Comparative activity of daptomycin and teicoplanin against enterococci isolated from blood and urine

KR FORWARD, P DEGAGNE, KR BARTLETT, GK HARDING. The comparative activity of daptomycin and teicoplanin against enterococci isolated from blood and urine. Can J Infect Dis 1992;3(4):173-178. The authors compared the activity of daptomycin with U1at of ampicilJ in. penicillin . teicoplanin and vancomycin against 304 strains of Enterococcus species isolated from blood and urine. Daptomycin was as active as penicillin against Enterococcusfaecal is: 90% of strains were inhibited by 2 mg/L. Daptomycin was more active than vancomycin (90% m in imal inhi bitory concentration [MlCgoJ 2 mg/L; 90% minimal bactericidal concen tration [MBCgoJ 8 mg/L) but was less active than teicoplan in (M!C5o 0.25: MBCgo 8 mg/L) or ampicillin (MICgo 1 mg/L: MBCso 2 mg/L) against Efaecalis. In time-kill studies daptomycin was not more rapidly bactericidal !.han ampicillin or penicillin but was s ignificanUy more rapidly bactericidal than either teicoplanin or vancomycin. In combination with genlamicin, daptomycin has activity similar to !.hat of penicillin, vancomycin and teicoplan in. Daptomycin may be a su itable alternative to penicillin in patients allergic to penicillins or for the treatment of enterococcal infections caused by beta-lactamaseproducing enterococci.

E TEROCOCCI ARE AN I CREASING LY IMPORTANT CAUSE OF serious infections in h o pitalized patients (l).This in c rease may be attributed in part to the widespread u e of third generation cephalospot-ins and monobactams in the past decade (2)(3)(4)(5).Accompanying this increase in prevalence has been the rapid emergence of enterococci which prod u ce aminocrlycoside-inactivating enzym es and are resistant to U1e synet-gistic interaction of a minoglycosides and pen icill in (6)(7)(8)(9) .Enterococcal stra ins resistant to penicill ins eillier by virtue of their ab ili ty to produce beta-lactarnase or by the development of altered penicillin-binding proteins are also being recovered with increasing frequ ency (10)(11)(12)(13)(14) .These stra ins a re also resistant to U1e synergistic interaction of a minoglycosides and penicillins.Recently.Leclercq et al ( 15) described two strains of Enterococcus faecium resistant to boU1 vancomycin and teicoplan in.
Because of tlle em erge nce of antibiotic resistance a mong enterococci, and since ma ny patients a re penicillin a lle rgic.alternate antib iotics a r e n eeded for the treatment of patients witll set;ous enterococcal infections.T he auU1ors compa red U1e activities of daptomycin.teicoplanin.penicillin.amp ic illin a nd vancomycin against Enterococcus species isolated from patients with bacteremia and urinary tract infec tions.

MATERIALS AND METHODS
Organisms: A total of 304 isolates from b lood and u t;ne cu ltures were collected over a five year period from patients seen at St Boniface Gen eral Hospital and tlle Health Sciences Centre in Winnipeg, Manitoba.Of these.141 were from blood cultures a nd 163 were urin e isolates.Organisms we re presumptive ly identified as Enterococcus species using esculin hydrolysis and 6.5% sodi um c hlotide tolerance (16).The organisms were s ubseq u ently identified to species level based on carbohydrate consumption (16).Al l stocks we re k ept at -70°C in skim milk.Antimicro bial a gents : Gentamicin.vancomycin.daptomycin.teicoplanin.penicillin and ampicillin we re su ppli ed as standa rd laborato ry powde rs by their respective m anufacturers .Al l solutions were prepa red according to manufacturers' instructions.Determina tio n of MICs/MBCs: Minimal inhibitory con centrations (MICs) were determined by the microtitre technique using cation-supplemented Mueller -Hinton broth (Difco Laboratories.Michigan) at pH 7.2 wiU1 a final volume of 0 . 1 mL per we ll.Antimicrob ial agents were tested over a range of twofold concentration increments from 0 .12 to 256 mg/L.Microtitre p lates were stored at -70°C a nd used wi.tllinU1ree wee ks of preparation.
The inoculum was standardized using a number 1 McFarland turbidity standard.This was diluted 1:10 using sterile water.A Dynatech MIC 2000 inoculator (Dynatech Laboratories, Virginia) was used to deliver 0.001 mL to each well .The final concentration of or-  •==============• Colony counts were performed by transferring 0.1 mL of broth to the surface of sheep blood agar plates.Plates were incubated at 37°C for 18 h and colony counts performed.
Time-kill synergy studies were performed on six blood culture isolates .Daptomycin, vancomycin, teicoplanin and penicillin were compared, each in combination with gentamicin against six E jaecalis strains not highly resistant to gentamicin.For the synergy studies, the vancomycin.teicoplanin and penicillin concentrations were 10 mo-;L: the daptomycin and gen tamicin concentrations were 5 mg/L.The method was otherwise as described for kill curves, with the exception that no sampling was done at 48 h.In order to compare bacterial survival in kill curve studies.a •repeated measures' analysis of variance comparing counts at each point using 'least square' means and a djusted for multiple comparisons via Bonferroni adjustments was used.

