A multicenter evaluation of linezolid antimicrobial activity in North America

Diagnostic Microbiology and Infectious Disease 43 (2002) 75– 83

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A multicenter evaluation of linezolid antimicrobial activity in North America Charles H. Ballowa,*, Ronald N. Jonesb, Douglas J. Biedenbachb, the North American ZAPS Research Group1 a

Buffalo Clinical Research Center, Buffalo, NY, USA b JMI Laboratories, North Liberty, IA, USA

Received 6 September 2001; accepted 30 October 2001

Abstract Overall, 141 centers in North America enrolled in this international surveillance study designed to evaluate the in vitro antimicrobial activity and spectrum of linezolid, a new oxazolidinone. Each participant tested the susceptibility of clinical isolates of staphylococcal species (n ⫽ 85) against 12 drugs, and enterococcal species (n ⫽ 40) against 6 drugs using reference broth microdilution trays; and of streptococcal species (n ⫽ 25) against 6 drugs using Etests (AB BIODISK, Solna, Sweden). Quality control testing was conducted using recommended strains, and verification of resistance to linezolid and select other agents was performed by a regional monitor. Of the 20,161 isolates collected from sites across the United States (US; n ⫽ 132) and Canada (n ⫽ 9), 18,307 were included in this analysis. Oxacillin resistance occurred in 38.7 and 70.6% of Staphylococcus aureus and coagulase-negative staphylococcal (CoNS) isolates, respectively. Vancomycin resistance was reported in 65.9 and 2.6% of Enterococcus faecium and E. faecalis, respectively. Penicillin resistance occurred in 37.2% of Streptococcus pneumoniae, 17.5% constituting high-level resistance (MIC, ⱖ2 ␮g/ml). The MIC90 for linezolid was 1 ␮g/ml for streptococci, 2 ␮g/ml for enterococci and CoNS isolates, and 4 ␮g/ml for S. aureus. Using the US FDA-recommended susceptible breakpoints for linezolid, there were no confirmed reports of linezolid resistance (i.e., MIC ⱖ8 ␮g/ml). The occurrence of linezolid MICs was unimodal and generally varied between, 1– 4 ␮g/ml for staphylococci (94% of recorded results), 1–2 ␮g/ml for enterococci (93%), and 0.5–1 ␮g/ml for streptococci (85%). Susceptibility to linezolid was not influenced by susceptibility to other antiicrobials such as vancomycin, ␤-lactams or macrolides. Only linezolid was universally active against essentially all tested Gram-positive specimens. The unimodal susceptibility pattern is indicative of excellent and near complete activity against key Gram-positive pathogens including multiply resistant strains, but surveillance for emerging resistances (rare) and the performance of routine susceptibility tests to guide patient therapy seems prudent.

1. Introduction Gram-positive bacteria play an important role in both community and hospital-acquired infections. In certain geographical regions across the globe, we are witnessing an * Corresponding author. Tel.: ⫹1-716-885-3580; fax: ⫹1-716-8853508. E-mail address: [email protected] (C.H. Ballow). 1 The North America ZAPS Research Group Investigators: Joan Abid, Jacksonville, FL; Mushtaq Ahmed, Gary, IN; Maria Alikahn, Chesterfield, MO; Robin Amirkhan, Dallas, TX; Peter Appelbaum, Hershey, PA; Ken Atwell, Cambridge, MA; Michel Bergeron, Sainte-Foy, PQ; Irene Betz, Rome, NY; Joseph Blondeau, Saskatoon, SK; Patty Bossert, Saint Joseph, MO; Paul Bourbeau, Danville, PA; John Boyle, New York, NY; Stephen Brecher, Boston, MA; William Brown, Detroit, MI; Robert Buck, Cleveland, OH; Cindy Calkins, Flint, MI; Karen Carroll, Salt Lake City, UT; Aida Casiano-Colon, Rochester, NY; Stephen Cavalieri, Omaha, NE; David Chesley, El Paso, TX; John Churak, Northbrook, IL; John Conly, Toronto, ON; Marie Coyle, Seattle, WA; Frank Cucchiara, South Wey-

