In Vitro and In Vivo Activities of a Novel Cephalosporin, BMS-247243, against Organisms other than Staphylococci

Abstract

BMS-247243, a novel cephalosporin inhibitory for methicillin-resistant staphylococci, primarily has activity against gram-positive bacteria. The activities of BMS-247243, cefotaxime, and ceftriaxone against streptococci and Streptococcus pneumoniae were similar. BMS-247243 inhibits Enterococcus faecalis but not Enterococcus faecium. BMS-247243 also inhibits many inherently vancomycin-resistant species (Leuconstoc, Lactobacillus, Pediococcus) and anaerobic gram-positive bacteria.

BMS-247243 is a bis-zwitterionic cephalosporin with good activity against methicillin-resistant (MR) staphylococci (2). This enhanced activity against MR Staphylococcus aureus (MRSA) is the result of its improved affinity for PBP 2a, the target enzyme responsible for methicillin resistance in staphylococci. Methicillin resistance rates among U.S. and European blood isolates are about 30% for S. aureus and 60% for other staphylococcal species (5).

In the past two decades, other key gram-positive pathogens, specifically, Streptococcus pneumoniae and enterococci, have developed resistance to the drugs indicated for use against them. Data from the 1997 U.S. SENTRY Surveillance Program indicated that 46% of S. pneumoniae bloodstream isolates were penicillin intermediate or resistant, with 16.5% being highly penicillin resistant (5). Penicillin-resistant S. pneumoniae strains and enterococci tend to be multidrug resistant. Vancomycin resistance occurred in 17% of enterococcal bloodstream isolates (5).

In the investigation described here, the in vitro and in vivo activities of BMS-247243 against nonstaphylococcal gram-positive bacteria and other organisms were determined.

MICs were determined by the NCCLS-recommended agar dilution method (3, 4). The test medium was Mueller-Hinton agar (MHA) except for the following: streptococci, Leuconostoc, Pediococcus, Lactobacillus, Corynebacterium, and Listeria monocytogenes, for which MHA with 5% defibrinated sheep blood was used; Haemophilus, for which hemophilus test medium was used; and anaerobes, for which brucella blood agar enriched with vitamin K was used. Bacterial inocula were adjusted to yield approximately 5 × 10⁴ CFU/spot. The culture plates were incubated aerobically (in 5% CO₂ for streptococci, listeria, Leuconstoc, Pediococcus, Lactobacillus, Corynebacterium, and Neisseria meningitidis) or anaerobically (for anaerobes) for 18 h, 24 h (for pneumococci), or 48 h (for Leuconstoc, Pediococcus, Lactobacillus, Corynebacterium, and anaerobes) at 35°C. The MIC endpoint was defined as the lowest drug concentration that inhibited all visible growth.

Time-kill studies were done with four Enterococcus faecalis strains and BMS-247243 (at 16 μg/ml), streptomycin (25 μg/ml), or the two drugs combined. Two of these strains were high-level streptomycin resistant (MICs, ≥2,000 μg/ml). Synergism was defined as a 2 log₁₀ enhanced killing with the combination compared to that with either drug alone.

S. pneumoniae strain A9585 was used to infect the right thigh muscle of immunosuppressed mice in the neutropenic thigh infection model described previously (2). Ceftriaxone and BMS-247243 were administered intravenously (i.v.) as a single dose of 2 mg/kg of body weight at 2 h postinfection, and bacterial counts in the infected thighs were determined at 24 h postinfection. The pharmacokinetics of BMS-247243 in mice after the administration of a 5-mg/kg i.v. dose are as follows: maximum concentration of drug in plasma, 21 μg/ml; plasma half-life (t_1/2), 1.7 h; and area under the concentration-time curve (AUC), 40 μg · h/ml. The 2-mg/kg dose was used in the neutropenic thigh model since this was the 50% effective dose (ED₅₀) of both cephalosporins for this pneumococcal strain in mice, as determined previously with this model by the use of three doses (0.5, 2, and 10 mg/kg). Due to its longer t_1/2 in the plasma of mice, exposure levels were higher with BMS-247243. Ceftriaxone administered at 20 mg/kg i.v. yielded a t_1/2 of 51 min and an AUC of 32 μg · h/ml, whereas for BMS-247243 the t_1/2 was 84 min and the AUC was 156 μ g· h/ml.

