a Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242; b Schering-Plough Research Institute, Kenilworth, NJ 07033, USA
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Abstract |
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Introduction |
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Clearly, new antimicrobials or modifications of existing compounds, are needed to treat the growing number of resistant Gram-positive pathogens effectively. The everninomicins are oligosaccharide antimicrobials, isolated from Micromonospora carbonacea.1113 The structure and stereochemistry of this class of antimicrobial have been studied extensively.1416 Evernimicin (SCH 27899), a novel oligosaccharide analogue of the everninomicin class, displays activity against a wide range of Gram-positive pathogens, including vancomycin-resistant enterococci (VRE), staphylococci and penicillin-resistant pneumococci.1721
In this era of rapidly changing antimicrobial susceptibilities, it is important to build a baseline database of sufficient size and geographical scope, early in the development of a new antimicrobial class.22 Future surveillance work can then, with confidence, be compared with early benchmark susceptibility test results. The purpose of the present study was to collect a large series of recent Gram-positive isolates and compare the antimicrobial activity of evernimicin against these isolates with those of several commonly used antimicrobial agents using the Etest (AB Biodisk, Solna, Sweden) method, a stable gradient MIC technology.23
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Materials and methods |
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Organisms tested
Where possible, each participating laboratory tested 50 isolates in each of four groups of organisms: (i) methicillin- or oxacillin-resistant S. aureus/coagulase-negative staphylococci (MRSA/MR-CoNS); (ii) enterococci; (iii) Streptococcus pneumoniae; and (iv) various non-pneumococcal streptococci. The latter group included a variety of streptococci considered of clinical importance, including ß-haemolytic streptococci, viridans group streptococci, Streptococcus bovis and Streptococcus milleri.
Organisms were identified to the species level by the routine methods available within each laboratory. Where species identification was not possible, the isolates were recorded as non-speciated isolates of the identified genus.
Generally, 50 recent clinical isolates in each group were collected and tested by each centre. In certain centres where collection of a particular group of organisms was problematic (as for MRSA in Scandinavia), participating laboratories were allowed to submit additional isolates from the three other groups of organisms.
Susceptibility testing methodology
The Etest allows a determination of MIC based on the inhibition created when a plastic carrier strip impregnated with a gradient antimicrobial concentration is placed on an agar plate inoculated with a particular organism and incubated overnight.23 The MIC is defined as the point where the zone of inhibition intersects the numerical scale (15 log2 dilution steps) on the edge of the strip. For the study drugs whose MICs were to be determined, the Etest strip method was used. The 80% inhibition endpoint was used for evernimicin as recommended by the manufacturer. The following antimicrobial agents were tested using Etest strips: ampicillin, ceftriaxone, chloramphenicol, ciprofloxacin, quinupristin/dalfopristin, erythromycin, oxacillin, penicillin G, evernimicin and vancomycin. Additional diffusion discs (BD Microbiology Systems, Cockeysville, MD, USA) were placed on the 150 mm agar plates to aid in the characterization of some clinical isolates. The following antimicrobial agents were tested using conventional discs: ceftizoxime (30 µg), clindamycin (2 µg), gentamicin (120 µg) and streptomycin (300 µg). The presence of an oxacillin Etest strip on the staphylococcal plate, along with the ceftizoxime disc, permitted greater accuracy in the identification of MRSA and MR-CoNS in the absence of salt-containing agar.24 A staphylococcal isolate was considered methicillin resistant if it was classified as intermediate or resistant' by either method.
The use of high-concentration gentamicin and streptomycin discs on the enterococcus test plate allowed the identification of high-level aminoglycoside resistance (synergy testing) among the enterococci. National Committee for Clinical Laboratory Standards (NCCLS) guidelines24,25 served as the method and interpretive criteria for each antimicrobial tested. The susceptibility criteria used for evernimicin were as follows: 4 mg/L, susceptible; 6 or 8 mg/L, intermediate; and
12 mg/L (
16 mg/L, rounded), resistant. These are proposed criteria and await regulatory approval.
Quality control
Each laboratory was provided with a set of study and manufacturer instructions for the Etest susceptibility method. To provide uniformity of testing, each laboratory was also asked to provide three replicates of MIC data on supplied quality control organisms (Enterococcus faecalis ATCC 29212, S. aureus ATCC 29213, and S. pneumoniae ATCC 49619.18,19,24 North American sites were additionally asked to provide data on seven challenge organisms (two staphylococci, three enterococci and two streptococci) chosen specifically to highlight potential susceptibility categorization problems. To pass the quality control phase of the study and proceed to the collection and testing of clinical isolates, each laboratory had to score 90% for quantitative and/or susceptibility categorical accuracy.
