Determining Linezolid’s Baseline In Vitro Activity in Canada Using Gram-Positive Clinical Isolates Collected prior to Its National Release

Abstract

All of the isolates of Staphylococcus aureus (n = 317), Enterococcus species (n = 315), Streptococcus pneumoniae (n = 282), and Staphylococcus epidermidis (n = 176) collected at 16 Canadian microbiology laboratories from October 2000 to April 2001 were susceptible to linezolid. Future studies will determine how linezolid clinical use in Canada affects its in vitro activity.

Oxazolidinones are synthetic protein synthesis inhibitors with a unique mechanism of action. They noncovalently bind to 23S rRNA residues of 50S ribosomal subunits and prevent the formation of functional 70S initiation complexes (2). The primary binding sites of oxazolidinones lie in the central loop of domain V of the 23S rRNA, clustering at the peptidyl transferase center (9, 10, 22, 24, 27). The presence of multiple copies of rRNA genes in clinically relevant bacterial species suggests that the selection of both in vitro (8, 28) and clinical mutants simultaneously resistant in each rRNA gene copy would be unlikely. Limited numbers of in vitro-selected, oxazolidinone-resistant enterococcal mutants (18) and resistant clinical isolates of enterococci and Staphylococcus aureus cultured posttherapy (4, 25; G. E. Zurenko, W. M. Todd, B. Hafkin, B. Meyers, C. Kauffman, J. Bock, J. Slightom, and D. Shinabarger, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 848, p. 118, 1999) have been reported. Changes to single copies of rRNA genes may be recessive, explaining the difficulty in selecting resistant mutants.

Linezolid has demonstrated clinical utility in treating infections due to aerobic gram-positive cocci; its in vitro spectrum of activity also includes certain gram-negative species, including anaerobes, and mycobacteria (2, 7, 26). Previous in vitro studies have demonstrated that, relative to susceptible isolates, the activity of linezolid is maintained against resistant and multidrug-resistant gram-positive cocci, including methicillin (oxacillin)-resistant staphylococci, penicillin-resistant pneumococci, and glycopeptide-resistant enterococci (1, 3, 8, 16, 26).

As a prelude to the May 2001 release of linezolid in Canada, a study was conducted to establish a baseline of activity for linezolid and its market competitors. Isolates were collected at 16 laboratories in Canada from October 2000 to April 2001. Fifteen laboratories served tertiary-care hospitals, and one was a community hospital laboratory. Six laboratories in Ontario participated, as did four laboratories in Quebec and two laboratories each in British Columbia, the prairie region (Alberta, Saskatchewan, and Manitoba), and the Atlantic region (New Brunswick, Nova Scotia, Prince Edward Island, and Newfoundland).

The isolates, limited to one per patient, were prospectively isolated from hospital inpatients and outpatients. Isolates were shipped to our central laboratory (Focus Technologies, Inc., Herndon, Va.), subcultured to purity on appropriate media, and stocked, and their identities were confirmed by standard laboratory methods. Of the isolates submitted, 317 S. aureus, 282 Streptococcus pneumoniae, 266 Enterococcus faecalis, 176 Staphylococcus epidermidis, 36 Enterococcus faecium, and 13 Enterococcus sp. (non-E. faecalis and non-E. faecium) isolates were viable and their identities were confirmed to the species level.

Isolate susceptibilities were determined by the NCCLS broth microdilution method (12) on frozen panels manufactured by TREK Diagnostics (Westlake, Ohio). MICs were interpreted in accordance with NCCLS guidelines (13, 14).

For isolates of methicillin (oxacillin)-susceptible S. aureus (MSSA) and methicillin (oxacillin)-resistant S. aureus (MRSA), the linezolid MIC for 90% of the isolates tested (MIC₉₀) was 4 μg/ml and the modal MIC was 2 μg/ml (Table 1). For all of the isolates collected in each of the five geographic regions of Canada (British Columbia, the prairie region, Ontario, Quebec, and the Atlantic region), the linezolid MIC₉₀ was 2 or 4 μg/ml and the modal MIC was 2 μg/ml. Linezolid MICs were distributed over narrow ranges for both MRSA (1 to 4 μg/ml) and MSSA (0.5 to 4 μg/ml) isolates. Specimen source (blood and cerebral spinal fluid; skin, soft tissue, and wound; urine; upper respiratory tract; and lower respiratory tract), patient gender, patient age (<18 years, 18 to 65 years, and >65 years), and inpatient or outpatient status did not influence linezolid MIC₉₀s, MIC ranges, or modal MICs (data not shown). Similarly, patient demographic characteristics did not influence linezolid MIC₉₀s, MIC ranges, or modal MICs for S. epidermidis, S. pneumoniae, or Enterococcus sp. isolates (data not shown).

