Comparative In Vitro Activity of Ceftobiprole against Staphylococci Displaying Normal and Small-Colony Variant Phenotypes

Abstract

The antistaphylococcal activity of ceftobiprole was compared with those of cefuroxime, linezolid, and moxifloxacin by using the agar dilution method. Apart from three strains with small-colony variant phenotypes, all Staphylococcus aureus isolates tested were inhibited by ≤2 μg/ml of ceftobiprole. This compound exhibited an excellent antistaphylococcal activity, comparable to that of linezolid.

Ceftobiprole (BPR) (formerly BAL9141) is a novel parenteral cephalosporin with activity against a broad range of pathogens, particularly against gram-positive bacteria (3, 5, 7). While the compound has been described to possess strong affinity for the mecA product PBP 2a, data on the activity of ceftobiprole against well-characterized staphylococci are limited. Therefore, aims of this study were (i) to challenge the ceftobiprole spectrum by evaluating its activity against well-defined staphylococcal strains of different species, including small-colony variants (SCVs), and (ii) to compare the in vitro antistaphylococcal activity of this cephalosporin with those of linezolid, moxifloxacin, and cefuroxime.

The 284 Staphylococcus aureus strains tested comprised 72 methicillin-susceptible S. aureus (MSSA) and 212 methicillin-resistant S. aureus (MRSA) strains, including 14 strains with stable SCV phenotypes. The 80 coagulase-negative staphylococci (CoNS) comprised 14 methicillin-susceptible and 21 methicillin-resistant Staphylococcus epidermidis strains, 17 methicillin-susceptible and 15 methicillin-resistant Staphylococcus haemolyticus strains, and 13 other CoNS belonging to eight different species.

All staphylococcal strains were freshly isolated from clinical material and were included in the testing only if they were considered etiologically relevant. Apart from isolates with SCV phenotypes, only one isolate per patient was tested. Eighty-one MRSA strains, each presenting a different spa type, were selected from our institutional collection (between 1996 and 2004). All other spa-typed MRSA isolates were collected during the course of a multicenter study in Germany also including community-acquired MRSA, with not more than five strains selected from each center. Overall, the MRSA strains tested represent more than 90 spa types and thus cover >90% of all registered European MRSA spa types within the SeqNet network (www.SeqNet.org) (6).

If the biochemical identification of staphylococcal isolates (ATB32 Staph; bioMerieux, Marcy l'Etoile, France) was ambiguous or categorized as unacceptable, 16S rRNA gene sequencing was performed as previously described (2). Isolates      were confirmed to be methicillin resistant by supplementation of the agar with 2% NaCl (read after incubation for 48 h at 30°C by using 5-μg oxacillin disks) and by detection of the mecA gene (10).

S. aureus SCVs (n = 14) were collected from patients with persistent and/or recurrent infections, such as chronic osteomyelitis or chronic skin and soft tissue infections (11-13). Isolates were recognized as SCVs and genotyped as previously described (11-13). Strains with SCV phenotypes were confirmed as S. aureus by testing for the S. aureus-specific nuc gene (9). For comparison, 13 isolates with normal morphotypes (clonally identical to the corresponding SCVs), which were recovered in the same or subsequent clinical specimens as the SCVs, were also included in this study.

The MICs were determined by using the agar dilution technique according to CLSI (4). Ceftobiprole, cefuroxime, linezolid, and moxifloxacin were obtained from their respective manufacturers. The test range was 0.03 to 128 μg/ml (up to 32 μg/ml for ceftobiprole). The results were read after 18 h of incubation at 36°C, and the results for SCVs and their parent strains were read also after 42 h and 66 h of incubation. Several reference strains were included as controls. Additionally, sterility and growth controls were always performed.

The MIC distribution data for the S. aureus strains with normal phenotypes as well as for the CoNS are shown in Table 1. Ceftobiprole exhibited excellent wide-spectrum antistaphylococcal activity. According to the MIC at which 50% of isolates were inhibited (MIC₅₀) and MIC₉₀ values, ceftobiprole was two- to eightfold more active than cefuroxime against methicillin-susceptible staphylococci, encompassing S. aureus and 10 different species of CoNS. While all S. aureus isolates with normal morphotypes, including MRSA isolates covering >90% of spa types registered in Europe, were inhibited by ≤2 μg/ml of ceftobiprole, cefuroxime MIC₉₀s for methicillin-resistant staphylococci (MRS) were all greater than 128 μg/ml. By comparison, activity of ceftobiprole was similar to linezolid against MRSA; however, ceftobiprole was slightly more active than the oxazolidinone against methicillin-susceptible strains. While moxifloxacin was the most active agent against MSSA strains, the in vitro activity of the fluoroquinolone against the MRS panel was poor.

TABLE 1.