In the pneumococcal pneumonia model in mice, female ICR mice were inoculated via the intratracheal route with 50 μl containing 1 × 10⁷ to 2.3 × 10⁷ CFU of an overnight culture of S. pneumoniae strains grown in Mueller-Hinton broth supplemented with 5% sheep blood. The test compounds were administered subcutaneously twice a day for 2 days starting at 1 h postinfection. Three doses (1.0, 3.16, and 10 mg/kg/day) of each compound were used, with 10 mice per group. Eighteen hours after the last treatment, the animals were euthanatized, their lungs were removed aseptically and homogenized in sterile water, and the number of viable organisms was determined by plating the tissue on blood agar plates. The ED₅₀ was determined on the basis of a 2 log₁₀ reduction in viable pathogen counts in the lungs recovered from treated animals compared to the counts in the lungs from the vehicle-treated infected control animals.

The MICs of BMS-247243 and the comparator drugs for the isolates tested in the present study are given in Tables 1 and 2. BMS-247243, cefotaxime, and ceftriaxone had similar activities against S. pneumoniae, with MICs at which 90% of isolates are inhibited (MIC₉₀s) of 0.03 to 0.06 μg/ml for penicillin-susceptible strains, 0.1 to 0.5 μg/ml for penicillin-intermediate strains, and ≈2 μg/ml for penicillin-resistant strains (Table 1). The MICs of the three cephalosporins were <2 μg/ml for 60 to 80% of the fully penicillin-resistant pneumococcal strains tested. Given that penicillin resistance is not common among beta-hemolytic streptococci, it was not unexpected to see that the BMS-247243, cefotaxime, and ceftriaxone MICs (≤0.12 μg/ml) were within the range of MICs of these cephalosporins for penicillin-susceptible pneumococci. The BMS-247243 MICs paralleled the penicillin MICs for viridans group streptococcal strains (Table 3). BMS-247243, cefotaxime, and ceftriaxone MICs were all ≤1 μg/ml for viridans group streptococcal strains for which penicillin MICs were ≤1 μg/ml and were ≥2 μg/ml for strains for which penicillin MICs were ≥2 μg/ml. Streptococcus sanguis and Streptococcus mitis are two species believed to have horizontally transferred penicillin resistance to S. pneumoniae via the transfer of penicillin-binding protein (PBP) genetic fragments (1), thus accounting for the similar BMS-247243 MICs for viridans group streptococci and fully penicillin-resistant pneumococci.

TABLE 1.