mouth, MA; Daniel Czerepuszko, New Britain, CT; Susan Davis, Memphis, TN; Thomas Day, Wynnewood, PA; Phyllis Della-Latta, New York, NY; Gerald Denys, Indianapolis, IN; Marc Desjardins, Johnson City, NY; Joseph DiPersio, Akron, OH; Ginny Dooley, Garland, TX; Lois Downing, Madison, WI; Ceil Duclon, Menomonee Falls, WI; William Dunne Jr., Detroit, MI; Steven Edberg, New Haven, CT; Patricia Ferreri, Minneapolis, MN; Kevin Forward, Halifax, NS; Bruce Foster, Modesto, CA; Thomas Fritsche, Seattle, WA; Deanna Fuller, Indianapolis, IN; Judy Fusco, Berkeley, CA; Sherry Gamble, Phoenix, AZ; Michael Gaydos, Dayton, OH; Mary George, Albany, NY; Peter Gilligan, Winston-Salem, NC; Jan Goodwin, Kansas City, MO; Larry Gray, Cincinnati, OH; George Hageage, Toledo, OH; Suleiman Hamway, East St. Louis, IL; Dwight Hardy, Rochester, NY; Mary Hayden, Chicago, IL; Ann Herring, Des Moines, IA; Daryl Hoban, Winnipeg, MB; William Hoehne, Winchester, MA; Thomas Huber, Temple, TX; Peter C. Iwen, Omaha, NE; Michael Jacobs, Cleveland, OH; Judith Johnson, Baltimore, MD; Sue Kehl, Milwaukee, WI; Vicki Kenyon, Tampa, FL; Wally Khalife, Lansing, MI; Renate Klein, Flushing, NY; Ronda Lancaster, Middletown, OH; Michel Laverdiere,

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alarming increase in antimicrobial resistance rates among key Gram-positive pathogens, and it is evident that new drug-resistant strains are emerging and proliferating (Jones, 1996). The incidence of vancomycin-resistant enterococci (VRE), for example, has been steadily increasing over the past 15 years, particularly in the United States (US). First reported in Europe 15 years ago (Leclercq et al., 1988), VRE resistance rates among nosocomial isolates was reported by the CDC National Nosocomial Resistance Surveillance (NNIS) system to be 0.3% in 1989, and 7.9% by 1993 (CDC, 1993). A study conducted by Pfaller et al. through the first half of 1997 reported VRE rates in blood stream infection isolates of 17.7% (Pfaller et al., 1998). Jones et al. (1998) and Edmond et al. (1999) both reported that approximately 50% of Enterococcus faecium isolates collected from sites across the US and Canada were found to be vancomycin-resistant. Certain European (Schmitz et al., 1999) and Western Pacific (Bell et al., 1998) nations have likewise reported a rising incidence of VRE, while Latin America and Canada (Pfaller et al., 1999; Karlowsky et al., 1999) do not commonly encounter this resistance phenotype. Rates of methicillin (oxacillin)-resistant Staphylococcus spp. in the medical centers of most countries around the world are significantly elevated (Jones, 1996), and the rate of oxacillin resistance among Staphylococcus aureus isolates collected throughout the US in 1997–99 was

Montreal, PQ; William Le Bar, Southfield, MI; Gary Lewis, Auburn, NY; Donald Low, Toronto, ON; Dawn Lumpkin, Kansas City, MO; Stephanie Magness, Darby, PA; Donna Main, Pensacola, FL; Mario Marcon, Columbus, OH; Mario Markovic, Cleveland, OH; Cherie Mattics, Ponca City, OK; James McLaughlin, Albuquerque, NM; Jan Monahan, Denver, CO; David Moroz, Atlanta, GA; Nahass, Somerville, NJ; Ike Northern, Moraine, OH; Sue Overman, Lexington, KY; Lee Padgett, Gainesville, FL; George Pankey, New Orleans, LA; Choong H. Park, Falls Church, VA; Anjali Pawar, Summit, NJ; Cynthia Pendowski, Traverse City, MI; Linda Perry, Boston, MA; Helen Phillips, Atlanta, GA; Carl Pierson, Ann Arbor, MI; Ted Ralph, London, ON; Russell A. Rawling, Liverpool, NY; Deborah Reardon, Burlington, VT; Kurt Reed, Marshfield, WI; Barbara Reisner, Galveston, TX; Robert Rennie, Edmonton, AB; Janet Reynolds, Indianapolis, IN; Mark Rice, Easton, PA; Gerald Riddle, Houston, TX; Kim Robertson, Moultrie, GA; Peggy Rogers, Seattle, WA; Susan Rossman, Houston, TX; Lois Rudzienski, Dayton, OH; Michael Saubolle, Tempe, AZ; Robert Sautter, Harrisburg, PA; Ron Savastano, Lowell, MA; Paul Schreckenberger, Chicago, IL; Carmen Sciortino, Louisville, KY; Timothy Sellen, Jacksonville, FL; Betsy Sentigar, Elmira, NY; David Sewell, Portland, OR; Lupe Simon, Inglewood, CA; Malcolm Slifkin, Pittsburgh, PA; Jan Snellings, Morgantown, NC; James Snyder, Louisville, KY; Patricia Somsel, Battle Creek, MI; Kenneth Sosnowski, Salem, VA; Carol Spiegel, Madison, WI; Mark Stanley, Berkley, CA; Gregory Steinkraus, Wilmington, NC; Bonnie Stewart, Greenville, TX; Barbara Strain, Charlottesville, VA; Dean Taubenheim, Hastings, NE; Terence Tay, Loma Linda, CA; David Thacker, Hickory, NC; John Thomas, Morgantown, WV; John Torresan, Warren, MI; Evelyn Totes, Hartford, CT; Monica Tritsch, Abington, PA; Kevin Tu, Charleston, WV; Nikki Turner, Coeure D’Alene, ID; Kenneth VanHorn, Valhalla, NY; David Velazco, Richmond, IN; Janet Verga, Neptune, NJ; Fordham VonReyn, Lebanon, NH; Rita Vora, Palm Beach Gardens, FL; William T. Walsh, Flint, MI; Audrey Wanger, Houston, TX; John Washington, Cleveland, OH; David Welch, Oklahoma City, OK; Carla Williams, Lufkin, TX; Debbie Wolf, Racine, WI.

