Antimicrobial activity of tigecycline against clinical isolates from Spanish medical centers. Second multicenter study

Diagnostic Microbiology and Infectious Disease 56 (2006) 437 – 444 www.elsevier.com/locate/diagmicrobio

Antimicrobial activity of tigecycline against clinical isolates from Spanish medical centers. Second multicenter study Carmen Betriua,4, Iciar Rodrı´guez-Aviala, Marı´a Go´meza, Esther Culebrasa, ´ lvarezb, Juan J. Picazoa, Fa´tima Lo´peza, Juan A the Spanish Tigecycline Group1 a Department of Clinical Microbiology, Hospital Clı´nico San Carlos, 28040 Madrid, Spain b Scientific Department, Wyeth Farma, 28700 Madrid, Spain Received 25 May 2006; accepted 10 July 2006

Abstract The antimicrobial activity of tigecycline, a novel glycylcycline, was evaluated against 1102 bacterial isolates from 20 centers in Spain. The MIC of tigecycline at which 90% (MIC90) of the pneumococci tested were inhibited was the lowest (0.06 Ag/mL) of all the antibiotics tested. The MICs of tigecycline against enterococci ranged from 0.03 to 0.125 Ag/mL. All staphylococci were inhibited by V 0.25 Ag/mL of tigecycline, and 99.6% of Enterobacteriaceae isolates were inhibited by V 2 Ag/mL of tigecycline. Tigecycline demonstrated good activity against Bacteroides fragilis group organisms with an MIC90 of 4 Ag/mL. The results of this study confirm the excellent activity of tigecycline against multiresistant Gram-positive and Gram-negative pathogens. D 2006 Elsevier Inc. All rights reserved. Keywords: Tigecycline; Activity; Bacterial isolates

1. Introduction During the last 2 decades, the emergence of Grampositive strains resistant to different antimicrobial agents (e.g., penicillin-resistant Streptococcus pneumoniae, glycopeptide-resistant enterococci, and methicillin-resistant staphylococci) has become a serious medical problem (Carmeli et al., 2002; Aspa et al., 2004; Schito, 2006). Furthermore, the prevalence of Enterobacteriaceae isolates that produce extended-spectrum h-lactamase (ESBL) or hyperproduce AmpC enzymes has increased worldwide (Bradford, 2001; Moland et al., 2002; Arpin et al., 2003). In addition, carbapenem-resistant Gram-negative bacilli, such as Pseudomonas aeruginosa and Acinetobacter baumannii, have emerged in many countries (Coelho et al., 2004; NNIS, 2004), and the emergence and dissemination of metallo-hlactamase–producing organisms has recently been described

4 Corresponding author. Tel.: +34-913303486; fax: +34-913303478. E-mail address: [email protected] (C. Betriu). 1 Members of the Spanish Tigecycline Group are listed in the Acknowledgments. 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.07.005

(Peleg et al., 2005). New compounds that are effective against infections caused by multiresistant Gram-positive and Gram-negative bacteria are urgently needed. Tigecycline (formerly GAR-936) is a new first-class glycylcycline antibiotic with broad-spectrum activity against aerobic and anaerobic Gram-positive and Gramnegative bacteria (Gales and Jones, 2000; Milatovic et al., 2003; Jacobus et al., 2004; Hoban et al., 2005; Sader et al., 2005). It acts by binding to the 30S ribosomal subunit and by blocking entry of amino-acyl tRNA molecules into the A site of the ribosome. The glycylcyclines overcome 2 common mechanisms that are responsible for tetracycline resistance—ribosomal protection and efflux pumps. The efficacy and safety of tigecycline in the treatment of hospitalized patients with complicated intra-abdominal infections and complicated skin-structure infections have been demonstrated (Babinchak et al., 2005; Ellis-Grosse et al., 2005). This antibiotic has displayed a favorable pharmacokinetic profile, with extensive tissue distribution and a long elimination half-life (Meagher et al., 2005). We previously reported (Betriu et al., 2002) on the antimicrobial activity of tigecycline and comparator agents