Antimicrobial activities of ceftobiprole and selected comparison drugs tested against staphylococci

Antimicrobial agent against indicated organism (no. of strains tested)a	MIC (μg/ml)b
	Range	50%	90%
MSSA (60)
Ceftobiprole	0.13-0.5	0.25	0.5
Cefuroxime	0.25-2	2	2
Linezolid	0.5-2	1	2
Moxifloxacin	≤0.03-4	≤0.03	0.06
MRSA (197)
Ceftobiprole	0.25-2	1	2
Cefuroxime	4-64	16	>128
Linezolid	0.25-1	1	1
Moxifloxacin	≤0.03-8	2	4
MSSE (14)
Ceftobiprole	0.13-1	0.25	0.5
Cefuroxime	0.25-4	0.5	1
Linezolid	0.5-1	0.5	1
Moxifloxacin	≤0.03-4	0.06	2
MRSE (21)
Ceftobiprole	0.5-4	1	2
Cefuroxime	0.5->128	8	>128
Linezolid	0.5-1	0.5	1
Moxifloxacin	≤0.03-4	1	2
MSSH (17)
Ceftobiprole	0.13-1	0.25	0.5
Cefuroxime	0.5-4	2	2
Linezolid	0.5-1	0.5	1
Moxifloxacin	≤0.03-4	≤0.03	2
MRSH (15)
Ceftobiprole	1-4	2	4
Cefuroxime	8->128	64	>128
Linezolid	0.5-1	0.5	1
Moxifloxacin	1-8	4	8
CoNS (13)
Ceftobiprole	≤0.03-1	0.5	1
Cefuroxime	0.25-8	2	4
Linezolid	0.5-2	1	1
Moxifloxacin	≤0.03-0.25	0.13	0.25

^aStrains exhibiting normal morphotype (results for SCVs and their parent strains are given in Table 2). MSSE, methicillin-susceptible S. epidermidis; MRSE, methicillin-resistant S. epidermidis; MSSH, methicillin-susceptible S. haemolyticus; MRSH, methicillin-resistant S. haemolyticus.

^bQuality control of all MIC determinations was performed by using the following reference strains: S. aureus subsp. aureus ATCC 25923, ATCC 29213, and ATCC 43300, S. epidermidis DSM 20044, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853. The MICs for these strains were within acceptable limits throughout testing.

The MICs of the agents tested against stable SCVs and their isogenic parent strains with normal morphotypes are shown in Table 2. While the number of SCV isolates tested is limited, it should be stressed that eight different clonal lineages of MRSA with SCV phenotypes and six different clonal lineages of MSSA with SCV phenotypes were included in this study (9). Of interest, antimicrobials were often less active against SCVs tested than their isogenic parent strains, with consistently low MICs for ceftobiprole. This phenomenon has been clearly described for aminoglycosides but has not been demonstrated for cephalosporins, fluroroquinolones, or linezolid (1). Nevertheless, apart from three strains (MICs, 4 μg/ml), all S. aureus isolates with SCV phenotypes, including those exhibiting methicillin resistance, were inhibited by ≤2 μg/ml of ceftobiprole.

TABLE 2.

Antimicrobial activities of ceftobiprole and selected comparison drugs tested against strain pairs of small-colony variants and their clonally identical parent strains with normal morphotype

PS/SCV strain pair (resistance type)a	MIC (μg/ml)
	Ceftobiprole		Cefuroxime		Linezolid		Moxifloxacin
	PS	SCV	PS	SCV	PS	SCV	PS	SCV
01 (MRSA)	1	2	64	>128	0.5	2	2	2
02 (MRSA)	1	4	>128	>128	0.5	2	2	8
03 (MRSA)	0.5	0.5	4	4	1	1	1	2
04 (MRSA)	2	4	>128	>128	0.5	4	4	8
05 (MRSA)	0.5	0.5	4	4	0.5	2	2	2
06 (MRSA)	0.5	1	4	2	1	1	1	2
07 (MRSA)	0.5	2	4	>128	1	4	2	8
08 (MRSA)	NAb	4	NA	>128	NA	2	NA	8
09 (MSSA)	0.13	0.13	1	1	1	4	<0.03	<0.03
10 (MSSA)	0.5	0.13	2	1	1	4	<0.03	0.06
11 (MSSA)	0.25	0.25	2	1	1	2	<0.03	0.13
12 (MSSA)	0.25	0.25	2	1	1	2	<0.03	0.13
13 (MSSA)	0.25	0.25	1	1	0.5	0.5	<0.03	0.06
14 (PSSA)	0.25	0.25	2	2	1	0.5	0.06	0.13

^aPS, parent strain; PSSA, penicillin-susceptible S. aureus.

^bNA, not available.

MRS, including MRSA isolates with SCV phenotypes, present a major challenge in terms of chemotherapy because effective antimicrobial treatment options for infections caused by these isolates are close to becoming exhausted. In this context, the ability of ceftobiprole to inhibit staphylococci with a methicillin resistance phenotype may be of major clinical importance. Except for three strains with SCV phenotypes, all S. aureus strains tested, including a large number of different spa types, were inhibited by ≤2 μg/ml of ceftobiprole. In fact, the antistaphylococcal activity of ceftobiprole was comparable to that of linezolid, the cephalosporin being even more active against methicillin-susceptible strains than the oxazolidinone. In previous studies testing the activity of ceftobiprole against staphylococci, smaller numbers of resistance phenotypes were tested to challenge the ceftobiprole spectrum and isolates from the same patient were not excluded (5, 7, 8). Of particular importance, multiple isolates of the same strain were not excluded by phenotypic or genomic typing. In contrast, we tested a large number of clonally unrelated strains, which is particularly important for MRSA. Furthermore, a broad spectrum of different, well-characterized staphylococcal species recovered from patients with overt infections, including isolates with SCV phenotypes, were encompassed in the testing. Thus, the range of MICs of antimicrobials documented in this study was broader than those observed in previous studies (7, 8, 14).

Overall, in our study, the newly developed agent ceftobiprole was highly active against both unrelated methicillin-susceptible and -resistant staphylococci, stimulating further evaluation of this agent for therapy of staphylococcal infections.