reported to be over 30% (Diekema et al., 2001). The rate of Streptococcus pneumoniae having high-level penicillin resistances in the US was summarized at 14.0% (Hoban et al., 2001), but only 6.8% in Canada. The incidence of penicillin-resistant pneumococci has generally increased yearly (Klugman & Feldman, 1999), possibly compromising the effectiveness of other ␤-lactam agents and macrolides via co-resistances. But perhaps the most serious consequence of this widespread increase in resistance phenotypes is that many of the strains from these species have become multiply resistant to several classes of Gram-positive focused or alternative drugs. Over the past five years, several new antimicrobial agents have been entered into clinical trials designed to determine their effectiveness in the treatment of infections caused by resistant Gram-positive organisms. One such agent is linezolid (Brickner et al., 1996), which is the first oxazolidinone FDA-approved in the US for the treatment of serious, often resistant Gram-positive infections, and which is available in both oral and i.v. formulations (Diekema & Jones, 2000). The oxazolidinones are a novel class of synthetic antimicrobial agents that act to inhibit the formation of the initiation complex required for bacterial protein synthesis (Shinabarger, 1999). This new class of agents has demonstrated very promising activity against multiply resistant Gram-positive organisms (Diekema & Jones, 2000; Jorgensen et al., 1997; Mason et al., 1996; Jones et al., 1996; Rybak et al., 2000; Zurenko et al., 1996). The in vitro antibacterial activity of linezolid against important Gram-positive pathogens, including multiply resistant strains, has been studied extensively (Jones et al., 1996; Jorgensen et al., 1997; Mason et al., 1996; Rybak et al., 2000; Zurenko et al., 1996). Collectively, linezolid MICs were determined to be unimodal and within a narrow range, and the linezolid MIC90 was 1 ␮g/ml for streptococci, 1 to 4 ␮mg/ml for staphylococci, and 0.5 to 4 ␮mg/ml for enterococci. The present study represents the North American component of an international surveillance study (Zyrox® Antimicrobial Potency Study; ZAPS) implemented throughout Europe, the Western Pacific region, and Latin America. The primary objective of this study was to establish a North American surveillance metwork of medical centers to evaluate and publish the in vitro activity of linezolid and selected other antimicrobial agents against a representative sample of common aerobic and facultatively anaerobic Gram-positive bacteria. Isolated strains came from wounds, the abdominal cavity, the respiratory tract, and blood cultures. A secondary objective was to characterize and quantitate the continued spread of resistance to new and conventional antimicrobials in the US and Canada. An earlier report summarized a similar study utilizing disk diffusion test (Jones et al., 2001), but this study uses reference dilution methods (NCCLS, 2000).

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Fig. 1. Location of North American ZAPS Research Participants.