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against groups of bacterial pathogens collected in Spain in 2001. The present report provides an overview of the results of a second multicenter study performed in our country in 2005. In addition, changes in the susceptibility patterns from 2001 to 2005 are documented. 2. Materials and methods In May 2005, 20 medical centers throughout Spain participated in the study. Eleven medical centers continued participation from the previous study in 2001 (Betriu et al., 2002) and 9 new centers joined the study. Each center was requested to collect up to 51 consecutive, nonduplicate aerobic isolates that included 6 penicillin-nonsusceptible Streptococcus pneumoniae, 6 methicillin-resistant Staphylococcus aureus (MRSA), 6 coagulase-negative staphylococci, 3 Enterococcus faecalis, 3 Enterococcus faecium, 3 ciprofloxacin-susceptible Escherichia coli, 3 ciprofloxacin-resistant Escherichia coli, 3 Klebsiella pneumoniae or Klebsiella oxytoca, 3 Enterobacter cloacae or Enterobacter aerogenes, 3 Citrobacter freundii or Citrobacter diversus, 3 Serratia marcescens, 3 Stenotrophomonas maltophilia, 3 A. baumannii, and 3 P. aeruginosa. The anaerobic strains were isolated in 2 of the 20 centers. Each collected up to 70 consecutive nonduplicate Bacteroides fragilis group strains and up to 25 Clostridium difficile isolates. Aerobic bacteria were collected from the urogenital tract (26%), respiratory tract (24.5%), blood (23.8%), skin and soft tissues (17.2%), abdomen (4.8%), devices (1.7%), joints (0.9%), cerebrospinal fluid (0.3%), and other sites (0.8%). Sources of the 137 B. fragilis group isolates included the abdomen (42.3%), skin and soft tissue (35.7%), blood (16%), female genital tract (3%), and other sites (3%). Isolates were identified at each participating laboratory using routine methodology and were sent on transport swabs (Culturette; Becton Dickinson Microbiology Systems, Sparks, MD) to the coordinating laboratory at the Hospital Clı´nico San Carlos, Madrid, Spain. Upon receipt, isolates were subcultured onto 5% sheep blood agar or brucella blood agar to ensure purity. Identification was confirmed with Slidex Staph, Slidex pneumo, Rapid ID 32 STREP, ID 32 STAPH, ID 32 GN, and Rapid ID 32A (bioMe´rieux, Marcy l’Etoile, France). MICs were determined by the agar dilution method according to the guidelines of the Clinical Laboratory Standards Institute (CLSI, formerly National Committee for Clinical Laboratory Standards 2003, 2004). Susceptibility testing of Streptococcus pneumoniae isolates was performed by agar dilution with Mueller–Hinton agar supplemented with 5% sheep blood. Agar dilution plates for tigecycline were prepared on the day of inoculation. The antimicrobials tested varied according to the bacterial species (see Tables 1–3). The following antimicrobial agents were provided by their respective manufacturers: tigecycline and piperacillin–tazobactam (Wyeth Farma, Madrid, Spain); amoxicillin, ampicillin, clavulanate, and

ceftazidime (GlaxoSmithKlineBeecham, Madrid, Spain); imipenem (Merck Sharp and Dohme, Madrid, Spain); levofloxacin, quinupristin–dalfopristin, teicoplanin, cefotaxime, and metronidazole (Sanofi Aventis, Barcelona, Spain); linezolid, sulbactam, and clindamycin (Pfizer, Madrid, Spain); and cefepime (Bristol Myers Squibb, Madrid, Spain). The remaining comparators were obtained from Sigma-Aldrich Quı´mica, Madrid, Spain. The following quality control strains were included: Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Enterococcus faecalis ATCC 51229, Streptococcus pneumoniae ATCC 49619, P. aeruginosa 27853, B. fragilis ATCC 25285, and Bacteroides thetaiotamicron ATCC 29741. Production of h-lactamase was tested for all enterococci isolates with nitrocefin disks (Cefinase; Becton Dickinson Microbiology Systems, Sparks, MD). The interpretive criteria were those of the CLSI (2005). Among erythromycin-resistant Streptococcus pneumoniae isolates, macrolide resistance phenotypes were determined by the double-disk method (Seppa¨la¨ et al., 1993). According to the CLSI (2005) criteria, Escherichia coli and Klebsiella spp. isolates with increased MIC results ( V 2 Ag/mL) for cefotaxime and/or cefepime were suspected of being ESBL-producing isolates. Phenotypic confirmation of these strains was performed using Etest ESBL strips (AB Biodisk, Solna, Sweden). Confirmation of a metallo-h-lactamase (MBL) was tested against A. baumannii and P. aeruginosa isolates with imipenem MICs of V 8 Ag/mL using Etest MBL strips (AB Biodisk). The results of this study were compared with those obtained in the previous study (Betriu et al., 2002) to detect any possible changes in the antibiotic susceptibility patterns. The data were analyzed with Epi Info 2005 software (version 3.3.2; CDC, Atlanta, GA). Statistical analysis of data was performed by the v 2 test, with Yates’ or applied Fisher exact results when necessary. A P value of less than 0.05 was used to determine statistical significance. 3. Results In total, 1102 bacterial isolates were processed, of which 915 were aerobic and 187 were anaerobic strains. The species represented are listed in Tables 1–3. Table 1 summarizes the MICs at which 50% and 90% of the isolates were inhibited (MIC50 and MIC90, respectively), MIC range, and percentage of resistance for the aerobic Grampositive cocci tested. Resistance for all drugs except tigecycline was based on CLSI (2005) breakpoints. Tigecycline was a highly active agent against those Streptococcus pneumoniae isolates tested, which were not susceptible to penicillin. The MICs of tigecycline at which 50% and 90% of the pneumococci tested were inhibited were the lowest of all the antibiotics tested. Tigecycline had an MIC90 of 0.06 Ag/mL compared with 1 Ag/mL for cefotaxime, linezolid, quinupristin–dalfopristin, and levofloxacin. All Streptococcus pneumoniae were inhibited by 0.125 Ag/mL of

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Table 1 In vitro activities of tigecycline and other antimicrobial agents against Gram-positive isolates Organism (no. of isolates)

Agent

Penicillin-intermediate and penicillin-resistant Streptococcus pneumoniae (84)

