2 h (efore nd fter dilution), t 3, 4, 5, 6, nd 8 h, nd then t 24 h nd if necessry diluted in phosphte-uffered sline. Three dilutions of ech smple were seeded on lood gr pltes nd colonies were counted to determine the numer of CFU. Only pltes with 50 to 500 colonies were counted. The PAE ws defined ccording to the following formul (3): PAE = T - C, where T is the time required for the vile counts of the cultures exposed to ntiiotics to increse y 1 log10 unit ove the counts oserved immeditely fter wshing nd C is the corresponding time for the controls. Determintion of the PA SME nd the SME. After wshing nd dilution, the remining three tues of the control cultures nd the cultures in the postntiiotic phse were exposed to 0.1, 0.2, nd 0.3x the MIC, respectively, of the ntiiotic used for the induction of the PAE nd reincuted t 37 C for nother 22 h. Smples were withdrwn, nd vile cteri were counted s descried ove. The PA SME ws defined ccording to the following formul (20): PA SME = TPA - C, where TPA is the time for the cultures previously exposed to ntiiotics nd therefter exposed to different su-mics to increse y 1 log10 unit ove the counts oserved immeditely fter wshing nd C is the corresponding time for the unexposed control. This definition ws chosen ecuse in clinicl sitution in which intermittent ntiiotic dosge is used, it is the comined effect of supr- nd suinhiitory concentrtions tht will determine the time until cteril regrowth. The SME ws defined s Ts - C, where Ts is the time for the cultures exposed only to the su-mics to increse y 1 log10 unit ove the counts oserved immeditely fter wshing nd C is the time descried ove (20). RESULTS MICs. The MICs were s follows: vncomycin with S. pyogenes group A, M12, NCTC P 1800, 0.5 mg/liter; vncomycin with S. pneumonie ATCC 6306, 0.5 mg/liter; roxithromycin with S. pyogenes group A, M12, NCTC P 1800, 0.13 mg/liter; roxithromycin with S. pneumonie ATCC 6306, 0.13 mg/liter; sprfloxcin with S. pyogenes group A, M12, NCTC P 1800, 0.25 mg/liter; sprfloxcin with S. pneumonie ATCC 6306, 0.13 mg/liter; mikcin with E. coli ATCC 25922, 8 mg/liter; nd mikcin with P. eruginos ATCC 27853, 2 mg/liter. The MICs for the ATCC strins re close to those found y other uthors except tht for mikcin with E. coli ATCC 25922, which ws higher in this study. However, the sme MIC ws otined on every occsion. PAEs, PA SMEs, nd SMEs. The results of ll experiments re given in Tle 1. The results re sed on the men CFU t ech smpling time from the three experiments. The rnge is from the individul experiments. Figure 1 shows the PA SME nd SME for roxithromycin with S. pyogenes M12, NCTC P The PA SME t 0.2 nd 0.3x the MIC ws more thn 9 h compred with the SME of only 0.1 h. Figure 2 shows the PA SME nd SME for sprfloxcin with the sme strin. Here lso, the PA SME ws sustntilly longer thn the SME (5.0 nd 1.4 h, respectively, t 0.3 x the MIC), even though the PA SME, s well s the PAE, ws shorter

3 1854 ODENHOLT-TORNQVIST ET AL. ANTimICROB. AGENTS CHEMOTHER. I 0 25 i -0- CmArol * QIC 0 Q2xMC * E3zMIC FIG " 2S5 PA SME () nd SME () for roxithromycin with S. pyogenes M12, NCTC P PAE ws induced with lox the MIC. for sprfloxcin compred with roxithromycin. Figure 3 shows the PA SME nd SME for mikcin with P. eruginos ATCC In this comintion, oth the PA SME nd the SME were short compred with the effect on E. coli ATCC DISCUSSION The MICs for cteri nd the phrmcokinetics of ntiiotics hve long een the min determinnts of ntiiotic dosge regimens used in clinicl prctice (11, 31). Normlly, one ims to mintin the concentrtion ove the MIC for most of the dosing intervl, often without tking protein inding, immune defense mechnisms, or phrmcodynmic prmeters into considertion (2, 9, 16). Since MIC determintions re red fter 18 to 24 h, they will not revel chnges in cteril numers during this period, such s, for exmple, n initil ctericidl effect followed y regrowth (9, 23). Phrmcodynmic prmeters such s the rte of cteril killing, the PAE, nd the PA SME my provide more ccurte description of the durtion of ntimicroil ctivity thn tht given y MIC determintions nd thus yield more informtion for the determintion of n optiml dosing regimen. A primry exposure to su-mics of ntiiotics my hve different effects on cteri. Beyond n inhiitory effect on the growth rte in vitro (12), su-mics my lter the cteril cell wll nd thus increse the susceptiility of the

4 VOL. 36, 1992 POSTANTIBIOTIC SUB-MIC EFFECTS Ei h I -0O-- Comtrd -U,- &1xMIC -0-- ILxMIC FIG I' 25 PA SME () nd SME () for sprfioxcin with S. pyogenes M12, NCTC P PAE ws induced with lox the MIC. cteri to the immune system (8, 15, 17, 22, 30), chnge the expression of cteril exoenzymes (10, 13), nd lso chnge the outcome of experimentl infections in nimls. Zk nd Krdolfer (35) hve shown in mouse model tht one-third of the MIC of mpicillin in peritonel fluid is enough to prevent regrowth of E. coli nd Stphylococcus ureus up to 18 h fter the injection. Roosendl et l. (24) showed tht su-mics of ceftzidime hd therpeutic effect in pneumonie model in mice, nd we hve demonstrted in rit model with implnted tissue cges tht one-third of the MIC of enzylpenicillin gives dely of severl hours efore regrowth of streptococci (19). However, these primry effects of su-mics of ntiiotics seem to e different from the effects of low concentrtions on cteri previously exposed to suprinhiitory concentrtions. We hve shown previously tht for streptococci nd pneumococci first exposed to suprinhiitory concentrtion, su-mics of 1-lctm ntiiotics prolong the inhiition of growth for severl hours. In contrst, su-mics lone did not lter the growth curves very much compred with those of the unexposed controls (18-20). Oshid et l. (21) demonstrted this PA SME for S. ureus with spoxicillin oth in vitro nd in thigh infection mouse model. Erlier, Snde et l. (25), in meningitis model in rits, demonstrted long inhiition of growth of pneumococci fter the concentrtion of mpicillin hd fllen elow the MIC. How-