2. Materials and Methods 2.1. Organisms Medical centers across the US and Canada were recruited to take part in this study. Each participating site was asked to test approximately 150 bacterial specimens isolated from either inpatients or outpatients, but with no more than one isolate per patient. The desired breakdown of these 150 isolates was as follows: 50 S. aureus (30 oxacillin-susceptible and 20 oxacillin-resistant); 35 CoNS (15 oxacillinsusceptible and 20 oxacillin-resistant); 40 enterococci (10 E. faecium and 30 E. faecalis, if the laboratory speciated enterococci); and 25 streptococci (20 S. pneumoniae and 5 other streptococci, including viridans group and/or ␤-hemolytic strains). All specimens were to be obtained from recent clinical isolates collected from infections in wound sites, the abdominal cavity, the respiratory tract, or blood cultures (though blood isolates were only accepted when regarded as clinically significant by local laboratory criteria). A total of 20,161 isolates were tested by 141 medical centers across the US (n ⫽ 132) and Canada (n ⫽ 9) (Fig. 1). The number of isolates included in this study was reduced to 18,307 by excluding all non-speciated or nonfaecium/faecalis enterococci. 2.2. Susceptibility testing methods Staphylococci and enterococci isolates were tested using broth microdilution method. Microdilution trays were obtained from Dade Behring MicroScan (Sacramento, CA). Streptococci isolates were tested using Etest (AB BIO-

DISK, Solna, Sweden). Study participants performed all susceptibility testing on-site using provided reagents. Categorical interpretations of susceptibility test results for each compound were assigned according to the National Committee for Clinical Laboratory Standards (NCCLS) documents (NCCLS, 2000 and 2002). The categorical interpretation for linezolid susceptibility was as follows: for staphylococci, ⱕ4 ␮g/ml was susceptible, and no other categories; for streptococci, ⱕ2 ␮g/ml was susceptible and no other categories; for enterococci, ⱕ2 ␮g/ml was susceptible, 4 ␮g/ml was intermediate, and ⱖ8 ␮g/ml was resistant (NCCLS, 2002). During the testing of clinical strains, quality control (QC) testing was also performed using S. aureus ATCC 29213. Only those sites with QC results within acceptable, NCCLS (2002) suggested MIC ranges had their test results included in the final analyses (Worth et al., 1996). Organisms demonstrating certain designated resistance patterns and/or unusual susceptibility phenotypes were forwarded to the microbiology reference monitor (RNJ) for further testing. The following organisms were requested for further testing for confirmation: (1) S. pneumoniae, S. pyogenes, other ␤-hemolytic or viridans group streptococci non-susceptible to linezolid (MIC, ⬎4 ␮g/ml) or trovafloxacin (MIC, ⬎1 ␮g/ml) or quinupristin/dalfopristin (MIC, ⬎1 ␮g/ml); (2) enterococci resistant to linezolid (MIC, ⬎4 ␮g/ml) or vancomycin (MIC, ⬎4 ␮g/ml) or teicoplanin (MIC, ⬎8 ␮g/ml) and any E. faecium resistant to quinupristin/dalfopristin (MIC, ⬎1 ␮g/ml); (3) staphylococci non-susceptible to linezolid (MIC, ⬎4 ␮g/ml) or vancomycin (MIC, ⱖ4 ␮g/ml) or teicoplanin (MIC, ⬎8 ␮g/ml) or quinupristin/dalfopristin (MIC, ⬎1 ␮g/ml). Re-

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Table 1 Antimicrobial activity of linezolid and eleven other compounds tested against 7,038 S. aureus strains categorized by oxacillin susceptibility Antimicrobial agent

Linezolid Vancomycin Teicoplanin Quinupristin/dalfopristin Erythromycin Clindamycin Chloramphenicol Doxycycline Ampicillin Cefazolin Cefotaxime Trovafloxacin a

Oxacillin-susceptible (n ⫽ 4,317)

Oxacillin-resistant (n ⫽ 2,721)

MIC (␮g/ml)

% by category

MIC (␮g/ml)