Tigecycline Penicillin Cefotaxime Erythromycin Vancomycin Linezolid Quinupristin – dalfopristin Levofloxacin Tigecycline Gentamicin Rifampin Levofloxacin Linezolid Quinupristin – dalfopristin Vancomycin Teicoplanin Tigecycline Gentamicin Rifampin Levofloxacin Linezolid Quinupristin – dalfopristin Vancomycin Teicoplanin Tigecycline Ampicillin Linezolid Quinupristin – dalfopristin Levofloxacin Vancomycin Teicoplanin Tigecycline Ampicillin Linezolid Quinupristin – dalfopristin Levofloxacin Vancomycin Teicoplanin

MRSA (112)

Coagulase-negative staphylococcib (106)

Enterococcus faecalis (54)

Enterococcus faecium (52)

% Resistant strainsa

MIC (Ag/mL) Range

50%

90%

V 0.015 – 0.125 0.2 – 4 V 0.06 – 1 V 0.06 – 256 V 0.06 – 0.5 V 0.06 – 1 0.25 – 2 0.25 – 8 0.03 – 0.125 0.25 to N 256 V 0.06 – 64 0.1 – 256 1 0.25 – 64 0.5 – 1 0.25 – 4 0.03 – 0.25 V 0.06 to N 256 V 0.06 to N 256 V 0.06 – 256 0.5 – 1 V 0.06 – 16 0.25 – 2 V 0.06 – 16 0.06 – 0.125 0.06 – 1 2 2 – 16 0.5 – 64 0.5 – 2 0.25 – 1 0.03 – 0.125 0.1 – 128 1 0.5 – 8 0.5 – 128 0.25 – 64 0.25 – 2

0.03 2 0.5 0.25 0.25 1 1 1 0.125 0.5 V 0.06 8 1 0.5 0.5 1 0.06 2 V 0.06 4 0.5 0.25 1 2 0.06 0.5 2 4 1 1 0.5 0.06 128 1 1 64 0.5 0.5

0.06 2 1 256 0.5 1 1 1 0.125 64 V 0.06 32 1 0.5 1 2 0.125 128 V 0.06 128 1 0.5 1 4 0.125 1 2 8 32 2 0.5 0.06 128 1 4 128 1 1

– 57.1 0 50 0 0 0 1.2 – 21.4 1.8 91.1 0 0.9 0 0 – 41.5 5.7 55.7 0 3.8 0 0 – 0 0 94.4 22.2 0 0 – 78.8 0 28.8 71.2 5.8 0

– , Breakpoints for tigecycline are not currently provided by the CLSI. a MICs for resistant isolates are those described in the CLSI. b Staphylococcus capitis (n = 1), Staphylococcus epidermidis (n = 57), Staphylococcus haemolyticus (n = 17), Staphylococcus hominis (n = 27), Staphylococcus saprophyticus (n = 2), Staphylococcus simulans (n = 1), and Staphylococcus warneri (n = 1).

tigecycline, and most strains (96.4%) were inhibited by 0.06 Ag/mL. Half of the pneumococci included in the study were resistant to erythromycin. The constitutive macrolide– lincosamide–streptogramin B resistance (cMLSB) phenotype was observed in the majority (76.2%) of erythromycinresistant isolates. By contrast, only 4 (9.5%) isolates displayed the inducible MLSB phenotype and 6 (14.3%) the M phenotype. Among Streptococcus pneumoniae, the rate of resistance to levofloxacin was low (1.2%), similar to that reported in the first multicenter study performed in 2001 (Betriu et al., 2002). Linezolid, vancomycin, and teicoplanin were uniformly active against all the staphylococci tested. Tigecycline was the most active agent against MRSA, with MIC50 and MIC90 values both of 0.125 Ag/mL. The activity of

tigecycline against MRSA was greater than that of vancomycin (MIC90, 0.125 and 1 Ag/mL, respectively). Resistance to gentamicin was detected in 21.4% of the MRSA isolates and more than 90% were resistant to levofloxacin. Resistance to quinupristin–dalfopristin was found in only 1 (0.9%) MRSA isolate and in 4 (3.8%) CNS isolates. By comparing the susceptibility patterns of MRSA from 2001 to 2005, we observed that the rate of resistance to levofloxacin increased significantly from 59.5% in 2001 to 91.1% in 2005 ( P b 0.00001). By contrast, resistance to gentamicin declined significantly from 38.7% in 2001 to 21.4% in 2005 ( P b 0.008). Among MRSA isolates, susceptibilities to the other agents evaluated have not changed significantly since 2001. On the basis of the MIC90, the activity of tigecycline against coagulase-negative

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Table 2 In vitro activities of tigecycline and other antimicrobial agents against Gram-negative isolates Organism (no. of isolates)

Agent

Ciprofloxacin-resistant Escherichia coli (58)

Tigecycline Ciprofloxacin Ampicillin Cefotaxime Cefepime Amoxicillin – clavulanate Piperacillin – tazobactam Gentamicin Tigecycline Ciprofloxacin Ampicillin Cefotaxime Cefepime Amoxicillin – clavulanate Piperacillin – tazobactam Gentamicin Tigecycline Ciprofloxacin Ampicillin Cefotaxime Cefepime Amoxicillin – clavulanate Piperacillin – tazobactam Gentamicin Tigecycline Ciprofloxacin Ampicillin Cefotaxime Cefepime Amoxicillin – clavulanate Piperacillin – tazobactam Gentamicin Tigecycline Ciprofloxacin Ampicillin Cefotaxime Cefepime Amoxicillin – clavulanate Piperacillin – tazobactam Gentamicin Tigecycline Ciprofloxacin Ampicillin Cefotaxime Cefepime Amoxicillin – clavulanate Piperacillin – tazobactam Gentamicin Tigecycline Imipenem Cefepime Piperacillin – tazobactam Ampicillin – sulbactam Sulbactam Levofloxacin Gentamicin Polymyxin B Tigecycline Trimethoprim – sulfamethoxazole Ticarcillin – clavulanate Piperacillin – tazobactam