Expanded evaluation of BMS-247243 against clinical isolates

Organism (no. of isolates tested) and compound	MIC (μg/ml)
	Range	50%	90%
Streptococcus pneumoniae
Penicillin susceptible (10)
BMS-247243	0.004-0.12	0.006	0.06
Vancomycin	0.25-0.5	0.5	0.5
Imipenem	0.008-0.016	0.008	0.008
Ceftriaxone	0.008-0.06	0.02	0.03
Cefotaxime	0.016-0.03	0.016	0.03
Cephalothin	0.12-0.25	0.12	0.25
Penicillin	0.016-0.03	0.03	0.03
Penicillin intermediate (10)
BMS-247243	0.016-0.12	0.06	0.12
Vancomycin	0.25-0.5	0.25	0.5
Imipenem	0.016-0.12	0.03	0.06
Ceftriaxone	0.06-0.5	0.25	0.5
Cefotaxime	0.06-2	0.25	0.25
Cephalothin	0.5-8	1	2
Penicillin	0.12-1	0.25	0.5
Penicillin resistant (48)
BMS-247243	0.25-4	0.5	2
Vancomycin	0.25-0.5	0.5	0.5
Ceftriaxone	1-4	1	2
Cefotaxime	0.5-8	1	2
Penicillin	2-16	2	4
Streptococcus sanguis (12)
BMS-247243	0.016-2	0.06	0.5
Vancomycin	0.5	0.5	0.5
Imipenem	0.008-1	0.03	0.06
Ceftriaxone	0.06-2	0.06	1
Cefotaxime	0.03-2	0.12	0.5
Cephalothin	0.25-16	0.5	8
Penicillin	0.03-4	0.12	1
Streptococcus mitis (10)
BMS-247243	0.004-0.5	0.05	0.25
Vancomycin	0.5	0.5	0.5
Imipenem	0.008-0.12	0.016	0.06
Ceftriaxone	0.008-1	0.06	0.5
Cefotaxime	0.008-1	0.06	0.5
Cephalothin	0.06-2	0.25	2
Penicillin	0.016-1	0.06	0.5
Streptococcus pyogenes (10)
BMS-247243	0.03	0.03	0.03
Vancomycin	0.5	0.5	0.5
Imipenem	0.004	0.004	0.004
Ceftriaxone	0.03-0.06	0.03	0.03
Cefotaxime	0.016	0.016	0.016
Cephalothin	0.06-0.12	0.06	0.12
Penicillin	0.016-0.03	0.016	0.016
Streptococcus agalactiae (10)
BMS-247243	0.016-0.12	0.06	0.12
Vancomycin	0.5	0.5	0.5
Imipenem	0.004-0.016	0.016	0.016
Ceftriaxone	0.016-0.06	0.03	0.06
Cefotaxime	0.016-0.12	0.03	0.06
Cephalothin	0.03-0.25	0.12	0.12
Penicillin	0.016-0.06	0.06	0.06
Enterococcus faecalis (18)
BMS-247243	0.5-8	1	4
Vancomycin	0.5-4	2	4
Imipenem	0.5-8	1	4
Cefotaxime	1->128	128	>128
Cephalothin	16-64	16	64
Ampicillin	1-4	1	2
Enterococcus faecium (11)
BMS-247243	0.06->64	16	>32
Vancomycin	0.5->64	1	4
Imipenem	≤0.25->128	4	32
Cefotaxime	≤0.25->128	32	>128
Cephalothin	4->128	8	>128
Ampicillin	0.25->64	2	64
Escherichia coli (10)
BMS-247243	>64	>64	>64
Imipenem	0.12-0.25	0.12	0.25
Cefotaxime	≤0.06-0.5	≤0.06	0.25
Cephalothin	4->128	8	>128
Ampicillin	4->128	>128	>128
Haemophilus influenaze
β-Lactamase negative (11)
BMS-247243	1-8	2	4
Vancomycin	≥64	64	64
Cefotaxime	0.016-0.03	0.016	0.03
Ceftriaxone	0.004-0.008	0.004	0.008
β-Lactamase positive (12)
BMS-247243	2-32	16	32
Vancomycin	≥64	>64	>64
Cefotaxime	0.016-0.03	0.016	0.03
Ceftriaxone	0.004-0.008	0.004	0.008
Neisseria meningitidis (16)
BMS-247243	32-64	64	>64
Vancomycin	32-64	32	64
Cefotaxime	0.001-0.008	0.002	0.008
Ceftriaxone	≤0.002	<0.002	<0.002
Penicillin	0.003-0.12	0.06	0.12
Clostridium perfringens (10)
BMS-247243	1-8	2	4
Vancomycin	0.5-1	0.5	1
Imipenem	≤0.06-0.12	≤0.06	0.12
Cefotaxime	0.13-8	4	4
Cephalothin	0.25-4	1	2
Penicillin	≤0.06-0.25	0.12	0.25
Peptostreptococcus spp. (10)
BMS-247243	0.06-2	1	2
Vancomycin	0.12-0.5	0.25	0.25
Imipenem	≤0.06-0.12	≤0.06	0.12
Cefotaxime	0.5-16	2	16
Cephalothin	≤0.06-4	0.12	2
Penicillin	≤0.06-0.5	≤0.06	0.12
Propionibacterium acnes (10)
BMS-247243	0.25-0.5	0.2	0.5
Vancomycin	0.5	0.5	0.5
Imipenem	≤0.06	≤0.06	≤0.06
Cefotaxime	0.06-0.5	0.12	0.25
Cephalothin	≤0.06-0.12	≤0.06	0.12
Penicillin	≤0.016-0.06	≤0.016	0.03

