Multicentre evaluation of the in vitro activity of linezolid in the Western Pacific

Jan M. Bell1,*, John D. Turnidge1, Charles H. Ballow2, Ronald N. Jones3,4,§ and the ZAPS Regional Participants

1 Department of Microbiology and Infectious Diseases, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, SA 5006, Australia; 2 Buffalo Clinical Research Center, Buffalo, NY; 3 The Jones Group/JMI Laboratories, North Liberty, IA; 4 Tufts University School of Medicine, Boston, MA, USA

Received 28 July 2002; returned 12 September 2002; revised 31 October 2002; accepted 4 November 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multiresistance to antimicrobial agents is common in staphylococci and pneumococci isolates in the Western Pacific region. The activity of linezolid, a new oxazolidinone, was evaluated against a spectrum of Gram-positive species collected in the region. Eighteen laboratories from six countries in the Western Pacific examined the linezolid susceptibility of 2143 clinical isolates of Staphylococcus aureus, coagulase-negative staphylococci (CoNS) and Enterococcus spp. using broth microdilution or disc diffusion methodology. For Streptococcus pneumoniae (n = 351) and other streptococci (n = 83), Etest (AB Biodisk, Solna, Sweden) strips were used. Results were compared with other common and important antimicrobials. Linezolid-resistant strains were not detected among streptococci or staphylococci, including a significant proportion of S. aureus strains that were multiresistant. Almost all enterococci, including 14 vancomycin-resistant Enterococcus faecium, were linezolid susceptible. A small proportion of enterococci (0.8%) were intermediate to linezolid, and one strain of Enterococcus faecalis had a zone diameter of 20 mm (resistant). The linezolid MIC ranges (MIC90) of those strains tested by broth microdilution or Etest were: 1–4 mg/L (2 mg/L) for S. aureus, 0.5–4 mg/L (2 mg/L) for CoNS, 0.5–4 mg/L (2 mg/L) for Enterococcus spp., 0.12–2 mg/L (1 mg/L) for S. pneumoniae and 0.25–2 mg/L (1 mg/L) for Streptococcus spp. There was no difference in linezolid susceptibility between countries or between multiresistant and susceptible strains of each species monitored.

Keywords: linezolid, Western Pacific region, ZAPS


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gram-positive bacteria are important causes of both community- and hospital-acquired infections. Most countries in the Western Pacific region have reported significantly elevated rates of methicillin (oxacillin)-resistant Staphylococcus aureus1 and penicillin-resistant Streptococcus pneumoniae.2,3 Vancomycin-resistant Enterococcus spp. and vancomycin-intermediate Staphylococcus spp. have also emerged in some countries in the region, particularly in Australia4 and Japan,5 respectively. The emergence of these resistances has focused attention on both older and newer agents as alternative therapeutic regimens.

Linezolid is the first of a novel antimicrobial class, the oxazolidinones, which have a unique mechanism of action.6 Oxazolidinones affect bacterial protein synthesis at the ribosomal level by binding to a specific target on the 50S subunit of the ribosome and inhibiting the formation of the initiation complex. This new class of antimicrobial agents has very promising activity against multiply resistant Gram-positive organisms.7,8 It has been approved in the USA, Australia and some European countries for the treatment of serious Gram-positive infections, including those caused by strains resistant to multiple other antimicrobials. Linezolid also has the advantage of being available in both oral and intravenous forms.9

As part of the ZAPS (Zyvox Antimicrobial Potency Study) programme worldwide,10 the activity of linezolid, a new oxazolidinone, was evaluated against a range of clinically important Gram-positive species isolated in clinical laboratories across the Western Pacific region.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Participating laboratories

Eighteen hospital-based laboratories from six countries in the Western Pacific region participated in the study: Australia (six sites), China (Hong Kong; two sites), Korea (three sites), Japan (three sites), Singapore (two sites) and Taiwan (three sites).

Isolates

Each laboratory was required to collect prospectively, for up to 6 months in the years 1999–2000, isolates from wounds and soft tissue, abdominal cavity, bone or joint specimens, upper respiratory sinuses, middle ear, lower respiratory tract, body cavities, cerebrospinal fluid and blood cultures. The following species were collected at each site: S. aureus (oxacillin resistant, n = 20; oxacillin susceptible, n = 30); coagulase-negative Staphylococcus spp. (CoNS; oxacillin resistant, n = 20; oxacillin susceptible, n = 15); Enterococcus faecalis (n = 30), Enterococcus faecium (n = 10) or Enterococcus spp. unspecified (n = 40); S. pneumoniae (n = 20); and other Streptococcus spp. (n = 5). No repeat isolates were allowed from the same patient.