Ciprofloxacin-susceptible Escherichia coli (59)

Enterobacter spp.b (57)

Serratia marcescens (49)

Citrobacter spp.c (52)

Klebsiella spp.d (60)

A. baumannii (57)

Stenotrophomonas maltophilia (53)

MIC (Ag/mL) Range

50%

90%

% Resistant strainsa

0.03 – 4 2 – 128 1 to N 256 V 0.06 – 256 V 0.06 to N 256 V 0.06 – 64 0.5 – 128 0.25 – 64 0.06 – 0.25 V 0.06 – 1 1 to N 256 V 0.06 – 16 V 0.06 – 8 1 – 32 0.5 – 2 0.25 – 8 0.125 – 2 V 0.06 – 32 32 to N 256 V 0.06 to N 256 V 0.06 – 256 32 – 128 2 to N 256 0.25 – 256 0.25 – 2 V 0.06 – 64 32 to N 256 0.125 – 64 V 0.06 – 1 32 – 128 0.25 to N 256 0.25 to N 256 0.06 – 0.5 V 0.06 – 128 4 to N 256 V 0.06 – 256 V 0.06 – 16 2 – 128 0.125 to N 256 0.125 – 4 0.125 – 4 V 0.06 – 128 32 to N 256 V 0.06 – 128 V 0.06 – 64 1 – 32 4 to N 256 0.25 – 64 0.125 – 16 0.25 – 128 2 – 256 V 0.06 to N 256 2 – 256 0.5 – 64 V 0.06 – 128 V 0.06 to N 256 0.5 – 1 0.25 – 4 V 0.06 – 2 8 to N 256 2 to N 256

0.125 16 N 256 V 0.06 V 0.06 8 2 0.5 0.06 V 0.06 2 V 0.06 V 0.06 2 2 0.25 0.25 V 0.06 256 0.25 V 0.06 64 4 0.25 0.5 V 0.06 128 0.25 V 0.06 64 0.5 4 0.125 V 0.06 64 V 0.06 V 0.06 32 4 0.25 0.25 V 0.06 64 V 0.06 V 0.06 2 8 0.25 4 2 32 N 256 32 8 8 N 256 1 0.5 0.25 8 256

0.125 32 N 256 8 0.5 32 8 64 0.125 0.125 N 256 0.125 0.25 16 2 0.5 0.5 V 0.06 N 256 128 1 64 256 0.5 1 0.5 N 256 2 0.25 128 16 8 0.25 0.1 N 256 32 0.5 64 128 4 0.5 8 N 256 0.5 0.5 32 256 0.5 8 128 256 N 256 128 32 32 N 256 1 4 0.5 256 N 256

– 100 82.8 5.1 1.7 12.1 1.7 24.1 – 0 45.8 0 0 8.5 0 0 – 3.5 100 21.1 3.6 100 19.3 3.6 – 2 100 2 0 100 6.1 6.1 – 5.8 86.5 3.8 0 59.6 15.4 0 – 15 100 1.7 1.7 11.7 18.3 8.3 – 24.6 63.2 71.9 56.1 – 64.9 78.9 0 – 0 13.2 54.7

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441

Table 2 (continued) Organism (no. of isolates)

Agent

Stenotrophomonas maltophilia (53)

MIC (Ag/mL)

Ceftazidime Amikacin Levofloxacin Tigecycline Ceftazidime Cefepime Imipenem Piperacillin – tazobactam Gentamicin Amikacin Levofloxacin

P. aeruginosa (62)

Range

50%

90%

0.125 to N 256 1 to N 256 V 0.06 – 64 0.5 – 32 0.125 – 128 0.5 – 32 0.5 – 256 1 to N 256 V 0.06 to N 256 0.5 – 128 0.125 to N 256

64 256 1 8 0.5 4 2 32 1 4 0.5

N 256 N 256 16 16 16 16 32 N 256 N 256 32 32

% Resistant strainsa 73.6 81.1 15.1 – 6.5 4.8 21.0 27.4 22.6 8.1 25.8

– , Breakpoints for tigecycline and sulbactam are not currently provided by the CLSI. a MICs for resistant isolates are those described in the CLSI. b Enterobacter aerogenes (n = 13) and Enterobacter cloacae (n = 44). c Citrobacter freundii (n = 36) and Citrobacter koseri (n = 16). d K. oxytoca (n = 16) and K. pneumoniae (n = 44).

staphylococci was 8-fold higher than that of vancomycin. More than half of these strains were resistant to levofloxacin and 41.5% were resistant to gentamicin. We also observed

an increasing trend in the rates of resistance to levofloxacin among coagulase-negative staphylococci (from 26% in 2001 to 55.7% in 2005, P b 0.0003).

