1 Focus Technologies, 13665 Dulles Technology Drive, Suite 200, Herndon, VA 20171-4603; 2 Focus Technologies, Franklin, TN 37064, USA; 3 Focus Technologies, Koninginneweg 11, 1217 KP Hilversum, The Netherlands
Received 27 August 2002; returned 3 December 2002; revised 17 December 2002; accepted 20 December 2002
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Abstract |
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Keywords: daptomycin, Gram-positive pathogens, multidrug-resistant
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Introduction |
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Materials and methods |
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During 20002001, 5948 Gram-positive isolates were collected from patient specimens at 40 hospitals distributed throughout Europe. In total, 1222 S. aureus, 1040 coagulase-negative staphylococci, 1840 Enterococcus faecalis, 454 Enterococcus faecium, 160 Enterococcus species (defined as non-E. faecalis and non-E. faecium), 865 S. pneumoniae and 367 Streptococcus agalactiae were collected. Isolates were transported using Amies swabs (Technical Consultants Ltd, Lancashire, UK) to our laboratory (Focus Technologies, Herndon, VA, USA) for in vitro antimicrobial susceptibility testing. Upon receipt, isolates were subcultured on to 5% sheep blood agar and their identities confirmed. S. aureus were confirmed using the slide coagulase test; coagulase-negative staphylococci were confirmed by the tube coagulase test and Vitek (bioMérieux, Hazelwood, MO, USA). All enterococci were confirmed using the PYR test and speciated using Vitek. Enterococci with unique phenotypes (e.g. quinupristindalfopristin-resistant E. faecium) were verified through either supplemental biochemical testing or PCR using D-Ala-D-Ala ligase gene (ddl) primers specific for E. faecalis and E. faecium.10 S. pneumoniae were confirmed using optochin disc testing and S. agalactiae were confirmed by the observation of ß-haemolysis and the PathoDx Strep Grouping agglutination test (Remel, Lenexa, KS, USA).
Antimicrobial testing
All isolates were tested by broth microdilution according to NCCLS guidelines.11 Isolates were subcultured on to 5% sheep blood agar and grown overnight (2024 h). Previously frozen Sensititre microdilution panels manufactured by TREK Diagnostics (Westlake, OH, USA) were used with wells containing daptomycin supplemented to physiological levels (50 mg/L) of Ca2+. MICs were interpreted as susceptible, intermediate or resistant according to NCCLS guidelines,12 where available. S. aureus ATCC 29213, E. faecalis ATCC 29212 and S. pneumoniae ATCC 49619 were tested daily as quality control organisms.
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Results |
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Resistance to oxacillin among the 1222 isolates of S. aureus collected from the 15 countries was 27.3%. Resistance to oxacillin also resulted in cross-resistance to many other agents including ciprofloxacin, erythromycin, clindamycin and gentamicin. The activities of daptomycin and linezolid were not affected by resistance to oxacillin since the MIC90s of those agents were the same for both oxacillin-susceptible and -resistant isolates. Daptomycin and quinupristindalfopristin (MIC90 0.5 mg/L) were the most active agents against oxacillin-resistant S. aureus compared with linezolid (MIC90 2 mg/L). Among coagulase-negative staphylococci, the overall oxacillin resistance rate was even higher (53.3%) than for S. aureus. Oxacillin-resistant coagulase-negative staphylococci were also cross-resistant to many other agents such as erythromycin, gentamicin, ciprofloxacin and co-trimoxazole. For the oxacillin-resistant coagulase-negative staphylococci, quinupristindalfopristin, daptomycin and linezolid had MIC90s of 0.25, 0.5 and 2 mg/L, respectively.
Activity against enterococci
The overall rate of vancomycin resistance in E. faecium was 25.1%. However, the vancomycin-susceptible isolates were highly resistant to ampicillin and ciprofloxacin with resistance rates of 64.6% and 63.1%, respectively. Among the vancomycin-resistant E. faecium isolates, resistance to ampicillin and ciprofloxacin increased to 83.3% and 79.8%, respectively. The activity of teicoplanin was adversely affected by cross-resistance to vancomycin, as resistance increased from 0% among vancomycin-susceptible isolates to 78.1% among the vancomycin-resistant isolates. Sixteen isolates (3.5%) of E. faecium were resistant to quinupristindalfopristin; 10 of the isolates were vancomycin susceptible and six were vancomycin resistant. The activities of linezolid, quinupristindalfopristin and daptomycin were not affected by resistance to vancomycin since the MIC90s of all three agents were 2, 2 and 4 mg/L, respectively, for both vancomycin-susceptible and -resistant organisms. Among E. faecalis, the overall vancomycin resistance rate was 2.2%. Ciprofloxacin was the least effective agent against vancomycin-susceptible E. faecalis, with 521/1798 isolates (29.0%) being resistant. The vancomycin-resistant isolates of E. faecalis were also increasingly resistant to teicoplanin and ciprofloxacin with resistance rates of 85.0% and 75.0%, respectively. The activities of daptomycin and linezolid were unaffected by cross-resistance to vancomycin, and both agents demonstrated equivalent activity based on MIC90s (MIC90 2 mg/L). Amongst the other Enterococcus species, 10.6% of the isolates were resistant to vancomycin. The least effective agents were ampicillin and ciprofloxacin with resistance rates of 37.5% and 31.3%, respectively. Linezolid and daptomycin had MIC90s of 2 and 4 mg/L, respectively.