Susceptibility testing

All Streptococcus species, including S. pneumoniae, were tested by stable gradient diffusion (Etest) against ceftriaxone, erythromycin, linezolid, penicillin, quinupristin–dalfopristin and trovafloxacin. Staphylococci and enterococci were tested by either broth microdilution (Microscan; Dade Behring, Sacramento, CA, USA) or disc diffusion using NCCLS methods11,12 against ampicillin, cefazolin, ceftriaxone, chloramphenicol, clindamycin, doxycycline, erythromycin, linezolid, oxacillin, quinupristin–dalfopristin, teicoplanin, trovafloxacin and vancomycin. NCCLS breakpoint criteria (M100-S12, 2002)13 were used to define susceptibility categories for all strains, where available.

Confirmatory testing

All isolates with possible resistance to linezolid (MIC > 4 mg/L) or zone diameters of <=20 mm were referred to the regional coordinating laboratory (Women’s and Children’s Hospital) for confirmation of the organism identification and susceptibility test result. Strains of Streptococcus or Staphylococcus spp. with suspected resistance to vancomycin were also sent to the coordinating laboratory for confirmation. Streptococci with a trovafloxacin MIC > 1 mg/L, any species (except E. faecalis) with a quinupristin–dalfopristin MIC > 1 mg/L (<=18 mm), staphylococci with a vancomycin MIC >= 4 mg/L (<=14 mm) or teicoplanin MIC > 8 mg/L (<=10 mm), or enterococci with vancomycin MIC > 4 mg/L (<=14 mm) or teicoplanin MIC > 8 mg/L (<=10 mm) were also referred to the coordinating laboratory for confirmation. All referred staphylococci were tested for the presence of mecA and nuc genes by multiplex PCR.14 Enterococci were identified and vancomycin genotype determined using PCR techniques.15 All referred isolates were tested by both susceptibility test methods.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Results for all antimicrobials against individual species are listed in Tables 14. Overall, nearly 2600 strains were examined. No resistance (non-susceptibility) to linezolid was detected in 351 S. pneumoniae or 83 Streptococcus spp., 884 S. aureus strains or 629 CoNS. Nearly all enterococci were susceptible. Eleven of 481 E. faecalis (2.3%) and one of 149 E. faecium (0.7%) had intermediate susceptibility to linezolid (MIC 4 mg/L or zone diameter 21–22 mm). One E. faecalis strain from Hong Kong was resistant (zone diameter 20 mm). Significant strains against which linezolid was active (MIC <= 4 mg/L; zones >= 21 mm) included all 339 oxacillin-resistant S. aureus, the majority of which were multiresistant (90% in all countries except Australia, where 64% were resistant to two or more non-ß-lactam drug classes) and all 14 vancomycin-resistant E. faecium (three from Australia and 11 from Korea). Resistance to quinupristin–dalfopristin was also not detected in any species other than the naturally resistant non-E. faecium enterococcal species; however, strains with intermediate susceptibility were identified.


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Table 1.  Resistance rates of six antimicrobials tested against 351 isolates of S. pneumoniae from the Western Pacific region
 

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Table 4.  Antimicrobial susceptibility patterns for E. faecalis and E. faecium isolates (n = 630) tested against eight antimicrobials (Western Pacific region, 1999–2000)
 
Linezolid activity against streptococci

Table 1 lists the susceptibility patterns of 351 pneumococci from six countries. For the Western Pacific region, only 46.7% of isolates were penicillin susceptible (MIC <= 0.06 mg/L) and high-level resistance was observed in 16.2% of pneumococci. A wide susceptibility variation was noted between nations with a range of susceptibility from 12% (Taiwan) and 18% (Korea) to 78% (Australia). Similarly, the macrolide resistance rates paralleled the penicillin pattern, with >=80% resistance found in three countries (China, Korea and Taiwan), but only 20% in Australia. Resistance to ceftriaxone (1.7%), quinupristin–dalfopristin (0.0%) and trovafloxacin (0.0%) was very rare. Linezolid was active against all S. pneumoniae strains at <=2 mg/L and against 99.4% of these isolates at <=1 mg/L.

The 83 strains of other streptococci were also very susceptible to linezolid (Table 2) with a MIC range of 0.25–2 mg/L and an MIC90 of 1 mg/L. Only 7.2% of these Streptococcus spp. isolates were not susceptible to penicillin, and the erythromycin resistance was 4–24% depending on the nation sampled (data not shown).


