ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1991, p. 553-559

Vol. 35, No. 3

0066-4804/91/030553-07$02.00/0 Copyright © 1991, American Society for Microbiology

In Vitro Activity of RP 59500, a Semisynthetic Injectable Pristinamycin, against Staphylococci, Streptococci, and Enterococci ROBERT J. FASS Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210 Received 9 November 1990/Accepted 9 January 1991

The in vitro activity of RP 59500, a semisynthetic pristinamycin, was compared with the activities of vancomycin, oxacillin, ampicillin, gentamicin, ciprofloxacin, and rifampin against five Staphylococcus species, five Streptococcus species, and four Enterococcus species. For staphylococci, MICs were 0.13 to 1 ,ug/ml and the MICs for 90% of the strains tested (MIC90s) were 0.13 to 0.5 ,ug/ml; there were no differences between oxacillin-susceptible and -resistant strains. For streptococci, MICs were 0.03 to 4 ,ug/ml and MIC90s were 0.25 to 2 ,ig/ml; viridans group streptococci were the least susceptible streptococci. For enterococci, MICs were 0.25 to 32 ,ug/ml and MIC90s were 2 to 4 ,ug/ml; Enterococcusfaecalis was the least susceptible. Vancomycin was the only comparative drug with consistent activity against all species of gram-positive cocci. With RP 59500, raising the inoculum 100-fold, lowering the pH of cation-adjusted Mueller-Hinton broth to 5.5, or omitting cation supplementation had little effect on MICs, but 50% serum increased MICs 2 to 4 dilution steps. The differences between MBCs and MICs were greater for staphylococci and enterococci than for streptococci. Time-kill studies with 24 strains indicated that RP 59500 concentrations 2-, 4-, and 16-fold greater than the MICs usually killed bacteria of each species at similar rates; reductions in CFU per milliliter were less than those observed with oxacillin or vancomycin against staphylococci and less than those observed with ampicillin against enterococci. RP 59500 antagonized the bactericidal activities of oxacillin and gentamicin against Staphylococcus aureus ATCC 29213 and that of ampicillin against E. faecalis ATCC 29212. Against the latter, combination with gentamicin was indifferent. RP 59500 has a broad spectrum of in vitro activity against grampositive cocci; combining it with other drugs is not advantageous.

Pristinamycin is a member of the streptogramin family of natural antibiotics. It consists of a complex of two main components of water-insoluble compounds, pristinamycin IA, a peptidic macrolactone belonging to the group B streptogramins, and pristinamycin IIA, a polyunsaturated macrolactone belonging to the group A streptogramins, which have synergistic antibacterial activities. Most clinically isolated strains of staphylococci, streptococci, and enterococci are susceptible, including strains resistant to macrolides, lincosamides, and streptogramin B, because susceptibility to factor A and A-B synergy are usually preserved (1, 5, 9). Pristinamycin also has in vitro activity against other grampositive organisms, Haemophilus influenzae, Neisseria sp., Pasteurella multocida, Gardnerella vaginalis, anaerobes, Mycoplasma sp., Ureaplasma sp., Chlamydia sp., and Rickettsia sp. but not against members of the family Enterobacteriaceae and nonfermentative gram-negative bacilli (2, 3, 6-8, 16). Pristinamycin IA and IIA have been chemically modified to provide a well-defined mixture (30:70) of water-soluble semisynthetic derivatives suitable for parenteral administration (1). In this study, the in vitro activity of this product, RP 59500, was compared with those of other antimicrobial agents against staphylococci, streptococci, and enterococci.

blood) at the Ohio State University Hospitals. Duplicate isolates from the same patients were excluded. Antimicrobial agents. RP 59500 was obtained from RhonePoulenc Pharmaceuticals, Princeton, N.J.; vancomycin was from Eli Lilly & Co., Indianapolis, Ind.; oxacillin was from Bristol-Myers Squibb Co., Syracuse, N.Y.; ampicillin was from Wyeth-Ayerst Research, Princeton, N.J.; gentamicin was from Schering Corp., Kenilworth, N.J.; ciprofloxacin was from Miles Inc., West Haven, Conn.; and rifampin was from Merrell-Dow Research Institute, Cincinnati, Ohio. Laboratory standards were diluted in accordance with manufacturer recommendations and dispensed into microdilution plates by using an MIC-2000 dispensing machine (Dynatech Laboratories, Inc., Chantilly, Va.) in log2 dilution steps from 0.015 to 32 p.g/ml. Plates were stored at -70°C until used. Susceptibility tests. MICs were determined by a standardized microdilution method (11) in 0.1-ml volumes of cationadjusted Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.). Oxacillin-containing medium was supplemented with 2% NaCl to distinguish oxacillin-resistant (OR) from oxacillin-susceptible (OS) staphylococci (11). For Streptococcus pyogenes, S. pneumoniae, and viridans group streptococci, the medium was Schaedler broth (Difco) supplemented with 1% heat-inactivated horse serum and 0.5 ,ug of vitamin K1 per ml. Recommended control strains (11) were used. Microdilution plates were inoculated with disposable inoculators (Dynatech) so that the final inoculum was approximately 5 x 105 CFU/ml. For five strains of each species, MICs were simultaneously determined by using an inoculum