Table 3 In vitro activities of tigecycline and other antimicrobial agents against anaerobes Organism (no. of isolates)

Agent

B. fragilis group (137)

Tigecycline Cefoxitin Imipenem Amoxicillin – clavulanate Piperacillin – tazobactam Clindamycin Metronidazole Moxifloxacin Tigecycline Cefoxitin Imipenem Amoxicillin – clavulanate Piperacillin – tazobactam Clindamycin Metronidazole Moxifloxacin Tigecycline Cefoxitin Imipenem Amoxicillin – clavulanate Piperacillin – tazobactam Clindamycin Metronidazole Moxifloxacin Tigecycline Cefoxitin Imipenem Piperacillin – tazobactam Metronidazole Quinupristin – dalfopristin Linezolid Moxifloxacin

B. fragilis (85)

Other B. fragilis group speciesb (52)

Clostridium difficile (50)

MIC (Ag/mL) Range

50%

90%

V 0.06 – 16 V 0.06 – 128 V 0.06 to N 256 V 0.06 – 16 V 0.06 to N 256 V 0.06 to N 256 V 0.06 – 4 0.125 – 128 V 0.06 – 16 2 – 128 V 0.06 to N 256 V 0.06 – 16 V 0.06 to N 256 V 0.06 to N 256 V 0.06 – 2 0.125 – 64 V 0.06 – 8 1 – 256 V 0.06 – 0.5 0.125 – 16 V 0.06 – 256 V 0.06 to N 256 V 0.06 – 4 0.125 – 128 V 0.06 – 1 64 to N 256 V 0.06 – 32 0.125 – 16 V 0.06 – 1 V 0.06 – 32 V 0.06 – 8 V 0.06 – 128

1 16 0.125 0.5 4 2 1 1 1 8 V 0.06 0.5 1 1 1 0.5 0.5 16 0.25 1 8 2 1 2 V 0.06 128 8 4 0.25 4 2 16

4 64 0.5 4 16 N 256 2 8 4 16 0.5 4 8 N 256 2 8 4 64 0.5 16 16 N 256 4 8 0.125 N 256 16 8 0.5 8 8 32

% Resistant strainsa – 16.8 1.5 2.2 2.9 39.4 0 – – 14.1 2.4 2.4 2.4 36.5 0 – – 21.2 1.9 1.9 3.8 44.2 0 – – 100 40 0 0 – – –

– , Breakpoints for tigecycline, moxifloxacin, quinupristin – dalfopristin, and linezolid are not currently provided by the NCCLS for anaerobes. a MICs for resistant isolates are those described in the NCCLS. b B. thetaiotamicron (23), Bacteroides distasonis (6), Bacteriodes caccae (5), Bacteriodes uniformis (5), Bacteriodes vulgatus (5), and Bacteroides spp. (8).

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Tigecycline has also demonstrated good activity against enterococci; it showed the lowest MICs of all the comparators tested. Among Enterococcus faecium isolates, 3 strains (5.8%) were vancomycin-resistant. Resistance to ampicillin was detected in 41 (78.8%) isolates, none of which produced h-lactamase. Tigecycline had very low MIC50 and MIC90 values against Enterococcus faecium (both of 0.06 Ag/mL) and was 16-fold more active than linezolid, vancomycin, and teicoplanin. Resistance to quinupristin–dalfopristin was 28.8%. All Enterococcus faecalis isolates were susceptible to vancomycin and ampicillin. The MICs of tigecycline against the 54 isolates of Enterococcus faecalis ranged from 0.06 to 0.125 Ag/mL, which were lower than those of ampicillin and the glycopeptides tested. Susceptibility to the agents evaluated has not changed significantly since 2001. The antimicrobial activity results from testing 507 aerobic Gram-negative isolates are listed in Table 2. Tigecycline showed excellent activity against Enterobacteriaceae with 90% of the strains inhibited at between 0.125 and 1 Ag/mL. Tigecycline was highly active against both ciprofloxacin-susceptible Escherichia coli and ciprofloxacin-resistant Escherichia coli. Against the 117 Escherichia coli isolates, tigecycline showed the best activity of all the agents tested (the MIC90 was 0.125 Ag/mL). The rate of resistance to amoxicillin–clavulanate among ciprofloxacinresistant Escherichia coli declined from 29.3% in 2001 to 12.1% in 2005 ( P b 0.04). Susceptibilities to the remaining antibiotics did not change significantly over the 5 years of testing, although the MIC90 of cefotaxime increased from 2 Ag/mL in 2001 to 8 Ag/mL in 2005. The prevalence of confirmed ESBL-positive isolates in Escherichia coli and Klebsiella spp. was 11.9% and 11.6%, respectively. The activity of tigecycline against ESBL producers was excellent (MIC range, 0.06–1 Ag/mL). Among Citrobacter spp., tigecycline MICs were in the range of 0.06–0.5 Ag/mL and 98% of these isolates were inhibited at V 0.25 Ag/mL. Tigecycline showed the lowest MIC50/MIC90 (0.5/1 Ag/mL) with Serratia marcescens and inhibited all strains at 2 Ag/mL. Against A. baumannii, the MIC50 of tigecycline was 4 Ag/mL and the MIC90 was 8 Ag/mL. Based on the MIC90, tigecycline was 4 dilutions more active than imipenem and 5 dilutions more active than cefepime. We detected 18 imipenem-resistant A. baumannii isolates and 3 of these gave a positive result in the Etest MBL assay, suggesting the presence of metallo-h-lactamases. The most active agent against A. baumannii was polymyxin B, and all isolates were inhibited at V 1 Ag/mL. Among A. baumannii, an increasing incidence of resistance to various agents was observed by comparing these results with those obtained in 2001 (Betriu et al., 2002). The rate of resistance to piperacillin– tazobactam rose significantly from 39.1% in 2001 to 71.9% in 2005 ( P b 0.0006), and the rate of resistance to cefepime increased from 37.5% to 63.2% ( P b 0.009). Most Stenotrophomonas maltophilia isolates were resistant to multiple antibiotics. The principal finding regarding