TABLE 1.

MICs of linezolid and comparative agents and their interpretations^a for 317 isolates of S. aureus and 176 isolates of S. epidermidis

Anti- microbial	All S. aureus (n = 317)						Oxacillin-susceptible S. aureus (n = 246)						Oxacillin-resistant S. aureus (n = 71)						All S. epidermidis(n = 176)						Oxacillin-susceptible S. epidermidis (n = 66)						Oxacillin-resistant S. epidermidis (n = 110)
	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R
Oxacillin	>16	0.25	≤0.06->16	77.6		22.4	0.5	0.25	≤0.06-2	100		0	>16	>16	4->16	0		100	>16	>16	≤0.06->16	37.5		62.5	0.12	≤0.06	≤0.06-0.25	100		0	>16	>16	0.5->16	0		100
Vancomycin	1	0.5	0.5-2	100	0	0	1	0.5	0.5-2	100	0	0	1	1	0.5-1	100	0	0	2	1	≤0.25-2	100	0	0	2	1	≤0.25-2	100	0	0	2	1	0.5-2	100	0	0
Teicoplanin	1	0.5	≤0.12-4	100	0	0	1	0.5	≤0.12-4	100	0	0	1	0.5	0.25-2	100	0	0	4	2	0.25-16	98.3	1.7	0	4	1	0.25-4	100	0	0	4	2	0.25-16	97.3	2.7	0
Gentamicin	2	0.5	≤0.06->32	93.1	0.6	6.3	1	0.5	≤0.06->32	97.6	0	2.4	>32	0.5	0.12->32	77.5	2.8	19.7	>32	≤0.06	≤0.06->32	58.5	8.0	33.5	16	≤0.06	≤0.06->32	86.4	3.0	10.6	>32	≤0.06	≤0.06->32	41.8	10.9	47.3
Erythromycin	>32	>32	0.25->32	63.1	2.5	34.4	>32	0.25	0.25->32	78.0	3.3	18.7	>32	>32	0.25->32	11.3	0	88.7	>32	>32	≤0.06->32	34.1	0.6	65.3	>32	0.25	0.12->32	63.6	1.5	34.8	>32	>32	≤0.06->32	16.4	0	83.6
Ciprofloxacin	>16	0.25	≤0.06->16	70.0	3.2	26.8	8	0.25	≤0.06->16	85.8	3.2	11.0	>16	>16	≤0.06->16	15.5	2.8	81.7	>16	>16	≤0.06->16	44.3	1.1	54.6	>16	0.25	≤0.06->16	80.3	0	19.7	>16	>16	0.12->16	22.7	1.8	75.5
Clindamycin	>8	0.12	0.06->8	78.2	0.3	21.5	0.12	0.12	0.06->8	92.7	0.4	6.9	>8	>8	0.06->8	28.2	0	71.8	>8	>8	≤0.03->8	56.3	0.6	43.2	>8	0.06	≤0.03->8	84.8	0	15.2	>8	>8	0.06->8	39.1	0.9	60.0
SXTb	0.25	≤0.12	≤0.12->8	96.5		3.5	≤0.12	≤0.12	≤0.12-2	100		0	>8	≤0.12	≤0.12->8	84.5		15.5	8	≤0.12	≤0.12->8	55.7		44.3	8	≤0.12	≤0.12->8	74.2		25.8	8	4	≤0.12->8	44.5		55.5
Quinupristin- dalfopristin	0.5	0.25	0.12-1	100	0	0	0.5	0.25	0.12-1	100	0	0	0.5	0.5	0.25-1	100	0	0	0.5	0.25	≤0.06-2	98.9	1.1	0	0.25	0.12	≤0.06-0.5	100	0	0	0.5	0.25	≤0.06-2	98.2	1.8	0
Linezolidc	4	2	0.5-4	100			4	2	0.5-4	100			4	2	1-4	100			1	1	0.25-4	100			1	1	0.25-4	100			1	1	0.5-2	100

^aS, susceptible; I, intermediate; R, resistant. MICs are in micrograms per milliliter.

^bSXT, trimethoprim-sulfamethoxazole.

^cThe 2002 NCCLS linezolid susceptibility breakpoint (MIC, ≤4 μg/ml) for Staphylococcus spp. was applied to the data (14).