Activity against streptococci
Among the 865 isolates of S. pneumoniae tested, 619 (71.6%) were penicillin susceptible, 165 (19.1%) were penicillin intermediate and 81 (9.4%) were penicillin resistant. Penicillin resistance correlated with reduced susceptibility to the other ß-lactams (co-amoxiclav, cefuroxime, ceftriaxone), erythromycin, clindamycin and co-trimoxazole. The penicillin susceptibility status of the isolates had no effect on the activity of vancomycin, levofloxacin or the newer directed spectrum agents, daptomycin, quinupristindalfopristin and linezolid, as the MIC90 of each agent was the same for both penicillin-susceptible and -resistant isolates. Based on MIC90s, daptomycin was the most active agent against penicillin-resistant S. pneumoniae (MIC90 0.25 mg/L) compared with quinupristindalfopristin (MIC90 0.5 mg/L) and linezolid (MIC90 1 mg/L). Among S. agalactiae, clindamycin (6.5%) and erythromycin (10.4%) displayed the highest rates of resistance compared with all other agents tested. The ß-lactam agents (penicillin, co-amoxiclav, cefuroxime, ceftriaxone) were very active, with MIC90s of 0.12 mg/L. Daptomycin and quinupristindalfopristin demonstrated equally potent activities against S. agalactiae (MIC90s 0.25 mg/L) compared with linezolid (MIC90 1 mg/L).
Prevalence of MDR
Table 2 shows the prevalence of multidrug-resistant (MDR; defined as concurrent resistance to three or more agents from different antimicrobial classes) phenotypes in S. aureus, E. faecium and S. pneumoniae collected in this study. Among S. aureus isolates, the most prevalent MDR phenotype included resistance to ciprofloxacin, erythromycin and oxacillin, and was demonstrated by 10.9% of the isolates tested. In addition, 10.1% of S. aureus isolates were also resistant to four agents, and 2.0% of isolates were resistant to five agents (ciprofloxacin, erythromycin, gentamicin, oxacillin, co-trimoxazole). Among the 454 isolates of E. faecium tested, 89 (19.6%) were MDR, with the most prevalent phenotype being resistance to ampicillin, ciprofloxacin and vancomycin. In S. pneumoniae, resistance to penicillin, erythromycin and co-trimoxazole was the most common MDR phenotype and was present in 3.4% of the isolates. The results in Figure 1 show that there were no major differences in the MIC distributions (modal MICs one-doubling dilution) for daptomycin against MDR and non-MDR isolates of S. aureus, E. faecalis, E. faecium and S. pneumoniae.
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The numbers of resistant isolates submitted by each European country are shown in Table 3. Oxacillin-resistant S. aureus, vancomycin-resistant enterococci and penicillin-resistant S. pneumoniae were prominent among isolates collected from most of the European countries. However, there were significant differences in the numbers of resistant isolates collected from the various countries that were used to profile the activity of daptomycin. Among S. aureus, 4.0% of the isolates collected were resistant to oxacillin in the Netherlands compared with 36.9% of the isolates from Portugal. The percentage of E. faecium isolates resistant to vancomycin ranged from 0% in Switzerland and Scandinavia (Sweden, Denmark and Finland) to 60.6% in Italy. Among S. pneumoniae, no penicillin-resistant isolates were collected from the Netherlands compared with 20.7% of isolates from Portugal. There is also considerable countrywide variation in MDR phenotypes for the different Gram-positive organisms. For example, MDR S. aureus ranges from 2.0% in the Netherlands to 36.7% in Ireland. There was little or no variation in the activity of daptomycin (based on MIC ranges and MIC90s) regardless of phenotype, national origin of isolates or varying numbers of resistant isolates per country.