For each laboratory, if the geometric mean MIC result for a particular group of organisms was four-fold, or more, greater or less than the geometric mean of the total study population, the entire group was excluded from the final analysis. Furthermore, laboratories were asked to send isolates with evernimicin MICs above previously specified levels (MRSA/MR-CoNS, 1 mg/L; enterococci and non-pneumococcal streptococci, 0.5 mg/L; and S. pneumoniae, 0.25 mg/L) to a central reference laboratory [University of Iowa College of Medicine (UICOM), Iowa City, IA, USA] for confirmation of susceptibility and further characterization. Reference laboratory values were substituted in the analysis of data where strains were repeated.
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Results |
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Quality control and validation of organisms
Data from three laboratories were eliminated from the survey because they failed to reach the 90% quantitative accuracy threshold18,19,24 specified in the study protocol using ATCC control strains. Data from two laboratories (a single group of organisms each) were also excluded from the final analysis because the geometric mean MIC was either four times higher (MRSA/MR-CoNS) or four times lower (MRSA/MR-CoNS) than the all-sites geometric mean MIC for that group of organisms. Data from two other laboratories approached, but did not reach, these exclusion values. When compared with the geometric mean MIC for the group, 17 of the remaining 30 laboratories had higher geometric means and 13 had lower geometric mean results. A review of the non-pneumococcal streptococci results by the reference laboratory (UICOM) led to the elimination of 80 isolates, each of whose susceptibility pattern was not consistent with the strain as it was identified (each clearly an enterococcus).
Forty-four of 52 isolates (84.6%), originally determined to have MICs above the evernimicin MIC values specified previously, were forwarded for retesting of susceptibility by UICOM. All except one were found to be fully susceptible to evernimicin (MIC 1 mg/L). The exception was an isolate of S. bovis against which the MIC of evernimicin was consistently 1.5 mg/L. Evernimicin MICs against two strains of MRSA, when retested, were 1 mg/L. In most cases, the discrepancy seems to have arisen as a result of difficulty in reading the 80% inhibition breakpoint for haemolytic isolates. All of the remaining isolates having values greater than specified MICs could not be confirmed and were eliminated from the analysis.
Of the 44 strains for which repeat MICs were performed, 25 were isolated in North America and 19 were from Europe and South Africa. These isolates consisted of 22 MRSA/MR-CoNS, 16 enterococci and six streptococci.
Susceptibility of MRSA and MR-CoNS to evernimicin
MIC50 and MIC90 values (concentrations inhibiting 50 and 90% of tested strains, respectively) for MRSA and MR-CoNS for all tested antimicrobial agents are displayed in Table I. Not all laboratories identified staphylococcal isolates to the species level. The MR-CoNS group included seven major species or groups: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Staphylococcus warnerii, Staphylococcus spp. and CoNS. Methicillin resistance was assessed by determining susceptibility to oxacillin and ceftizoxime and then analysing three subsets of isolates: (i) those susceptible to both oxacillin and ceftizoxime (dropped from the analysis); (ii) those resistant to both agents; and (iii) those intermediate or resistant to either cited ß-lactam alone.
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Examining the susceptibility of MRSA and MR-CoNS isolates according to the geographical distribution of study centres showed that resistance to quinupristin/dalfopristin in Scandinavia and North America was greater than that in western or eastern Europe (Table II). Resistance to erythromycin was widespread throughout the study centres, although noticeably less in Scandinavia. High susceptibility to evernimicin was maintained across all regions.
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MIC50 and MIC90 values were 1.0 mg/L and consistently lower for evernimicin than for all other tested agents against unspeciated enterococcus isolates, Enterococcus faecium and E. faecalis (Table III). A susceptibility rate of 100% for evernimicin was recorded for all three groups. Whereas the Enterococcus spp. group and E. faecalis were both highly susceptible to vancomycin and ampicillin, susceptibility rates were markedly reduced for E. faecium (59.5 and 17.3%, respectively) indicating that this species has developed widespread resistance. Of the antimicrobials tested, the quinupristin/dalfopristin combination produced the lowest susceptibility rates for all three enterococcus groups tested, with E. faecalis being almost totally resistant to this newer agent.
Ampicillin-resistant, quinupristin/dalfopristin-susceptible enterococci (E. faecium) were analysed according to their vancomycin-susceptibility phenotype (data not shown). This purer population of highly probable E. faecium isolates26 was fully susceptible to evernimicin, with MIC50 and MIC90 below 0.5 mg/L, regardless of glycopeptide susceptibility pattern.
Table II lists the worldwide patterns of resistance in isolates of Enterococcus spp. to the antimicrobials tested in this survey. There was uniform reporting of high resistance rates to quinupristin/dalfopristin throughout Europe and North America (10.314.4% susceptibility overall; all among E. faecium isolates). Significant variations in the pattern of susceptibility to chloramphenicol were noted for eastern European isolates, where only 42.5% of isolates were susceptible (57.365.4% for other regions).