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Table 2.  Distribution of linezolid MIC values tested against 1048 Gram-positive coccia
 
Linezolid activity against staphylococci

Table 3 summarizes the in vitro activity of linezolid and nine comparison agents tested against 1520 isolates of staphylococci. Among the oxacillin-susceptible S. aureus, the most common resistance pattern was erythromycin resistance usually without concurrent clindamycin resistance (so-called M-phenotype). The exception was the isolates from Taiwan, where most (59–73%) macrolide-resistant isolates were co-resistant to clindamycin and chloramphenicol. In contrast, oxacillin-resistant S. aureus were often resistant to erythromycin (82–100%), clindamycin (54–87%) and the tetracyclines (8–87%). As observed with oxacillin-susceptible strains, the oxacillin-resistant S. aureus were more likely to be resistant to chloramphenicol in Taiwan. Linezolid, quinupristin–dalfopristin and the two tested glycopeptides inhibited all S. aureus strains; no so-called VISA strains were discovered.5


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Table 3.  Antimicrobial susceptibility patterns for 10 agents tested by two methods against 1520 isolates of staphylococci from the Western Pacific region
 
The oxacillin-susceptible CoNS (224 strains) were most frequently resistant to erythromycin (19.2% overall; range 12–26%). Resistance to other agents was most often observed for chloramphenicol (6.7%), clindamycin (5.4%) and doxycycline (6.7%). Constitutive MLSB resistance (that is, resistance to both erythromycin and clindamycin) was discovered in more than one-half of isolates in Japan; all other nations were dominated by the M-phenotype. Greater levels of resistance were common among oxacillin-resistant CoNS (Table 3; 405 strains). Two- to 10-fold higher rates of resistance were documented for erythromycin (70.1%), clindamycin (39.8%), chloramphenicol (17.8%) and doxycycline (14.8%) compared with oxacillin-susceptible CoNS. Trovafloxacin was also less active and two Staphylococcus haemolyticus strains with elevated teicoplanin MICs were confirmed from Australia.

Linezolid, quinupristin–dalfopristin and vancomycin were active against all CoNS isolates and the activity was not influenced by resistance to oxacillin or other monitored agents (Table 3). The linezolid MIC range for the staphylococci was 0.5–4 mg/L with an MIC50 and MIC90 result of 2 mg/L for both S. aureus and CoNS (Table 2).

Linezolid activity against enterococci

Table 4 shows the activity of linezolid tested against 630 isolates of E. faecalis and E. faecium from the Western Pacific region. Resistance to glycopeptides was not observed among the E. faecalis isolates, but 14 vancomycin-resistant E. faecium were confirmed from Australia [three strains (7%); both a vanB pattern] and Korea [11 strains (27%); nine with a vanA phenotype]. Ampicillin was effective in vitro against E. faecalis (98.3% susceptible), but not against E. faecium (17.4% susceptible). Quinupristin–dalfopristin was active only against E. faecium; however, 9.6% of vancomycin-susceptible isolates had intermediate in vitro testing results.

The activity of other alternative agents against E. faecium or E. faecalis included chloramphenicol (8.7% and 26.0% resistance, respectively), doxycycline (22.4% and 29.5% resistance, respectively) and new fluoroquinolones such as trovafloxacin (15.8% and 50.3% resistance, respectively). All E. faecium tested were susceptible to linezolid and only one isolate of E. faecalis was resistant (equates to 0.2% resistance). This isolate had a zone diameter of 20 mm, being only 1 mm below the NCCLS breakpoint.13 Only 0.8% of enterococci had an intermediate result for linezolid, all were E. faecalis strains. The range of linezolid MICs among the enterococci was 0.5–4 mg/L (MIC90 2 mg/L; Table 2).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As the oxazolidinones represent an entirely new class of antimicrobials,9 and this study was conducted before registration and marketing of linezolid anywhere in the world, it is not surprising to find that there was complete (100%) susceptibility among staphylococci and streptococci and ~99% susceptibility among the enterococci. It confirms the lack of cross-resistance with other classes of drugs, including those that work at the level of the ribosome.16 Similar findings to those presented here have been reported in other geographic regions including North America,10,17 Latin America18 and Europe.1921

Enterococci were largely susceptible as well, although a few strains of enterococci fell into the intermediate range. This was not unexpected and was understood at the time that the NCCLS breakpoints for enterococci were established.13

The study provides a valuable baseline for linezolid for the Western Pacific region before its clinical usage. It confirms the potency of linezolid against the major community and nosocomial Gram-positive pathogens, including activity against strains that have become resistant to other oral or parenteral agents. Oxacillin-resistant S. aureus is a particular problem in many countries in the Western Pacific region,1 and the availability of an agent that can be administered for serious infections to in-patients, or for ongoing outpatient therapy orally, represents a major advance in the management of infections caused by this pathogen and glycopeptide refractory species.4,5,9