50%

90%

Susceptible

Resistant

50%

90%

Susceptible

Resistant

2 1 0.25 0.25 0.25 0.25 8 2 4 0.25 1 0.06

4 1 0.5 0.5 ⬎16 0.5 8 2 ⬎16 0.5 1 0.12

100.0 100.0 100.0 99.9 72.7 92.2 96.3 98.8 14.7 99.7 99.5 97.9

0.0 0.0 0.0 0.0 25.4 5.5 0.6 0.3 85.3 0.1 0.1 1.4

2 1 0.25 0.5 ⬎16 ⬎16 8 2 ⬎16 32 ⬎32 2

4 1 1 1 ⬎16 ⬎16 16 4 ⬎16 32 ⬎32 8

100.0 100.0 99.9 99.4 6 21.8 83.8 94.4 0.0 0.0 0.0 46.0

0.0 0.0 0.0 0.4 93.5 76.4 1.9 1.2 100.0 100.0 100.0 38.8

a

% by categorya

NCCLS [2002] broth microdilution method and interpretive criteria. ¶ar

slightly higher than reported rates from 1997–99 (Diekema et al., 2001), but protocol design dictated 40% oxacillin resistances, e.g., excellent compliance. All of the S. aureus isolates, regardless of oxacillin susceptibility, were susceptible to linezolid (MICs, ⱕ4 ␮g/ml) and vancomycin (ⱕ4 ␮g/ml), and virtually all were susceptible to teicoplanin (⬎99.9%) and quinupristin/dalfopristin (99.4 –99.9%). Similar results were obtained when these same 12 antimicrobial agents were tested against 4,633 CoNS (Table 2), of which 70.6% were oxacillin-resistant (protocol target at 57%). These CoNS isolates were all susceptible to linezolid (MIC90, 2 ␮g/ml), and a very small percentage (0.5%) of the CoNS displayed vancomycin non-susceptible MICs at 8 ␮g/ml. The quantitative susceptibility results confirm the earlier results by Jones et al. (2001) that showed all US isolates of staphylococci (1778 strains) had zone diameters of ⱖ21 mm

ferral strains not received by the reference center and confirmed were removed from further analysis.

3. Results and discussion 3.1. Linezolid activity against staphylococci Table 1 shows the antimicrobial activity of linezolid and 11 other antimicrobial agents against 7,038 S. aureus isolates. Antibacterial activity MIC results were categorized as susceptible, intermediate or resistant (though only susceptible and resistant categories are shown), and S. aureus isolates were grouped by oxacillin-susceptibility patterns. Furthermore, non-susceptible categories for linezolid were listed as resistant (NCCLS, 2002). Of the 7,038 S. aureus isolates, 2,721 (38.7%) were oxacillin-resistant, which was

Table 2 Antimicrobial activity of linezolid and eleven other compounds tested against 4,633 coagulase-negative Staphylococcus spp. strains categorized by oxacillin susceptibility Antimicrobial agent

Oxacillin-susceptible (n ⫽ 1,360) MIC (␮g/ml)

Linezolid Vancomycin Teicoplanin Quinupristin/dalfopristin Erythromycin Clindamycin Chloramphenicol Doxycycline Ampicillin Cefazolin Cefotaxime Trovafloxacin a

Oxacillin-resistant (n ⫽ 3,273) MIC (␮g/ml)

a

% by category

% by categorya

50%

90%

Susceptible

Resistant

50%

90%

Susceptible

Resistant

1 1 0.5 0.25 0.25 0.25 4 2 0.25 0.25 0.5 0.06

2 1 2 0.5 ⬎16 2 8 2 4 0.5 1 1

100.0 100.0 99.8 99.6 60.7 87.6 98.8 95.4 56.1 99.5 99.6 91.6

0.0 0.0 0.0 0.1 37.9 10 0.6 1.6 43.9 0.4 0.4 6.5

1 1 2 0.25 ⬎16 16 4 2 8 2 8 1

2 2 4 0.5 ⬎16 ⬎16 8 4 ⬎16 32 ⬎2 8

100.0 99.5 98.2 98.3 16.7 44.7 92.1 92.1 0.0 0.0 0.0 52.8

0.0 0.5 0.3 0.6 82.2 52.3 5.1 5.1 100.0 100.0 100.0 35.0

NCCLS [2002] broth microdilution method and interpretive criteria.

C.H. Ballow et al. / Diagnostic Microbiology and Infectious Disease 43 (2002) 75– 83

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Table 3 Antimicrobial activity of linezolid and five other compounds tested against 908 E. faecium strains categorized by vancomycin susceptibility Antimicrobial agent

Vancomycin-susceptible (n ⫽ 310) MIC (␮g/ml)

Linezolid Teicoplanin Quinupristin/dalfopristin Chloramphenicol Doxycycline Ampicillin a

Vancomycin-resistant (n ⫽ 598) MIC (␮g/ml)

a

% by category

% by categorya

50%

90%

Susceptible

Resistant

50%

90%

Susceptible

Resistant

2 0.25 1 4 2 ⬎16

2 0.5 4 8 8 ⬎16

95.5 99.7 70.3 91.3 77.7 45.2

0.0 0.0 13.2 3.2 6.8 54.5

2 ⬎16 1 4 2 ⬎16

2 ⬎16 1 8 8 ⬎16

97.7 21.1 90.6 97.2 76.1 1.7

0.0 63.0 3.8 1.3 8.5 98.3

NCCLS [2002] broth microdilution method and interpretive criteria.