There were very few reports of vancomycin-resistant enterococci (13 isolates) from centres in Europe (six nations) and South Africa. In contrast, each centre in the USA reported at least one VRE isolate (111 strains altogether). Of the North American VRE strains, 86.5% were E. faecium and the remaining strains were unspeciated. Only one vancomycin-resistant E. faecalis strain was documented from a medical centre in Russia. Evernimicin was highly active (MIC90 0.751.0 mg/L) against enterococci throughout all geographical regions; no resistant strains were reported.
Susceptibility of S. pneumoniae to evernimicin
S. pneumoniae isolates were divided for analyses according to their susceptibility to penicillin (Table IV). In total, only 66.9% of S. pneumoniae isolates tested were susceptible to penicillin in this worldwide random sample. Patterns of resistance to penicillin did not affect the susceptibility of S. pneumoniae to evernimicin, quinupristin/dalfopristin or vancomycin. For evernimicin, the MIC50 and MIC90 were <0.1 mg/L in all groups. Penicillin-intermediate and -resistant isolates of S. pneumoniae also had marked cross-resistance to erythromycin, with only 54.9 and 35.0% of isolates, respectively, susceptible. Ceftriaxone retained some activity against penicillin-intermediate strains of S. pneumoniae (67.3% susceptible) but had virtually no activity against penicillin-resistant strains (4.3%).
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Penicillin non-susceptible strains were more prevalent in North America (39.6%) and among the strains reported from one site in Scandinavia (34.0%). All other geographical areas had <30% penicillin-intermediate or -resistant isolates combined.
Evernimicin activity against non-pneumococcal streptococci of clinical significance
In total, 1394 strains of streptococci (other than S. pneumoniae) that were of clinical significance were isolated during this study (Table IV). The distribution of species was as follows: Streptococcus agalactiae (serogroup B; 379 strains); Streptococcus pyogenes (serogroup A; 455 strains); other ß-haemolytic streptococci (serogroup C; 41 strains); serogroup F (nine strains); serogroup G (64 strains); other non-specified ß-haemolytic streptococci (nine strains) and S. bovis (19 strains). Remaining streptococci were classified in the viridans group.
Bacterial isolates within this non-pneumococcal streptococci group were highly susceptible to evernimicin, regardless of their susceptibility to penicillin. The evernimicin MIC50 and MIC90 were 0.5 mg/L in all tabulated groups. All other antimicrobials showed lower in vitro potency against penicillin-intermediate or -resistant strains, with the quinupristin/dalfopristin results heavily and negatively influenced by the resistances among S. bovis strains.
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Discussion |
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This global study was designed to assess the in vitro antimicrobial activity of evernimicin against Gram-positive pathogens. The results were compared with those of other commonly used antimicrobial agents, including vancomycin, and a new streptogramin combination, quinupristin/ dalfopristin.10,26,28,29 The worldwide distribution of the survey centres in this surveillance programme also permitted analysis of global patterns of resistance to the studied compounds.
MRSA and MR-CoNS are an increasing therapeutic problem in both the USA17,26 and Europe.2,58,30 The rates of susceptibility to evernimicin among both MRSA and MR-CoNS were excellent during this study throughout both cited regions, with MIC90s 1.0 mg/L for each species group. While the activity of quinupristin/dalfopristin against MRSA was also good, with results consistent with those published previously,26 the frequency of strains with quinupristin/dalfopristin MICs > 1 mg/L (17.3%) appears to have increased, but only 0.7% of strains were resistant (
4 mg/L).
Although initially susceptible to the fluoroquinolones, many strains of staphylococci, particularly MRSA and MR-CoNS, now show high levels of resistance to these agents.2,30,31 During this study, MRSA and MR-CoNS co-resistant to ciprofloxacin or erythromycin were common throughout Europe and North America. These results are in accordance with those of earlier studies with fluoroquinolones conducted in the USA and Europe.5,26,30,32 In a CDC-based USA study, the reported resistance among MRSA to ciprofloxacin was as high as 80% (see Table I).30
The susceptibility results for S. aureus, analysed according to susceptibility to oxacillin and ceftizoxime, indicate that only the oxacillin- and ceftizoxime-susceptible population differed significantly (were more susceptible) in the rates of co-resistances. The recently modified oxacillin breakpoint criteria for CoNS24 and the ceftizoxime susceptibility data were used to identify accurately mecA gene expression. The results indicate that ceftizoxime resistance may be an additional sensitive indicator of the mecA gene in these pathogens when compared with, or used with, oxacillin tests.