5 1856 ODENHOLT-TORNQVIST ET AL. 10r ANTimICROB. AGENTS CHEMOT-HER. 91 I Contrd 0 I. 2 ' PAE -0-- QlimC -*- 0.2xMIC 0* 3xMIC h I I *U-- C0 LIXMIC 02xMIC 0.3xMIC FIG If 25 h PA SME () nd SME () for mikcin with P. eruginos ATCC PAE ws induced with lox the MIC. ever, when penicillinse ws given intrcistemlly, this effect disppered nd the long inhiition of growth ws considered to e due to residul suinhiitory concentrtions (27). The longer PAEs seen in some in vivo models compred with in vitro effects my thus e explined y the PA SME, since the in vivo PAE normlly is mesured from the time the concentrtion flls elow the MIC nd does not tke into considertion the possile effect of the PA SMEs (7, 25). Evidently, the PA SME exists lso for non-,b-lctm ntiiotics. Winstnley et l. (34) reported recently PA SME for Enterococcusfeclis with teicoplnin nd showed PA SME of pproximtely 10 h compred with n SME of only 0.3 h. In the present study, we hve shown tht the PA SME t 0.3 x the MIC of vncomycin does not llow ny regrowth of streptococci or pneumococci for 24 h. The PA SME of roxithromycin for streptococci t 0.3 x the MIC ws more thn 9 h, compred with n SME of 0.1 h t the sme concentrtion. For pneumococci, the PA SME t 0.3x the MIC resulted in complete killing of the cteri, while the SME ws 4 h. Also, for sprfloxcin there ws gret difference etween the PA SME nd the SME, lthough the PA SME ws somewht shorter thn for roxithromycin nd vncomycin. Amikcin ws shown to hve n effect on E. coli quite different from tht on P. eruginos. In spite of high MIC

6 VOL. 36, 1992 for E. coli, the killing during the induction exceeded tht of P. eruginos y 3 log1o CFU. Also, there ws considerly longer PAE for E. coli thn for P. eruginos, nd the PA SME t 0.3 x the MIC gve complete killing of E. coli, compred with PA SME of 3.3 h for P. eruginos. In contrst to the other ntiiotic-cterium comintions, there ws lso pronounced primry effect of su-mics for E. coli t oth 0.2 nd 0.3 x the MIC. We hve demonstrted this for imipenem with P. eruginos, wheres no such effect ws seen for imipenem with E. coli (20). The SME of mikcin with P. eruginos t 0.2 nd 0.3 x the MIC did not differ much from tht of the unexposed control, which grees with the work of Zhnel et l., who did not find ny ctericidl effect t one-fourth of the MIC of mikcin for the sme strin (36). The mechnism ehind the PA SME is not known. For,3-lctm ntiiotics, we hve hypothesized tht the mechnism ehind this effect is due to continuous cteril production of penicillin-inding proteins during the exposure to penicillin nd tht only smll mount of penicillin is necessry to ind these newly produced penicillin-inding proteins, if suprinhiitory concentrtion hs een given first (20, 29). It seems lso tht for drugs with other mechnisms of ction, such s inding to DNA gyrse or RNA, only smll mount of the drug is necessry to prevent norml cell growth once the cteri hve een dmged y suprinhiitory concentrtion of the ntiiotic. In generl, it seems tht long PAE nd pronounced rte of killing promote long PA SME, which t 0.3x the MIC cn e ctericidl for 24 h for certin ntiioticcterium comintions. Determintions of MICs nd the phrmcokinetics of ntiiotics hve een mjor fctors in deciding the dosge used in clinicl trils. However, phrmcodynmic prmeters such s the rte of cteril killing, PAE, nd PA SME will proly influence the optiml dosing intervl. The PAE hs in the lst few yers roused gret interest in regrd to understnding the success of spced dosing (3), ut in humns nd in experimentl niml models suprinhiitory ntiiotic concentrtion lwys will e followed y su- MICs. There is pronounced difference etween the PA SMEs nd the SMEs. Our experiments hve shown tht for ntiiotic-cterium comintions for which PAE exists, very long inhiition of cteril growth cn e chieved with su-mics fter suprinhiitory dose hs een given. This cn proly influence the dosing schedules of ntiiotics nd my lter current dosing regimens, which my not lwys e optiml in terms of efficcy, toxicity, nd cost. REFERENCES 1. Altemeier, W. A Penicillin therpy with prolonged intervl dosge schedules. Ann. Surg. 128: Brz, M A critique of niml models in ntiiotic reserch. Scnd. J. Infect. Dis. Suppl. 14: Crig, W. A., nd S. Gudmundsson The postntiiotic effect, p In V. Lorin (ed.), Antiiotics in lortory medicine. The Willims & Wilkins Co., Bltimore. 4. Egle, H., R. Fleischmn, nd M. 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