linezolid (MIC, ⱖ8 ␮g/ml), although a small percentage (2.3– 4.5%) of both vancomycin-susceptible and -resistant E. faecium and E. faecalis displayed linezolid intermediate susceptibility (MIC, 4 ␮g/ml) (NCCLS, 2002). Teicoplanin was, as expected, highly effective against vancomycin-susceptible E. faecium and E. faecalis, but was of modest or little value against vancomycin-resistant strains depending upon the van A or van B genotype of glycopeptide resistance. Vancomycin-resistant E. faecium isolates showed only 3.8% resistance to quinupristin/dalfopristin, while a significantly reduced proportion (86.8%) of vancomycin-susceptible E. faecium isolates were quinupristin/dalfopristin susceptible or -intermediate (NCCLS, 2002). Of the latter isolates, 13.2% displayed actual quinupristin/dalfopristin resistance, a feature observed before (Jones et al., 1998; 2001a and 2001b). Over 90% of E. faecalis isolates, regardless of vancomycin susceptibility, were resistant to quinupristin/dalfopristin. Chloramphenicol was generally more active against E. faecium (91.3–97.2% susceptible) than E. faecalis (77.4 – 84.3%), a pattern also noted for doxycycline. Ampicillin was usually very active against E. faecalis and approximately one-half of vancomycin-susceptible E. faecium, but was rarely (1.7% susceptible) effective versus vancomycin-resistant E. faecium. Linezolid-resistant enterococci have been documented in the clinical trials for patients receiving extended regimens of linezolid and in early clinical application (Gonzales et al.,

or susceptible (NCCLS, 2002). Results from Europe (Gemmell et al., 2001) demonstrated identical MIC90 results for linezolid, but one Dutch site had a MIC90 of 8 ␮g/ml repeated by the monitor to be 2 ␮g/ml. Therefore, in vitro experience dictates that non-susceptible linezoid MIC results (⬎4 ␮g/ml) remain very rare and should be confirmed by experienced, reference laboratories (Jones et al., 2001). Such real events have occurred recently in the US, where a dialysis patient strain mutation was discovered with a linezolid MIC of ⬎32 ␮g/ml following extended linezolid therapy. These linezolid-resistant isolates (three from one patient) showed a G2576T mutation in DNA encoding 23S rRNA (Tsiodras et al., 2001). Other mutations in the central loop of domain V have also been observed in enterococci (G2528U, G2576U; the latter from a strain detected here [RNJ]) [Data on file, Pharmacia]. 3.2. Linezolid activity against enterococci Tables 3 and 4 demonstrate the results of reference MIC susceptibility testing of linezolid and five other antimicrobial agents against 908 E. faecium and 2,369 E. faecalis isolates (categorized by vancomycin susceptibility), respectively. Vancomycin resistance was detected in 65.9% of E. faecium isolates (usually a Van A pattern) and 2.6% of E. faecalis isolates (usually a Van B pattern). None of the isolates for either enterococcal species were resistant to

Table 4 Antimicrobial activity lf linezolid and five other compounds tested against 2,369 E. faecalis strains categorized by vancomycin susceptibility Antimicrobial agent

Vancomycin-susceptible (n ⫽ 2,308) MIC (␮g/ml)

Linezolid Teicoplanin Quinupristin/dalfopristin Chloramphenicol Doxycycline Ampicillin a

Vancomycin-resistant (n ⫽ 61) MIC (␮g/ml)

a

% by category

% by categorya

50%

90%

Susceptible

Resistant

50%

90%

Susceptible

Resistant

2 0.25 16 4 4 1

2 0.25 ⬎16 ⬎16 8 2

96.5 100.0 2.9 84.8 60.4 97

0.0 0.0 92.5 12.3 9.7 3

2 0.25 16 8 4 1

2 ⬎16 ⬎16 ⬎16 8.0 ⬎16

95.6 64.5 3.2 77.4 58.1 79.0

0.0 21.0 96.8 19.4 8.1 21.0

NCCLS [2002] broth microdilution method and interpretive criteria.