RESULTS
The authors determined susceptibilities of 30 4 Enterococcus species strains: 141 from blood and 163 from urine.1\vo hundred and eighty-one strains were E faecalis.18 were E jaecium and five were other Enterococcus species.1\venty-seven E jaecalis isolates CAN J INFECT DIS VOL 3 No 4 JULY/ AUGUST 1992 (19.0%) from blood were highly resistant to gentamicin (MIC greater than or equal to 500 m g/L).as were 12 isolates from urine.Table l shows the comparative activity of daptomycin , teicoplanin and other agents against the 304 strains tested.Teicoplanin was the most active agent with an M!Cgo of0.25 mg/L: however, the MBCgo was 8 mg/L and 256 strains were tolerant of teicoplanin.The MIC5o.MICgo and MBCgo for daptomycin were l. 2 and 8 mg/L and compared favorably with those of penicillin (2. 2 and 8 mg/L.respectively) .No strain was tolerant of daptomycin and only one strain was tolerant of ampicillin and penicillin.Vancomycin was the least active of the agents tested wiU1 an M!Cgo of 4 mg/L and an MBCgo of 64 mg/L.Thirty strains were tolerant of vancomycin .The comparative activities of the agents tested determined by time-kill method are shown in Figure 1.Daptomycin (2 mg/L).vancomycin and teicoplanin were relatively bacteriostatic, resulting in a one log10 fall in count from the original inoculum.Penicillin.ampicillin and daptomycin at 5 mg/L were more rapidly bactericidal.each resulting in 1000-fold redu ctions in counts .After 24 and 48 h, killing of enterococci was not different between penicillin and daptomycin (5 mo)L) (P>0.05) but was significantly greater than that of vancomycin.teicoplanin or daptomycin (2 mg/L) (P<0.05).

Time (Hours)
Figure 2) Time-lcill synergy curves performed in six strains ojEnterococcus faecalis not highly resistant to gentamicin.Daptomycin and gentamicin were tested at 5 mg/ L. Other antibiotics were tested at 10 mg/L.CFU Colony forming units The results of time-kill synergy studies are shown in Figure 2.After 4 h, daptomycin (5 mg/L) in combination with gentamicin (5 mg/L) was most rapidly bactericidal, although the counts were not statistically significantly different from penicillin, vancomycin or teicoplanin each in combination with gentamicin.The daptomycingentamicin combination was the only one which resulted in a sterile broth after 4 h incubation.Teicoplanin , penicillin and vancomycin each combined with gentamicin also resulted in a synergistic interaction, ie, the number of viable cells was 10-fold lower tl1an the more effective antibiotic used alone.The geometric mean counts after 4 h were 48, 14 and 3 CFU/ mL, respectively.After 24 h.daptomycin , penicillin and vancomycin each combined with gentamicin were sterile.The teicoplanin -gentamicin combination was not; the geometric mean count was 4 CFU/mL.After 24 h, daptomycin (5 mg/L) alone was not statistically different from teicoplanin, penicillin or vancomycin in combination with gentamicin (P>0.05).