Linezolid had an MIC₉₀ and a modal MIC of 1 μg/ml for both methicillin (oxacillin)-susceptible S. epidermidis and methicillin (oxacillin)-resistant S. epidermidis (MRSE) isolates. For all of the isolates collected in each of the five geographic regions of Canada, the linezolid MIC₉₀ was 1 or 2 μg/ml and the modal MIC was 1 μg/ml. Linezolid MICs were distributed over narrow ranges for both MRSE (0.5 to 2 μg/ml) and methicillin (oxacillin)-susceptible S. epidermidis (0.25 to 4 μg/ml) isolates.

Linezolid had an MIC₉₀ and a modal MIC of 1 μg/ml for penicillin-susceptible, -intermediate, and -resistant S. pneumoniae isolates, and the linezolid MICs were distributed over a narrow range for all of the isolates (≤0.25 to 1 μg/ml) (Table 2). For all of the isolates collected in each of the five geographic regions of Canada, the linezolid MIC₉₀ and the modal MIC were 1 μg/ml.

TABLE 2.

MICs of linezolid and comparative agents and their interpretations^a for 282 isolates of S. pneumoniae

Anti- microbial	All S. pneumoniae (n = 282)						Penicillin susceptible (n = 227)						Penicillin intermediate (n = 27)						Penicillin resistant (n = 28)
	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R
Penicillin	1	≤0.03	≤0.03-4	80.5	9.6	9.9	≤0.03	≤0.03	≤0.03-0.06	100	0	0	1	0.12	0.12-1	0	100	0	4	2	2-4	0	0	100
Amoxicillin- clavulanate	1	≤0.015	≤0.015-8	97.9	0.3	1.8	≤0.015	≤0.015	≤0.015-0.06	100	0	0	1	1	≤0.015-1	100	0	0	8	2	0.5-8	78.5	3.6	17.9
Ceftriaxone	0.5	≤0.015	≤0.015-1	94.7	5.3	0	≤0.015	≤0.015	≤0.015-0.12	100	0	0	0.5	0.5	≤0015-1	96.3	3.7	0	1	0.5	0.5-1	50.0	50.0	0
Cefuroximeb	4	≤0.12	≤0.12-8	86.2	2.5	11.3	≤0.12	≤0.12	≤0.12-0.5	100	0	0	4	0.25	≤0.12-4	59.3	22.2	18.5	8	4	2-8	0	3.6	96.4
Vancomycin	0.5	0.25	0.12-0.5	100	0	0	0.5	0.25	0.12-0.5	100	0	0	0.5	0.25	0.25-0.5	100	0	0	0.5	0.25	0.25-0.5	100	0	0
Erythromycin	>8	0.06	≤0.015->8	81.9	0.4	17.7	0.06	0.06	≤0.015->8	92.5	0	7.5	>8	0.06	0.03->8	59.3	3.7	37.0	>8	>8	0.06->8	17.9	0	82.1
Levofloxacin	1	0.5	≤0.25-16	99.3	0	0.7	1	0.5	≤0.25-16	99.1	0	0.9	0.5	0.5	≤0.25-1	100	0	0	0.5	0.5	0.5-1	100	0	0
SXTc	4	0.12	≤0.06->8	74.1	7.5	18.4	1	0.12	≤0.06-8	87.2	7.9	4.9	8	4	0.12->8	33.3	11.1	55.6	>8	4	0.5->8	7.1	0	92.9
Clindamycin	0.06	0.03	≤0.015->4	92.2	0.4	7.4	0.06	0.03	≤0.015->4	96.5	0	3.5	>4	0.03	≤0.015->4	74.1	3.7	22.2	>4	0.03	0.03->4	75.0	0	25.0
Quinupristin- dalfopristin	0.25	0.25	≤0.12-1	100	0	0	0.25	0.25	≤0.12-0.5	100	0	0	0.5	0.25	≤0.12-1	100	0	0	1	≤0.12	≤0.12-1	100	0	0
Linezolidd	1	1	≤0.25-1	100			1	1	≤0.25-1	100			1	1	≤0.25-1	100			1	1	0.5-1	100

^aS, susceptible; I, intermediate; R, resistant. MICs are in micrograms per milliliter.

^bCefuroxime MICs were interpreted by using cefuroxime axetil (oral) breakpoints (13).

^cSXT, trimethoprim-sulfamethoxazole.

^dThe 2002 NCCLS susceptibility breakpoint (MIC, ≤2 μg/ml) for S. pneumoniae were applied to the data (14).

Linezolid had MIC₉₀s and modal MICs of 1 μg/ml for E. faecalis and 2 μg/ml for E. faecium (Table 3). The linezolid MIC for all of the other Enterococcus sp. isolates tested was ≤2 μg/ml (data not shown). For the E. faecalis isolates collected in each of the five defined geographic regions of Canada, the linezolid MIC₉₀ was 1 or 2 μg/ml and the modal MIC was 1 μg/ml.