MATERIALS AND METHODS

Organisms. The organisms studied included 477 bacterial strains arbitrarily selected from recent isolates (60% from 553

554

ANTIMICROB. AGENTS CHEMOTHER.

FASS

TABLE 1. In vitro activities of RP 59500 and comparative antimicrobial agents Organism (no.)

OS Staphylococcus aureus (30)

OR Staphylococcus aureus (30)

OS Staphylococcus epidermidis (30)

OR Staphylococcus epidermidis (30)

OS Staphylococcus haemolyticus (29)

OR Staphylococcus haemolyticus (30)

Antimicrobial agent

Rangea

RP 59500 Vancomycin Oxacillin Ampicillin Gentamicin

0.13-0.5 0.5-1 0.25-2 0.25->32 S0.5, 2 0.13-2

Ciprofloxacin Rifampin RP 59500 Vancomycin Oxacillin Ampicillin Gentamicin Ciprofloxacin Rifampin RP 59500 Vancomycin Oxacillin Ampicillin Gentamicin Ciprofloxacin Rifampin RP 59500 Vancomycin Oxacillin Ampicillin Gentamicin Ciprofloxacin Rifampin RP 59500 Vancomycin Oxacillin Ampicillin Gentamicin Ciprofloxacin Rifampin RP 59500

Vancomycin Oxacillin Ampicillin

OS Staphylococcus hominis (30)

Gentamicin Ciprofloxacin Rifampin RP 59500 Vancomycin Oxacillin

Ampicillin Gentamicin Ciprofloxacin Rifampin OR Staphylococcus hominis (6)

RP 59500

Staphylococcus saprophyticus (30)

Vancomycin Oxacillin Ampicillin Gentamicin Ciprofloxacin Rifampin RP 59500 Vancomycin Oxacillin Ampicillin Gentamicin Ciprofloxacin Rifampin

.0.015 0.25-0.5 0.25-2 .32 4->32 S0.5, .2 1, .16 s0.015, >8 0.13-0.25 0.5-2 0.06-0.25 0.06-2 .0.06, 8 0.25-1

(,ug/ml)

MIC 50%

0.25 1 0.5 NAb 0.25 0.5 0.015 0.25 1 RC R 0.5 0.5 50.015 0.13 1 0.13 NA 0.03 0.25

90%

0.5 1 1 NA 0.5 1 16

sO:0.015 0.13 2 0.25 NA 0.06 0.5

s0.015

0.015

s.-0015

0.13-0.25 0.5-2 1->32 1-32 s0.06, .1 .2, 8 .0.03, .4 0.13-0.25 0.25-4 0.13-2 0.06->32 .0.06, 4 0.13-0.5

0.13 1 R R 2 0.25

0.13 2 R R 16

s0.015

.0.015

s0.015

0.13-0.5 0.5-4

.32 16->32 0.03, .0.5 0.25-0.5 0.015 0.13-0.5 0.5-2 0.06-1 0.06-8 -O.13, 2 0.03-0.5

s0.015-0.03 0.13-1 0.5-1

4->32 2-32 0.03, 21 .0.25, >16 s0.015 0.25-1 0.5-2 0.25-2 0.25-0.5 '0.015-0.03 0.13-1 s0.015-0.03