DISCUSSION
The emergence of high level resistance to both aminoglycosides and penicillins makes treatment of serious enterococcal infections increasingly difficult.The present authors observed, as have others, that vancomycin was relatively bacteriostatic against a significant proportion of strains of enterococci (17 -19) .However, combined with gentamicin, vancomycin was as effective as the combination of penicillin and gentamicin.Although vancomycin alone may cure many serious enterococcal infections including endocarditis, many physicians recommend its use in combination with an aminoglycoside (20)(21)(22)(23)(24).In addition, vancomycin use may be associated witl1 adverse reactions; less toxic antibiotics are needed (23).
Teicoplanin is a new cell -wall-active glycopeptide antibiotic witl1 activity against Gram-positive bacteria including Enterococcus species (25)(26)(27).Although MICs were usually lower than for other agents tested, most strains were tolerant.The MBCgo was still below serum levels achieved with intravenous administration and lower than the concentration used in other kill curve studies (28) .In time-kill studies the concentration tested did not result in significant bactericidal activity.Therapy with teicoplanin may be complicated somewhat by the observation that serum levels may not be predictable in seriously ill patients (29) .In a rabbit model of enterococcal endocarditis teicoplanin is no more effective ilian ampicillin in the sterilization of vegetations (30).Since ampicillin alone is frequently inadequate for the treatment of serious enterococcal infections. it seems likely that mon otherapy with teicoplanin vvill be no more efficacious.
The in vitro activity of daptomycin determined using the microtitre method was similar to that of penicillin.The MBC was usually one or two dilutions higher than the MIC. and tolerance was not observed.In time-kill studies this agent was rapidly bactericidal when tested at a concentration of 5 mg/L.Whether the dosage to maintain this serum level will be tolerated in seriously ill patients r emains to be detem1ined.The results of synergy testing were also promising.Daptomycin in combination with gentamicin was more rapidly bactericidal than any other combination tested, although this difference did not reach statistical significance.On the othe r hand, Min iter et al (31) observed that daptomycin -gentamicin was less frequently synergistic than penicillin -gentamicin using the checkerboard techn ique of measuring antibiotic interaction .They also observed that in a mouse pyelonephritis model, daptomycin alone or in combination with gentamicin was less active than ampicillin alone or in combination .
Clearly.otl1er anin1al studies and subsequent human trials are necessary to detem1ine the role to be p layed by eitl1er daptomycin or teicoplanin in the antibiotic armamentarium.Whether physicia ns will choose to use these agen ts in serious enterococcal infections will depend upon tl1e results of studies in an imal models .

Figure 1 )
Figure 1) Time-kill curves of 25 isolates of Enterococcus faecalis.Daptomycin was tested at 2 and 5 mg/L.Other antibiotics were tested at 10 mg/ L. CFU Colony forming units

TABLE 1
Comparative susceptibilities of daptomycin, teicoplanin and three other agents against Enterococcus species MBC Minimal bactericidal concentration: MIC Minimal inhibitory conc entrationganism was 10 5 colony forming units (CFU)/mL.Plates were incubated at 35°C for 18 to 24 h in ambient air.The MIC was considered to be tlle lowest concentration of antibiotic resulting in no visible growU1 in tlle microtitre well.Minimal bacte ricidal concentrations (MBCs) we re performed by subculturing 0.01 mL from each well and spreading it onto U1e surface of a sheep blood agar plate.The MBC was defined as the lowest antib iotic concentration resulting in a 99 .9%reduction in U1e original inoculum.Strains were considered tolerant if the MBC/ MIC ratio was 32 or greater.Staphy lococcus aureus ATCC 25923, 29213 and Staphy lococcus jaecalis ATCC 29212 were used a s control strains .a ntibiotics was included in each run.The inoculum (mid logariUlmic phase) was standardized against a 0.5 Mc-Farland standard and diluted 1:10 in sterile broth .The inoculum (0.1 mL) was added to each tube to achieve a final concentration of 10 5 CFU /mL.The stationary tubes were incubated at 37°C in ambient air and were sampled at 0, 4 , 24 and 48 h.CAN J INFECT D IS VOL 3 No 4 JULY/ AUGUST 1992 Daptomycin versus teicoplanin for enterococci