the changes in susceptibility patterns of Stenotrophomonas maltophilia from 2001 to 2005 was the emergence of resistance to ticarcillin–clavulanate. In 2001, all isolates were susceptible to ticarcillin–clavulanate, and in 2005 we found 7 strains resistant to this agent. Tigecycline had good activity against Stenotrophomonas maltophilia; all isolates were inhibited at V 4 Ag/mL. In contrast, tigecycline showed poor activity against P. aeruginosa (MIC90, 16 Ag/mL). P. aeruginosa isolates showed high levels of resistance to most of the antimicrobials tested. Rates of resistance to imipenem, piperacillin–tazobactam, gentamicin, and levofloxacin were around 25%, and resistance to cefepime, ceftazidime, and amikacin ranged from 4.8% to 8.1%. Of the 13 imipenem-resistant isolates, 4 (30.7%) were screenpositive for metallo-h-lactamase. Results of MIC studies for anaerobes are presented in Table 3. Against B. fragilis group organisms, tigecycline inhibited 89.8% of the isolates at 4 Ag/mL with an MIC range of V 0.06 to 16 Ag/mL. Resistance to clindamycin was observed in 39.4% of the B. fragilis group organisms. No differences in the activities of tigecycline between clindamycin-susceptible and clindamycin-resistant isolates tested were noted. Only 2 strains were resistant to imipenem. Comparing the resistance percentages for the B. fragilis group over the 5 years of the study revealed that the incidence of resistance to both cefoxitin and clindamycin rose from 7.5% and 26.7% in 2001 to 16.8% and 39.4% in 2005, respectively ( P b 0.04). In addition, tigecycline showed good activity against Clostridium difficile. More than 90% of Clostridium difficile isolates were inhibited by 0.25 Ag/mL of tigecycline. No resistance was detected to metronidazole. Tigecycline was found to be more active than metronidazole; the tigecycline MIC50/MIC90 values were V 0.06/0.125 Ag/mL, compared with those of metronidazole, which were 0.5/1 Ag/mL. 4. Discussion Tigecycline showed excellent activity against the multidrug-resistant Gram-positive and Gram-negative pathogens tested, including MRSA, penicillin-resistant pneumococci, vancomycin-resistant enterococci, ESBL-producing Enterobacteriaceae, A. baumannii, Stenotrophomonas maltophilia, and B. fragilis group organisms. The MICs of tigecycline ranged from V 0.015 to 0.25 Ag/mL for all Gram-positive aerobes tested and from 0.03 to 4 Ag/mL for Enterobacteriaceae isolates. The previously described high prevalence of the constitutive phenotype among erythromycin-resistant Streptococcus pneumoniae isolates (Betriu et al., 2000) indicates that neither clindamycin nor 16-membered macrolides could be considered as alternative therapeutic option. Tigecycline was very active against pneumococci presenting the cMLSB phenotype of macrolide resistance, with an MIC90 of 0.06 Ag/mL. Recent studies (Bouchillon et al., 2005; Hoban et al., 2005) on the in vitro activity of tigecycline against

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Enterobacteriaceae have reported an MIC range of 0.03 to 16 Ag/mL with MIC50 and MIC90 results ranging from 0.125 to 1 and 0.5 to 2 Ag/mL, respectively. In the present study, tigecycline MIC values are generally lower than these. This could be because other studies performed susceptibility testing using broth microdilution. By contrast, we determined MICs by the agar dilution method. The MIC values of tigecycline, like those of other agents, are 1 or 2 dilutions higher when the broth microdilution method is used (Hope et al., 2005). The increasing incidence of ESBL-producing Enterobacteriaceae has been described in many countries (Bradford, 2001; Moland et al., 2002; Arpin et al., 2003). A nationwide epidemiologic study conducted in 40 Spanish hospitals revealed that the prevalence of ESBL-producing isolates among ciprofloxacin-resistant Escherichia coli increased from 1.1% in 2001 to 11.3% in 2004 (Picazo et al., 2002, 2004). In the present study, this rate is 17.2%. The activity of tigecycline was not significantly affected by the presence of ESBLs. As has been published elsewhere (Gales and Jones, 2000; Milatovic et al., 2003; Bouchillon et al., 2005; Sader et al., 2005), tigecycline is inactive against P. aeruginosa. Tigecycline demonstrated good activity against the B. fragilis group strains and Clostridium difficile isolates tested. Our results agree with those reported by other authors (Edlund and Nord, 2000; Gales and Jones, 2000; Jacobus et al., 2004). Recent studies have revealed that the activity of tigecycline was affected by the amount of dissolved oxygen in the media. When tested in fresh broth media, tigecycline was 2 to 3 dilutions more active than aged media (Bradford et al., 2005; Hope et al., 2005; Petersen and Bradford, 2005). If we compare the results of the present study with those described in the first study performed by our group in 2001, we observe that tigecycline MICs generally appear lower than those previously reported (Betriu et al., 2002). One explanation may be that, in the present study, the activity of tigecycline was tested on plates prepared on the day of use, whereas in the previous study we used agar media prepared 24–48 h before use. Tigecycline susceptibility breakpoint interpretations as defined by the Food and Drug Administration (FDA) indicate that V 2 Ag/mL is susceptible for Enterobacteriaceae, V 0.5 Ag/mL for Staphylococcus aureus (including methicillin-resistant isolates), V 0.25 Ag/mL for Enterococcus faecalis (vancomycin-susceptible isolates only) and Streptococcus spp. other than Streptococcus pneumoniae, and V 4 Ag/mL for anaerobes. According to these criteria, all MRSA isolates, all Enterococcus faecalis, 99.4% of the Enterobacteriaceae isolates, and 89.8% of B. fragilis group and all Clostridium difficile isolates included in the study are susceptible to tigecycline. These results confirm the findings of previous studies that have documented the excellent activity of tigecycline against a variety of Gram-positive and Gram-negative bacteria (Gales and Jones, 2000; Milatovic et al., 2003; Bouchillon et al.,