0.25 1 0.25 NA 0.03 0.25

0.5 sO0.015

0.25 2 R R 4 0.25

0.25 1 0.5 NA 0.03 0.5 '99.9% killing (14). For five strains of each nonfastidious species, MICs of RP 59500 were also determined in cation-adjusted MuellerHinton broth at pH 5.5, in Mueller-Hinton broth without cation supplementation, and in 50% cation-adjusted broth50% heat-inactivated pooled human serum. Killing kinetics. Killing kinetic studies were performed with RP 59500 and comparative drugs against 24 bacterial strains by using drug concentrations 2-, 4-, and 16-fold greater than the MICs. The organism-drug combinations were as follows: OS Staphylococcus aureus with RP 59500 (MIC, 0.25 ,ug/ml) and oxacillin (MIC, 0.5 ,ug/ml); OR S. aureus with RP 59500 (MIC, 0.25 ,ug/ml) and vancomycin (MIC, 1 ,ug/ml); OS S. epidermidis with RP 59500 (MIC, 0.13 ,ug/ml) and oxacillin (MIC, 0.13 jig/ml); OR S. epidermidis with RP 59500 (MIC, 0.13 ,ug/ml) and vancomycin (MIC, 1 ,ug/ml); Enterococcusfaecalis with RP 59500 (MIC, 2 ,ug/ml) and ampicillin (MIC, 1 ji.g/ml); and E. faecium with RP 59500 (MIC, 1 p.g/ml) and ampicillin (MIC, 2 ,ug/ml). Studies were performed in 50-ml volumes of cation-adjusted Mueller-Hinton broth with an inoculum of approximately 106 CFU/ml. Incubation was at 35°C in a shaker water bath. Quantitative subcultures were performed after 0, 3, 6, and 24 h of incubation to determine CFU per milliliter. At those times, 0.1-ml volumes of undiluted and serially diluted 1:10 portions were dispersed into 10-ml MuellerHinton agar pour plates. The antimicrobial dilution was 1:1,000 in the 10-2 plate, which ensured that a 4 log1o or greater reduction in CFU/ml could be detected for all drugorganism combinations despite antibiotic carryover. The plates were incubated at 35°C for 48 h before the colonies were counted. Similar killing kinetic studies were performed with fixed

Rb R R R R R R R 0-1 0-1 0-1 0-1 0-2 0 0 0-1 0-1 0 x

0-2 0-2 0-2

Ciprofloxacin

0-2 0-3 0-2

2

0-2

1-2 2-4 1-2 1-2 1-2 0-1 1-2 0-1 0-1 0-2 0 0-2 0-1 1-2

0-2 1 0-2 1 1

0-1 1-2 0 0-1 0-1 0-5 0-4 0-1 0-1

Rif-

ampin Sb S S S S S S S S 0 1 0 0 1 -1 .2 20 2

105 CFU/ml.

concentrations of RP 59500 (8 p.g/ml), oxacillin (8 pLg/ml), and gentamicin (4 [Lg/ml), alone or in combinations, against S. aureus ATCC 29213 (MICs, 0.25, 0.5, and 0.25 ,ug/ml, respectively) and with fixed concentrations of RP 59500 (8 p.g/ml), ampicillin (8 pLg/ml), and gentamicin (4 pLg/ml), alone and in combinations, against E. faecalis ATCC 29212 (MICs, 2, 1, and 8 ,ug/ml, respectively). For S. aureus, the drug concentrations were 16- to 32-fold greater than the MICs; for E. faecalis, the drug concentrations were 4- to 8-fold greater than the MICs, except for that of gentamicin, which was one-half the MIC. RESULTS The MICs of RP 59500 and comparative antimicrobial agents for the 477 study strains are shown in Table 1. RP 59500 MICs for all staphylococci were 0.13 to 1 pLg/ml; there were no differences between OS and OR strains. Vancomycin was the only other drug with consistent antistaphylococcal activity; MICs were 0.25 to 4 pg/ml. RP 59500 MICs for all streptococci were 0.03 to 4 [ig/ml; viridans group streptococci were the least susceptible. Ampicillin, oxacillin, vancomycin, and rifampin also had consistent antistreptococcal activity, except against occasional strains in the viridans group. RP 59500 MICs for enterococci were 0.25 to 32 ji.g/ml; E. faecalis was the least susceptible species. However, other enterococci were all inhibited by 2 .2 .2

3-4 3-6 1-3 2-5 0-2 0-4 0-4 0-4 1-4 0 0 xc

1-4 .2 >2 4

557

(no.

Organismb Organismb

pH 5.5

OS S. aureus OR S. aureus OS S. epidermidis OR S. epidermidis OS S. haemolyticus OR S. haemolyticus OS S. hominis OR S. hominis S. saprophyticus S. agalactiae S. bovis E. faecalis E. faecium E. durans E. avium

3-4

Increase in MICs log2 dilution steps) Without 50% cations serum

of

0-1 -1-0 -1i-1-0 -1-1 0-1 0 0 0 0 0-1 0-1 0-1

-1-0

-1-0 0 0 -1-0 0-1 0 0 0-1 0 0-1 0 0-1 0 0

0 0-1

2-3 2-3 2-3 3 2-3 3-4 2-4 2-3 3-4 2-3 2-4 23 2-3 3 2-3

a pH 7.3. b Five strains of each were used.

a Five strains of each were used. b Differences indeterminate because resistant (R) or susceptible (S). c X, Trailing endpoints.