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Table 5 Antimicrobial activity lf linezolid and four other commonly-used antimicrobial agents tested against 2,598 S. pneumoniae strains categorized by penicillin susceptibility Antimicrobial agent

Linezolid

Quinupristin/dalfopristin

Erythromycin

Ceftriaxone

Trovafloxacin

Penicillin categorya

MIC (␮g/ml)b 50%

90%

Susceptible

Resistant

S (n ⫽ 1,631) I (n ⫽ 512) R (n ⫽ 455) S I R S I R S I R S I R

0.75 0.75 0.75 0.5 0.5 0.5 0.125 0.19 4 0.016 0.19 0.75 0.125 0.125 0.125

1 1 1 1 1 1 0.25 ⬎64 ⬎256 0.047 0.5 1.5 0.25 0.19 0.19

100.0 100.0 100.0 96.0 94.7 93.0 91.7 54.8 20.4 99.7 94.3 27.9 99.3 99.4 98.9

0.0 0.0 0.0 0.2 0.2 1.1 6.2 41.7 77.4 0.1 0.4 9.0 0.7 0.4 0.9

% by categoryc

Susceptible (S) ⱕ0.06 ␮g/ml; Intermediate (I) 0.12–1 ␮g/ml; and Resistant (R) ⱖ2 ␮g/ml. Testing performed using Etest method (AB BIODISK, Solna, Sweden). c NCCLS [2002] criteria for interpretation. a

b

2001), a phenomenon also noted with quinupristin/dalfopristin (Chow et al., 1997). Most oxazolidinone resistance in enterococci has a modestly elevated linezolid MIC (8 or 16 ␮g/ml) and an associated mutation in the 23S rRNA. Isolates with zone diameters of ⱕ20 mm (Jones et al., 2001) or MICs reproducibly at ⱖ8 ␮g/ml should be referred to an experienced laboratory or the manufacturer for confirmation (NCCLS, 2002). Our previous study in the US (Jones et al., 2001) detected only 0.4% of disk diffusion test results in the linezolid-resistant category, none confirmed by monitor retesting. A similar experience was published by European investigators (Gemmell et al., 2001). 3.3. Linezolid activity against streptococci The activity of linezolid and four other antimicrobial agents against 2,598 S. pneumoniae isolates categorized by their penicillin susceptibility patterns is reported in Table 5. Of the 2,598 pneumococcal isolates, 37.2% were resistant to penicillin (MIC, ⬎0.06 ␮g/ml), with 17.5% displaying

high-level (ⱖ2 ␮g/ml) resistance. All of the S. pneumoniae isolates were susceptible to linezolid, regardless of the penicillin susceptibility level. Both quinupristin/dalfopristin and trovafloxacin were highly effective (ⱖ98% of susceptible) against S. pneumoniae, but a small amount of resistance to both these agents was found in each of the three penicillin-susceptibility categories. The fluoroquinolone resistance as an emerging problem has been documented (Chen et al., 1999) and resistance rates to a newer, potent agent such as trovafloxacin at 0.4 – 0.9% is an alarming statistic. Ceftriaxone was very effective against penicillinsusceptible and -intermediate S. pneumoniae (94.3–99.7%), but of lower potential clinical value against penicillin-resistant S. pneumoniae (9.0% resistance). The erythromycin in vitro spectrum showed a direct relationship, co-resistance, with that of ␤-lactams. Only 54.8 and 20.4% of S. pneumoniae were macrolide-susceptible among penicillin-intermediate and -resistant isolates, respectively. Linezolid was also highly effective against the nonpneumoniae streptococci isolates (Table 6). All strains were

Table 6 Antimicrobial activity lf linezolid and five other commonly-used antimicrobial agents tested against 761 other Streptococcus spp. strains Antimicrobial agent

Linezolid Quinupristin/dalfopristin Penicillin Erythromycin Ceftriaxone Trovafloxacin a

MIC (␮g/ml)a

% by category

50%

90%

Susceptible

Resistant

0.75 0.5 0.064 0.19 0.064 0.125

1.5 1 0.19 8 0.38 0.25

100.0 95.4 86.9 67.0 94.9 97.8

0.0 2.7 1.8 24.0 3.3 1.7

Testing performed using Etest method (AB BIODISK, Solna, Sweden) and interpreted by NCCLS [2002] criteria..