TABLE 2.

MIC range for additional bacterial species of which less than 10 strains were tested

Organism (no. of strains)	MIC (μg/ml)
	BMS-247243	Vancomycin	Imipenem	Cefotaxime	Penicillin or ampicillina
Enterococci, VanAb (7)	1->16	>128	NTc	NT	1->64
Enterococcus faecium, VanB (3)	>16	16->64	NT	NT	≥64
Enterococi, VanCd (8)	0.12-16	4-8	NT	NT	0.25-8
Micrococcus spp. (5)	0.06-4	0.25-1	≤0.007-0.25	≤0.12-16	≤0.007-4
Leuconstoc spp. (5)	0.25-1	>128	0.25-8	4-64	1
Pediococcus spp. (2)	0.25, 0.5	>128	0.12, 0.25	8, 16	0.5, 2
Lactobacillus spp. (2)	0.12, 0.5	>128	0.03, 1	16, 128	2, 32
Cornyebacterium jeikium (6)	0.5->64	0.5-1	0.25->32	2-16	>64
Corynbacterium sp. strain D2 (2)	>64	1	>32	>128	>64
Corynebacterium spp. (6)	0.06-8	0.5-1	≤0.015-32	≤0.25-128	0.12-16
Listeria monocytogenes (5)	0.25-1	1	0.06-0.12	32->128	0.12-0.5
Clostridium difficile (2)	32	2	4	>64	1, 2
Klebsiella pneumoniae (5)	>64	NT	0.12	≤0.06-0.5	16->128
Enterobacter cloacae (5)	>64	NT	0.25-0.5	0.25-64	>128
Serratia marcescens (5)	>64	NT	0.25-0.5	0.5-2	>128
Salmonella enteritidis (5)	>64	NT	0.12-0.5	≤0.06-0.25	4->128
Pseudomonas aeruginosa (5)	>64	NT	1-16	16-32	>128
Moraxella catarrhalis
β-Lactamase negative (7)	0.5-16	NT	NT	0.03-0.25	0.015-0.03
β-Lactamase positive (8)	16->32	NT	NT	0.06-1	1->16
Bacteroides fragilis (2)	32, >64	NT	≤0.06	>64	16, 32

^aPenicillin for gram-positive bacteria and Moraxella catarrhalis; ampicillin for enterococci, C. difficile, and gram-negative bacteria.

^bFive E. faecium and two E. faecalis strains.

^cNT, not tested.

^dFour E. gallinarium and four E. casseliflavus strains.

TABLE 3.

Activities of BMS-247243 against penicillin-intermediate and -resistant strains of viridans group streptococci

Streptococcus species and strain	MIC (μg/ml)
	Penicillin	BMS-247243	Cefotaxime	Ceftriaxone
S. mitis
A28580	0.5	0.5	0.5	0.5
A28588	0.5	0.5	1	0.5
A28600	0.5	0.5	1	1
A28602	0.5	0.25	1	1
A28545	0.5	0.12	0.12	0.25
S. sanguis
A28558	0.5	0.5	1	1
A28548	1	0.5	2	2
A28550	1	0.5	0.5	0.5
A28551	1	0.5	1	1
S. mitis A28589	1	0.12	2	2
S. sanguis A28562	2	2	2	2
S. salivarius
A29053	2	1	1	1
A29055	4	2	2	2
S. mitis
A29054	4	4	4	4
A29056	8	4	4	4
A29057	8	2	8	4
A29058	16	4	8	8