The effects of modifying cation-adjusted Mueller-Hinton broth on RP 59500 MICs for nonfastidious organisms are shown in Table 4. Reducing the pH to 5.5 or eliminating cation supplementation had no effect on MICs. Addition of 50% serum consistently increased MICs 2 to 4 dilution

drugs are shown in Table 3. The bactericidal activity of RP 59500 varied by species. MBCs were 0 to 1 dilution step higher than the MICs for streptococci but were often >2 dilution steps higher for staphylococci and enterococci. This tended to be true for the comparative antimicrobial agents as well.

steps.

The results of the killing kinetic studies with RP 59500 and

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FIG. 1. Killing kinetics (in log1o increments) of RP 59500 (P), oxacillin (0), vancomycin (V), ampicillin (A), and gentamicin (G) at concentrations 2-, 4-, and 16-fold higher than the MICs (see text) and no drug (C) with staphylococci and enterococci. Solid lines represent the means for four strains, excluding those which demonstrated breakthrough growth; the latter (numbers in parentheses) are indicated by dashed lines.

ANTIMICROB. AGENTS CHEMOTHER.

FASS

558

P8/08P P8/A8

-1°

8

P8/G4

P8

~~~~~~~~~~~A8 P8/G4 08

A8/G4

-4

G4 J /G4

-5-J 0

3

6

24

0

3

6

24

HOURS

FIG. 2. Killing kinetics (in log1o increments) of RP 59500 (P), oxacillin (0), ampicillin (A), and gentamicin (G) at fixed concentrations and no drug (C) with control strains. Solid lines represent the means of two identical experiments. For MICs, see the text.

comparative drugs are shown in Fig. 1. Against staphylococci, RP 59500, vancomycin, and oxacillin had stereotypic rates of killing at concentrations 2-, 4-, and 16-fold higher than the MICs. However, lesser degrees of killing or breakthrough growth were sporadically observed with all three drugs at 2x concentrations. With S. aureus, RP 59500 typically reduced CFU per milliliter by 1 to 2 log10 increments at 24 h, whereas oxacillin and vancomycin typically reduced CFU per milliliter by 2 to 4 log1o increments. With S. epidermidis, RP 59500 typically reduced CFU per milliliter by 1 to 3 log1o increments, whereas oxacillin and vancomycin typically reduced CFU per milliliter by 3 to 5 log1o increments. Against enterococci, different drug concentrations had various effects. With RP 59500, concentrations 2-fold greater than the MICs often resulted in breakthrough growth; concentrations 4- or 16-fold greater than the MICs typically reduced CFU per milliliter by 0.5 to 1 log1o increments. With ampicillin, higher concentrations had a lesser killing effect; lower concentrations typically reduced CFU per milliliter by 3 to 4 log1o increments for E. faecalis and 1 to 2 log1o increments for E. faecium. The killing kinetics of RP 59500 and the comparative drugs, alone and in combinations, against S. aureus ATCC 29213 and E. faecalis ATCC 29212 are shown in Fig. 2. Against S. aureus, the rank order of killing rates was gentamicin > oxacillin > RP 59500. RP 59500 antagonized the killing effects of both gentamicin and oxacillin. Against E. faecalis, the rank order of killing rates was ampicillin > RP 59500 > gentamicin. RP 59500 antagonized the killing effect of ampicillin. While gentamicin was synergistic with ampicillin, it was indifferent with RP 59500. DISCUSSION

RP 59500 have not been studied, MICs of .2 ,ug/ml have been considered to represent susceptibility for orally administered natural pristinamycin (7, 9). Among the comparative drugs tested, only vancomycin had more consistent activity; all isolates except one strain of S. anginosus were inhibited by c4 ,ug/ml. Although we found that single strains of viridans group streptococci were resistant to RP 59500 or vancomycin, another study reported that 50 strains of oral streptococci were uniformly susceptible to both pristinamycin and vancomycin (10). The in vitro activity of RP 59500 was not affected by increasing the inoculum size, lowering the pH of the medium, or reducing the cation concentration but was reduced in the presence of serum. It was less bactericidal than oxacillin or vancomycin against staphylococci and less bactericidal than ampicillin against enterococci. It antagonized the killing effects of those drugs and showed no synergy with gentamicin against either genus. Nonetheless, the intravenous preparation may provide a needed alternative to available agents for treatment of serious infections caused by gram-positive cocci. ACKNOWLEDGMENT

This work was supported by a grant from Rh6ne-Poulenc, Princeton, N.J.

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10.

agents active against apparent with the

11.

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12.

The need for new antimicrobial

gram-positive cocci has become

more

13.

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