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2005; Fritsche et al., 2005; Hoban et al., 2005; Sader et al., 2005). Emerging antimicrobial resistance remains an important concern worldwide, and the treatment of infections caused by multiresistant pathogens is becoming increasingly limited. There is an urgent need for new therapeutic options. The results of this study suggest that tigecycline could be an alternative in the treatment of nosocomial infections caused by Gram-positive and Gram-negative bacteria, including multiresistant bacteria. Tigecycline was recently approved by the FDA as a parenteral agent for the treatment of complicated skin and skin structure infections and intraabdominal infections. As tigecycline is not yet available in Spain, it would be interesting to monitor the in vitro activity of tigecycline compared with several agents to assess the evolution of MICs over time.

Acknowledgments This study was supported by a financial grant from Wyeth Farma. Members of the Spanish Tigecycline Group (Coordinator, J.J. Picazo, Madrid) are as follows: J. Aznar (Sevilla), J. Blanco (Badajoz), E. Bouza (Madrid), R. Cisterna (Bilbao), J.A. Garcı´a Rodrı´guez (Salamanca), M. Gobernado (Valencia), J.L. Go´mez Garce´s (Madrid), A. Guerrero (Alcira), A. Gutie´rrez (Madrid), M.T. Jime´nez de Anta (Barcelona), J. Maiquez (Valencia), R. Martı´n (Barcelona), E. Perea (Sevilla), B. Regueiro (Santiago de Compostela), M.J. Revillo (Zaragoza), J. Rodrı´guez Otero (Madrid), C. Rubio (Zaragoza), J. Ruiz (Murcia), and R. Villanueva (La Corun˜a).

References Arpin C, Dubois V, Coulange L, Andre C, Fischer I, Noury P, Grobost F, Brochet JP, Jullin J, Dutilh B, Larribet G, Lagrange I, Quentin C (2003) Extended-spectrum beta-lactamase–producing Enterobacteriaceae in community and private health care centers. Antimicrob Agents Chemother 47:3506 – 3514. Aspa J, Rajas O, Rodrı´guez de Castro F, Blanquer J, Zalacaı´n R, Fenoll A, de Celis R, Vargas A, Rodrı´guez Salvanes F, Espana PP, Rello J, Torres A (2004) Drug-resistant pneumococcal pneumonia: clinical relevance and related factors. Clin Infect Dis 38:787 – 798. Babinchak T, Ellis-Grosse E, Dartois N, Rose GM, Loh E (2005) The efficacy and safety of tigecycline for the treatment of complicated intraabdominal infections: analysis of pooled clinical trial data. Clin Infect Dis 41(Suppl. 5):S354 – S367. Betriu C, Redondo M, Palau ML, Sa´nchez A, Go´mez M, Culebras E, Boloix A, Picazo JJ (2000) Comparative in vitro activities of linezolid, quinupristin–dalfopristin, moxifloxacin, and trovafloxacin against erythromycin-susceptible and -resistant streptococci. Antimicrob Agents Chemother 44:1838 – 1841. ´ lvarez J, Picazo JJ Betriu C, Rodrı´guez-Avial I, Sa´nchez BA, Go´mez M, A (2002) In vitro activities of tigecycline (GAR-936) against recently isolated clinical bacteria in Spain. Antimicrob Agents Chemother 46:892 – 895. Bouchillon SK, Hoban DJ, Johnson BM, Johnson JL, Hsiung A, Dowzicky MJ (2005) In vitro activity of tigecycline against 3989 Gram-negative