Enterococci colonize the bowel of >90% of healthy adults with E. faecalis accounting for >80% of enterococci in clinical infections.3,5 The incidence of E. faecalis infections has also increased in recent years, probably because of widespread use of antimicrobial agents inactive against the enterococci.2,3 Furthermore, enterococci, one of the leading causes of hospital-acquired infections, have become increasingly resistant to ß-lactams primarily as a consequence of altered penicillin-binding proteins (PBPs). They are generally regarded as intrinsically resistant to cephalosporins and more recently have developed resistance to vancomycin. In contrast to the incidence of clinical enterococcal species, vancomycin resistance is more common in E. faecium (>50% of strains) than in E. faecalis (<5% of strains).3,5
During this study, evernimicin was highly effective in vitro against all Enterococcus spp. tested, regardless of their vancomycin resistance phenotype. In contrast, isolates of E. faecalis were almost completely resistant to quinupristin/dalfopristin.26 Activity against E. faecium was also lower for quinupristin/dalfopristin than for evernimicin. The activity and bactericidal action of quinupristin/dalfopristin can be greatly influenced by macrolide co-resistance in E. faecium,28 and resistance has emerged on chemotherapy.29 Examination of the susceptibility rates for E. faecium in relation to the quinupristin/dalfopristin and ampicillin-resistance phenotypes, revealed potential misidentification of this species in approximately 20% of cases (i.e. E. faecalis identified as E. faecium).26 The vast majority of vancomycin resistance among enterococci, usually E. faecium, was reported from centres in North America.
Penicillin susceptibility or resistance had no effect on the potency of evernimicin, quinupristin/dalfopristin or vancomycin in vitro when tested against S. pneumoniae. The absence of co-resistance with these cited agents and penicillin confirms earlier reports.26 In contrast, penicillin resistance was associated with varying degrees of co-resistance to third-generation cephalosporins (ceftriaxone), erythromycin and clindamycin, which may be geographically specific. When a similar analysis was performed on the non-pneumococcal streptococci subset of strains, evernimicin also maintained its activity against those pathogens regardless of associated penicillin resistance.
Owing to their broad spectrum of activity against contemporary Gram-positive pathogen isolates, compounds like evernimicin may be a suitable alternative to glycopeptides. However, like vancomycin, evernimicin has limited activity against Gram-negative strains;1721 thus, when mixed bacterial infections are detected, combination therapy would be required. Nevertheless, these results are encouraging in that they suggest that agents of the everninomicin class may prove to be candidates for managing the growing number of resistant Gram-positive pathogens.
For all the species tested, the relative potencies or activities among the three most effective antimicrobials were consistent: evernimicin > quinupristin/dalfopristin > vancomycin, whereas the spectrum rank order was: evernimicin > vancomycin > quinupristin/dalfopristin. These results confirm previous findings which showed that evernimicin displayed potent activity against Gram-positive organisms,17,20,21 exceeding the current activity documented for vancomycin. Evernimicin was clearly more active by weight than vancomycin against all tested species, including enterococci (MIC90 1.0 and 4.0 mg/L for evernimicin and vancomycin, respectively) and S. pneumoniae (MIC90 0.047 and 0.75 mg/L, respectively).
This study also confirmed previous findings by showing the Etest to be a reliable, reproducible and possibly the preferred method for assessing the evernimicin MIC.3,19,23 Evernimicin MIC readings for staphylococci were occasionally more difficult to determine when the edges of the zones of inhibition were diffuse, but application of the 80% inhibition criteria recommended by AB Biodisk enhanced accuracy. Overall, this study confirms that the Etest was suitable for use in microbiological oligosaccharide activity surveillance programmes.18,19 In addition to providing data on the geographical patterns of pathogen incidence, worldwide monitoring of bacterial resistance patterns (as reported in this paper) can provide early recognition of evernimicin resistance in specific bacteria,32 which may reduce the inappropriate use of certain antibacterial agents and/or direct epidemiological interventions.5,22
In conclusion, the results of this in vitro surveillance study demonstrate that evernimicin exhibits consistent, complete antimicrobial activity against tested contemporary Gram-positive bacterial isolates. The in vitro efficacy of evernimicin compares favourably with that of alternative antibacterial treatments, including vancomycin and the streptogramin combination, quinupristin/dalfopristin. These results support the continued investigation of the clinical success and tolerability of compounds in the everninomicin class and provide a benchmark for future surveillance programmes of these important agents having potency against the troublesome Gram-positive cocci.
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Acknowledgments |
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Notes |
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Members of the Ziracin Susceptibility Testing Group are listed in the Acknowledgements.
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References |
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Received 3 April 2000; returned 5 July 2000; revised 8 August 2000; accepted 31 August 2000