C.H. Ballow et al. / Diagnostic Microbiology and Infectious Disease 43 (2002) 75– 83

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Fig. 2. Distribution of linezolid MICs.

susceptible to linezolid (MIC90, 1.5 ␮g/ml by Etest), and of the six antimicrobial agents tested, linezolid alone showed no resistance. Resistance rates of non-pneumoniae streptococci to trovafloxacin, quinupristin/dalfopristin, and ceftriaxone were 1.7%, 2.7% and 3.3%, respectively. Previous reports of linezolid activity against streptococci (Gemmell et al., 2001; Jones et al., 1996 and 2001a; Jorgensen et al., 1997; Mason et al., 1996; Zurenko et al., 1996) have rarely discovered a strain with an MIC of ⱖ4 ␮g/ml (non-susceptible). Again such strains should be referred to reference-quality laboratories (NCCLS, 2002). Essentially all linezolid in vitro evaluations reported a MIC90 of 1 or 2 ␮g/ml and a range of 0.12– 4 ␮g/ml, best illustrated by Gemmell et al. (2001). A disk diffusion study in 106 US medical centers observed 0.6% of zone diameters in the non-susceptible range (ⱕ20 mm; none confirmed) (Jones et al., 2001a). This latter study recommended that clinical laboratories should practice the following related to linezolid in vitro susceptibility testing: 1) if a zone of ⱕ20 mm was observed, repeat the test to confirm the results and purity of the culture; 2) if confirmed, forward the strain to a reference or public health laboratory for further investigations that could include molecular-level studies; and 3) notify the physician/hospital record of the detection of a possible linezolid non-susceptible isolate (Jones et al., 2001a). Other agents with similarly complete spectrum against some Gram-positive species would also require identical routine laboratory practices (Jones et al., 1998 and 2001b).

4. Conclusions These results from 1999 –2000 in the US illustrate the broad occurrence of isolates resistant to oxacillin (staphylococci), glycopeptides (enterococci), penicillin (streptococci) and macrolides (streptococci). Only among the enterococci and streptococci could frequency rates be determined

in this suveillance study as follows: 1) VRE in E. faecalis at 2.6% and in E. faecium at 65.9%; 2) penicillin resistance in S. pneumoniae at 37.2% (high-level, 17.5%); 3) erythromycin resistance in pneumococci at 28.0%; 4) 0.8% resistance among S. pneumoniae to trovafloxacin; and 5) 13.1% resistance among the combined viridans group and ␤-hemolytic streptococci to penicillin. The rates compare favorably to other reports of isolates for 1999 –2000 (Hoban et al., 2001; Jones et al., 2001a and 2001b). The reduced activity of the highly effective fluoroquinolone (trovafloxacin), affirms the presence (Chen et al., 1999) and possible increased rates of resistance in S. pneumoniae in the US. Trovafloxacin has a potency versus pneumococci that should be comparable to gemifloxacin and moxifloxacin (Deshpande & Jones, 2000). The overall distribution of linezolid MICs for S. aureus, enterococci, and S. pneumoniae is displayed in Fig. 2. Linezolid MICs were generally unimodal and ranged between 1– 4 ␮g/ml for staphylococci (94% of strains), 1–2 ␮g/ml for enterococci (93% of strains), and 0.5–1 ␮g/ml for streptococci (85% of strains). Overall, linezolid either matched or out-performed all other antimicrobial agents tested in this in vitro study for spectrum of activity. In comparison to quinupristin/dalfopristin, another relatively new and promising agent for use against Gram-positive infections (Chow et al., 1997; Jones et al., 1998), linezolid had: (1) slightly greater activity against S. aureus, CoNS, S. pneumoniae, and other streptococci; (2) considerably greater activity against E. faecium, especially vancomycin-susceptible isolates, and (3) far greater activity against E. faecalis. The activity of linezolid against all streptococci isolates tested was equal to, or superior to, the highly effective fluoroquinolone, trovafloxacin. Although in vitro results must be clinically substantiated before the overall effectiveness of an antimicrobial agent may be properly assessed, this study’s results demonstrated linezolid to be a very promising new agent and class against the growing problem of antimicrobial-resistant Gram-positive organisms (Diekema & Jones, 2000). And given the

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constantly increasing incidence of vancomycin resistance among enterococci and staphylococci (Fridkin et al., 2001), linezolid may provide a valuable alternative to glycopeptide agents for resistant Gram-positive infections. Lately, laboratories must be alert to the appearance of oxazolidinoneresistant Gram-positive isolates and initiate procedures to refer and/or confirm these organisms in the same manner as vancomycin-intermediate S. aureus (Fridkin et al., 2001).

Acknowledgments Presented in part at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Ontario, September 20, 2000. Supported by an educational/research grant from Pharmacia Corporation.

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