BMS-247243 has activity against enterococci, with the MIC₉₀s of BMS-247243, cefotaxime, and ceftriaxone being 4 μg/ml for E. faecalis. BMS-247243 was inactive against ampicillin-resistant enterococcal strains, and since most Enterococcus faecium isolates are ampicillin resistant, BMS-247243 should be considered inactive against this species. Due to the predominance of E. faecium among vancomycin-resistant enterococcal strains, BMS-247243 was, for the most part, inactive against strains of the VanA and VanB phenotypes (Table 2). BMS-247243 was inhibitory for the inherently low-level vancomycin-resistant (VanC) species Enterococcus gallinarum and Enterococcus casseliflavus. Since enterococci are not generally killed by β-lactams at levels achievable in serum, the bactericidal activity of BMS-247243 against E. faecalis was determined. Like other β-lactams, BMS-247243 was not cidal against enterococci. However, in combination with streptomycin, BMS-247243 killed enterococcal strains that did not have high-level resistance to the aminoglycoside (i.e., streptomycin MICs, <2,000 μg/ml) (data not shown).

BMS-247243 was active against Micrococcus spp. and Listeria monocytogenes (Table 2). Along with imipenem, BMS-247243 was among the β-lactams most active against the intrinsically vancomycin-resistant species Pediococcus, Leuconstoc, and Lactobacillus spp.; however, BMS-247243 and imipenem lacked activity against some strains of Corynebacterium spp. BMS-247243 was inactive against gram-negative aerobic bacteria and had limited activity against β-lactamase-nonproducing Haemophilus species and Moraxella strains (Tables 1 and 2). The hydrophobic nature of BMS-247243 likely accounts for its inactivity against gram-negative bacteria. Hydrophobicity, along with compound charge and size, can influence the permeation of the drug across the outer membrane porin channels of gram-negative bacteria (6). BMS-247243 was active against gram-positive anaerobic bacteria, such as Peptostreptococcus spp. and Propionibacterium acnes, although it was less active against Clostridium spp. (Tables 1 and 2).

In the soft tissue muscle infection model in neutropenic mice, BMS-247243 was effective in sterilizing infected thighs by 24 h. While ceftriaxone was also effective, the infected thighs of animals in this treatment arm had significant numbers of viable pneumococci at 24 h (Fig. 1). Three S. pneumoniae strains were used in the lung infection model. BMS-247243 and ceftriaxone demonstrated comparable efficacies in this model (Table 4), regardless of the penicillin susceptibility or resistance of the strain.

FIG. 1.

Comparative efficacies of BMS-247243 and ceftriaxone against penicillin-susceptible S. pneumoniae strain A9585 in the neutropenic murine thigh infection model.

TABLE 4.

Efficacies of BMS-247243 and ceftriaxone in S. pneumoniae lung infections in mice

S. pneumoniae strain (penicillin susceptibility)	BMS-247243		Ceftriaxone
	MIC (μg/ml)	ED50 (mg/kg/day)	MIC (μg/ml)	ED50 (mg/kg/day)
A9585 (sensitive)	0.008	<1	0.03	<1
A28312 (intermediate)	0.06	<1	0.06	<1
A28212 (resistant)	0.5	1.4	1	1.8

In summary, the spectrum of activity of the anti-MRSA cephalosporin BMS-247243 is directed primarily against gram-positive bacteria. Its activities against streptococci and pneumococci are comparable to those of cefotaxime and ceftriaxone. While BMS-247243 has activity against E. faecalis, killing of enterococci requires a combination of BMS-247243 with an aminoglycoside, and this combination is active only against strains not highly resistant to aminoglycosides.