444

C. Betriu et al. / Diagnostic Microbiology and Infectious Disease 56 (2006) 437 – 444

and Gram-positive clinical isolates from the United States Tigecycline Evaluation and Surveillance Trial (TEST Program; 2004). Diagn Microbiol Infect Dis 52:173 – 179. Bradford PA (2001) Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 14:933 – 951. Bradford PA, Petersen PJ, Young M, Jones CH, Tischler M, O’Connell J (2005) Tigecycline MIC testing by broth dilution requires use of fresh medium or addition of the biocatalytic oxygen-reducing reagent oxyrase to standardize the test method. Antimicrob Agents Chemother 49:3903 – 3909. Carmeli Y, Eliopoulos G, Mozaffari E, Samore M (2002) Health and economic outcomes of vancomycin-resistant enterococci. Arch Intern Med 162:2223 – 2228. Clinical and Laboratory Standards Institute (2005) Performance standards for antimicrobial susceptibility testing. Fifteenth informational supplement Document M100-S15. Wayne (PA)7 CLSI. Coelho J, Woodford N, Turton J, Livermore DM (2004) Multiresistant Acinetobacter in the UK: how big a threat? J Hosp Infect 58:167 – 169. Edlund C, Nord CE (2000) In-vitro susceptibility of anaerobic bacteria to GAR-936, a new glycylcycline. Clin Microbiol Infect 6:159 – 163. Ellis-Grosse EJ, Babinchak T, Dartois N, Rose G, Loh E (2005) The efficacy and safety of tigecycline in the treatment of skin and skinstructure infections: results of 2 double-blind phase 3 comparison studies with vancomycin–aztreonam. Clin Infect Dis 41(Suppl 5):S341 – S353. Fritsche TR, Strabala PA, Sader HS, Dowzicky MJ, Jones RN (2005) Activity of tigecycline tested against a global collection of Enterobacteriaceae, including tetracycline-resistant isolates. Diagn Microbiol Infect Dis 52:209 – 213. Gales AC, Jones RN (2000) Antimicrobial activity and spectrum of the new glycylcycline, GAR-936 tested against 1,203 recent clinical bacterial isolates. Diagn Microbiol Infect Dis 36:19 – 36. Hoban DJ, Bouchillon SK, Johnson BM, Johnson JL, Dowzicky MJ (2005) In vitro activity of tigecycline against 6792 Gram-negative and Grampositive clinical isolates from the global Tigecycline Evaluation and Surveillance Trial (TEST Program, 2004). Diagn Microbiol Infect Dis 52:215 – 227. Hope R, Warner M, Mushtaq S, Ward ME, Parsons T, Livermore DM (2005) Effect of medium type, age and aeration on the MICs of tigecycline and classical tetracyclines. J Antimicrob Chemother 56:1042 – 1046. Jacobus NV, McDermott LA, Ruthazer R, Snydman DR (2004) In vitro activities of tigecycline against the Bacteroides fragilis group. Antimicrob Agents Chemother 48:1034 – 1036.

Meagher AK, Ambrose PG, Grasela TH, Ellis-Grosse EJ (2005) Pharmacokinetic/pharmacodynamic profile for tigecycline—a new glycylcycline antimicrobial agent. Diagn Microbiol Infect Dis 52:165 – 171. Milatovic D, Schmitz FJ, Verhoef J, Fluit AC (2003) Activities of the glycylcycline tigecycline (GAR-936) against 1,924 recent European clinical bacterial isolates. Antimicrob Agents Chemother 47: 400 – 404. Moland ES, Black JA, Ourada J, Reisbig MD, Hanson ND, Thomson KS (2002) Occurrence of newer beta-lactamases in Klebsiella pneumoniae isolates from 24 U.S. hospitals. Antimicrob Agents Chemother 46:3837 – 3842. National Committee for Clinical Laboratory Standards (2003) Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically; approved standard Document M7-A6. (6th ed.). Wayne (PA)7 NCCLS. National Committee for Clinical Laboratory Standards (2004) Methods for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard Document M11-A6. (5th ed.). Wayne (Pa)7 NCCLS. National Nosocomial Infections Surveillance (NNIS) (2004) System report, data summary from January 1992 through June 2004, issued October 2004. Am J Infect Control 32:470 – 485. Peleg AY, Franklin C, Bell JM, Spelman DW (2005) Dissemination of the metallo-beta-lactamase gene blaIMP-4 among Gram-negative pathogens in a clinical setting in Australia. Clin Infect Dis 41: 1549 – 1556. Petersen PJ, Bradford PA (2005) Effect of medium age and supplementation with the biocatalytic oxygen-reducing reagent oxyrase on in vitro activities of tigecycline against recent clinical isolates. Antimicrob Agents Chemother 49:3910 – 3918. Picazo JJ, Betriu C, Rodrı´guez-Avial I, Azahares E, Sa´nchez AB (2002) Surveillance for antimicrobial resistance: VIRA study. Enferm Infecc Microbiol Clin 20:503 – 510. Picazo JJ, Betriu C, Rodrı´guez-Avial I, Culebras E, Go´mez M (2004) Surveillance of antimicrobial resistance: VIRA study 2004. Enferm Infecc Microbiol Clin 22:517 – 525. Sader HS, Jones RN, Stilwell MG, Dowzicky MJ, Fritsche TR (2005) Tigecycline activity tested against 26,474 bloodstream infection isolates: a collection from 6 continents. Diagn Microbiol Infect Dis 52:181 – 186. Schito GC (2006) The importance of the development of antibiotic resistance in Staphylococcus aureus. Clin Microbiol Infect 12(Suppl. 1): 3 – 8. Sepp7l7 H, Nissinen A, Yu Q, Huovinen P (1993) Three different phenotypes of erythromycin-resistant Streptococcus pyogenes in Finland. J Antimicrob Chemother 32:885 – 891.