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Discussion |
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The in vitro antimicrobial activity of daptomycin is well known to be dependent on the physiological concentration of calcium in the test medium.14,2023 Many of the early studies to evaluate the activity of daptomycin, including one for proposed staphylococcal breakpoints,19 were conducted in cation-supplemented MuellerHinton broth (CSMHB) adjusted to physiological levels of Ca2+ (50 mg/L). The current medium recommended by the NCCLS is cation-adjusted MuellerHinton broth (CAMHB), which contains 2025 mg/L of Ca2+. Daptomycin is typically two- to four-fold more active when tested in media supplemented to contain the physiologically relevant concentration of calcium ions. The importance of the calcium content on testing the activity of daptomycin is also proving important in proposing breakpoints that are likely to be recommended by various agencies in Europe. A recent study conducted in the UK, testing the activity of daptomycin against 328 recent clinical isolates, showed that daptomycin had an MIC90 of 1 mg/L for staphylococci and streptococci, and an MIC90 of 2 mg/L for the enterococci.24 The effect of calcium concentration on daptomycin activity was highlighted and a tentative breakpoint of 2 mg/L for staphylococci, streptococci and enterococci was proposed using the methods of the British Society for Antimicrobial Chemotherapy (BSAC). However, it is clear that the results from the clinical trials will be important in establishing the final breakpoints that are adopted, especially for organisms such as E. faecium, for which the MIC90 of daptomycin was 4 mg/L.
Since many European countries use different susceptibility testing methods and interpretative criteria, it is often difficult to assess or compare resistance rates from studies generated and reported in different countries.25 In this study, all isolates collected in each country were tested at a central laboratory using NCCLS-recommended methodologies to compare resistance in each country. A few surveillance studies have focused on antimicrobial resistance in Gram-positive cocci in countries in Europe. Many of the European studies that have been conducted have investigated enterococcal resistance to the glycopeptides vancomycin and teicoplanin.2628 The results of this study confirm previous observations showing that resistance to vancomycin and teicoplanin is observed more frequently among E. faecium than among E. faecalis. For S. pneumoniae, penicillin resistance varies considerably within different countries in Europe. A surveillance study conducted in 19971998 showed a high prevalence of penicillin-resistant S. pneumoniae in Spain and France,29 and this was also true for the isolates collected in this study conducted in 20002001. There is also very little data on the prevalence of MDR Gram-positive bacteria in Europe. However, reports are now beginning to appear on multi-resistant S. pneumoniae in the USA and suggest its prevalence is increasing.30,31 There is a need to monitor antimicrobial resistance in Europe since increased travel and trade within the European Union make it more likely that the dissemination of resistant pathogens will increase. This is already apparent for methicillin-resistant S. aureus, which was once primarily a nosocomial pathogen and is now being detected in the community setting.32
The results of this study show that there is significant resistance to selected agents in many clinically important Gram-positive pathogens in many of the European countries studied. Even more alarming is the fact that resistance is not restricted to a single agent but may involve resistance to multiple agents. The new directed spectrum agents are effective against clinically important pathogens. As expected, these agents are also active against organisms that are multidrug resistant, since their use is currently not widespread.33 The ability of the newer directed spectrum agents to circumvent multiple resistance mechanisms is due to their novel targets and mechanisms of action, which it is to be hoped will provide these agents with a long period of therapeutic utility. For example, the ability of daptomycin to circumvent existing resistance mechanisms is due to its novel mode of action targeting the function of the bacterial plasma membrane.5,34 Unfortunately, the evolutionary ability of microbes to develop resistance mechanisms to counteract novel pharmacophores will always exist. The results of this study have already shown that there were 16 isolates of E. faecium from seven countries (Austria, Belgium, France, Germany, Greece, Portugal and Spain) that were resistant to quinupristindalfopristin. Linezolid-resistant isolates were not detected in this study; however, there have been reports on linezolid-resistant isolates of S. aureus and E. faecium in the USA3537 and recently the UK.38 In vitro studies to generate spontaneous daptomycin-resistant mutants of S. aureus, S. epidermidis, E. faecalis and E. faecium have proved unsuccessful.34 Serial passage of S. aureus in the presence of daptomycin did generate resistant mutants that had daptomycin MICs that were 8- to 32-fold higher than the parent strain; however, many resistant mutants had significant growth defects, alteration of membrane potential and reduced virulence.
Daptomycin was highly active against both MDR and non-MDR isolates of S. aureus, E. faecium and S. pneumoniae. This study confirms that resistance, including multidrug resistance, is prevalent in Gram-positive pathogens in many countries in Europe during 20002001. The results of the study will be important in benchmarking the antimicrobial activity of daptomycin against European isolates and will serve as a baseline for future studies to monitor changes in the susceptibility of these organisms to daptomycin following its approval and clinical use in European countries.
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Acknowledgements |
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Footnotes |
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