    Acknowledgements
 
This study was supported by an educational/research grant from Pharmacia and Upjohn (Kalamazoo MI, USA). ZAPS Western Pacific Regional Participants are as follows: Australia: J. Turnidge (Women’s and Children’s Hospital, Adelaide); R. Benn (Royal Prince Alfred Hospital, Sydney); L. Grayson (Monash Medical Centre, Melbourne); J. Faoagali (Royal Brisbane Hospital, Brisbane); K. Christiansen (Royal Perth Hospital, Perth). China: A. Cheng (Prince of Wales Hospital, Hong Kong); D. Tsang (Queen Elizabeth Hospital, Hong Kong). Japan: S. Kohno (Nagasaki University School of Medicine, Nagasaki); K. Yamaguchi (Toho University, Tokyo); M. Kahu (Tohoku University, Aoba-ku Sendai). Korea: J. H. Woo (University of Ulsan, Seoul); K. W. Choi (Seoul National University Hospital, Seoul); K. W. Lee (Yonsei University, Seoul). Singapore: M. Yeo (Singapore General Hospital); G. Kumarasinghe (National University Hospital). Taiwan: Po-Ren Hsueh (National Taiwan University Hospital, Taipei); Y. C. Liu (Veterans General Hospital, Kaohsiung); H. S. Leu (Chang-Gung Memorial Hospital, Taoyuan).


    Footnotes
 
* Corresponding author. Tel: +61-8-8161-6359; Fax: +61-8-8161-6051; E-mail: bellj{at}mail.wch.sa.gov.au Back

§ ZAPS Western Pacific Regional Participants are listed in the Acknowledgements. Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Bell, J. M., Turnidge, J. D. & the SENTRY APAC participants. (2002). High prevalence of oxacillin-resistant Staphylococcus aureus from hospitalised patients in the Asia-Pacific and South Africa: results from the SENTRY Antimicrobial Surveillance Program, 1998–1999. Antimicrobial Agents and Chemotherapy 46, 879–81.[Abstract/Free Full Text]

2 . Song, J. H., Lee, N. Y., Ichiyama, S., Yoshida, R., Hirakata, Y., Fu, W. et al. (1999). Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study. Clinical Infectious Diseases 28, 1206–11.[ISI][Medline]

3 . Turnidge, J. D., Bell, J. M. & Collignon, P. J. (1999). Rapidly emerging antimicrobial resistances in Streptococcus pneumoniae in Australia. Pneumococcal Study Group. Medical Journal of Australia 170, 152–5.[ISI][Medline]

4 . Bell, J., Turnidge, J., Coombs, G. & O’Brien, F. (1998). Emergence and epidemiology of vancomycin-resistant enterococci in Australia. Communicable Diseases Intelligence 22, 249–52.[Medline]

5 . Hiramatsu, K., Hanaki, H., Ino, T., Yabatu, K., Oguri, T. & Tenover, F. C. (1997). Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy 40, 135–6.[Free Full Text]

6 . Swaney, S. M., Aoki, H., Ganoza, M. C. & Shinabarger, D. L. (1998). The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrobial Agents and Chemotherapy 42, 3251–5.[Abstract/Free Full Text]

7 . Jones, R. N., Johnson, D. M. & Erwin, M. E. (1996). In vitro antimicrobial activities and spectra of U-100592 and U-100766, two novel fluorinated oxazolidinones. Antimicrobial Agents and Chemotherapy 40, 720–6.[Abstract]

8 . Rybak, M. J., Cappelletty, D. M., Moldovan, T., Aeschlimann, J. R. & Kaatz, G. W. (1998). Comparative in vitro activities and postantibiotic effects of the oxazolidinone compounds eperezolid (PNU-100592) and linezolid (PNU-100766) versus vancomycin against Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, and Enterococcus faecium. Antimicrobial Agents and Chemotherapy 42, 721–4.[Abstract/Free Full Text]

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10 . Jones, R. N., Ballow, C. H. & Biedenbach, D. H. (2001). Multi-laboratory assessment of the linezolid spectrum using the Kirby–Bauer disk diffusion method. Report of the Zyvox Antimicrobial Potency Study (ZAPS) in the United States. Diagnostic Microbiology and Infectious Disease 40, 59–66.[CrossRef][ISI][Medline]

11 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.

12 . National Committee for Clinical Laboratory Standards. (2000). Performance Standards for Antimicrobial Disk Susceptibility Tests: Approved Standard M2-A7. NCCLS, Wayne, PA, USA.

13 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing—Twelfth Informational Supplement: M100-S12. NCCLS, Wayne, PA, USA.

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20 . Henwood, C. J., Livermore, D. M., Johnson, A. P., James, D., Warner, M., Gardiner, A. et al. (2000). Susceptibility of Gram-positive cocci from 25 UK hospitals to antimicrobial agents including linezolid. Journal of Antimicrobial Chemotherapy 46, 931–40.[Abstract/Free Full Text]

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