TABLE 3.

MICs of linezolid and comparative agents and their interpretations^a for 266 isolates of E. faecalis and 36 isolates of E. faecium

Antimicrobial	All E. faecalis (n = 266)						All E. faecium (n = 36)
	MIC90	Modal MIC	MIC range	% S	% I	% R	MIC90	Modal MIC	MIC range	% S	% I	% R
Ampicillin	1	0.5	≤0.25-8	100	0	0	128	64	0.5-128	30.6	0	69.4
Vancomycin	2	<0.5	≤0.5-4	100	0	0	256	≤0.5	≤0.5-256	77.8	0	22.2
Teicoplanin	0.25	0.12	0.06-4	100	0	0	32	0.25	0.06-64	77.8	0	22.2
Tetracycline	64	32	≤2->64	29.3	0.4	70.3	32	≤2	≤2->64	58.3	5.6	36.1
Ciprofloxacin	64	0.5	≤0.12->64	61.7	1.5	36.8	>64	>64	0.25->64	19.4	5.6	75.0
Nitrofurantoin	8	8	2-32	100	0	0	64	32	8-64	72.2	27.8	0
Quinupristin-dalfopristin	8	4	0.25-32	5.3	5.3	89.4	2	0.5	0.25-2	83.3	16.7	0
Linezolidb	1	1	0.25-2	100			2	2	0.5-2	100

^aS, susceptible; I, intermediate; R, resistant. MICs are in micrograms per milliliter.

^bThe 2002 NCCLS susceptibility breakpoint (MIC, ≤2 μg/ml) for Enterococcus spp. was applied to the data (14).

The MIC profiles for the isolates were also examined by species for resistance to zero, one, two, and three or more antimicrobial agents. Linezolid MIC distributions and MIC₉₀s were essentially unchanged for pansusceptible and one-drug-, two-drug-, and multidrug-resistant (resistant to three or more antimicrobials) isolates of staphylococci, S. pneumoniae, and enterococci. The MICs of linezolid were not influenced by the resistance of staphylococci to β-lactams, fluoroquinolones, or macrolides; the resistance of pneumococci to penicillin, macrolides, or fluoroquinolones; or the resistance of enterococci to ampicillin or vancomycin.

Infections attributable to MRSA, MRSE, vancomycin-intermediate S. aureus, penicillin-resistant and multidrug-resistant S. pneumoniae, and vancomycin-resistant enterococci are increasingly being identified in Canada, the United States, and worldwide and underlie the need for therapeutic alternatives, particularly oral formulations (5, 11, 15, 19, 21, 23). Linezolid is the first systemic antimicrobial of a new chemical class introduced into clinical medicine in the last 30 years.

Previous studies detailing the activity of linezolid against clinically significant gram-positive bacteria were performed with largely U.S. and European isolates (3, 6, 8, 16, 17, 26, 28). Those studies demonstrated, as has the present study, that linezolid activity is maintained for MRSA compared with MSSA; penicillin-susceptible, -intermediate, and -resistant S. pneumoniae; and vancomycin-susceptible and -resistant E. faecalis, E. faecium, and other enterococci. Linezolid has also been shown to retain its activity against vancomycin-intermediate S. aureus (20). Previous studies reported that linezolid inhibited virtually all isolates of S. aureus at concentrations of ≤8 μg/ml; coagulase-negative staphylococci at concentrations of ≤4 μg/ml; streptococci, including S. pneumoniae, at concentrations of ≤4 μg/ml; and enterococci, including E. faecalis and E. faecium, at concentrations of ≤4 μg/ml (3, 6, 8, 16, 17, 26, 28). Linezolid has not shown cross-resistance with other antimicrobial classes in any study, as demonstrated in the present study. The present study also demonstrated, as have previous studies, narrow unimodal MIC distributions for gram-positive cocci. No differences in linezolid activity among multidrug-resistant isolates, compared with susceptible isolates, were noted by Patel et al. (17) or in the present study.

The present study demonstrated that, at the time of the national release of linezolid in 2001, linezolid-resistant isolates (14) of S. aureus, S. epidermidis, S. pneumoniae, E. faecalis, and E. faecium were not detected among the isolates collected in 16 Canadian hospitals. The lack of preexisting linezolid-resistant populations was readily apparent from the narrow MIC distributions for each gram-positive species studied. Linezolid activity was unaffected by methicillin resistance in S. aureus and S. epidermidis, penicillin resistance in S. pneumoniae, and vancomycin resistance in enterococci.