a Antibiotic Resistance Monitoring and Reference Laboratory, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT; b Pharmacia Corporation, Davy Avenue, Knowlhill, Milton Keynes MK5 8PH, UK
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Abstract |
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Introduction |
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In this study, 25 sentinel laboratories across the UK were selected to determine the prevalence of antibiotic resistance among Gram-positive pathogens. The organisms surveyed comprised S. aureus, coagulase-negative staphylococci (CNS), enterococci and pneumococci. The isolates were tested by each sentinel laboratory against a standard set of reference antibiotics and also linezolid, which is the first oxazolidinone agent to be licensed. Oxazolidinones inhibit the initiation of protein synthesis9 and linezolid shows considerable promise in treating severe infections caused by Gram-positive pathogens.10,11
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Materials and methods |
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Twenty-five sentinel laboratories with a wide geographical distribution in the UK were asked to collect and test consecutive clinically significant isolates (defined by an intention to treat), comprising: 40 pneumococci, 40 enterococci, 30 CNS and 30 S. aureus. Each laboratory was also requested to collect and test an additional 10 methicillin-resistant S. aureus (MRSA) isolates. Duplicate isolates from the same infection episode in the same patient were excluded. The laboratory completed a case record form for each isolate, giving the patient's age, gender, clinical diagnosis and the site of isolation.
Identification of bacteria by sentinel laboratories
Staphylococci were identified by Gram's stain and coagulase reaction; pneumococci on the basis of optochin susceptibility as tested on horse blood agar and enterococci with the API20-STREP or ATB32-STREP system (bioMérieux, La Balme les Grottes, France) or, in the case of one laboratory, using the BBL Crystal GP system (Becton Dickinson, Oxford, UK). CNS were not identified to species level.
Susceptibility testing by sentinel laboratories
Isolates were grown overnight on a non-selective medium and resuspended in 0.9% MuellerHinton broth (Oxoid, Basingstoke, UK) to the density of a 0.5 McFarland standard, except for mucoid pneumococci, which were resuspended to the density of a 1.0 McFarland standard. The suspensions were inoculated on to 15 cm diameter plates of MuellerHinton agar (Oxoid). This medium was supplemented with 5% lysed horse blood for pneumococci. Oxacillin was tested separately against staphylococci on 9 cm plates with 2% NaCl added to the agar. Etest strips (Cambridge Diagnostic Services, Cambridge, UK) were applied to the plates and the results were read according to the manufacturer's directions. Staphylococci and enterococci were incubated in ambient air at 37°C, whereas pneumococci were incubated in 5% CO2 at 3537°C. MIC results were read after 24 h incubation. Oxacillin, vancomycin and teicoplanin results for staphylococci were re-read after 48 h, with these latter results taken as definitive.
Quality assurance
Proficiency in testing was established by asking centres to test six reference strains from the Antibiotic Resistance Monitoring and Reference Laboratory (ARMRL) at the start of the study and to include these with each batch of isolates tested. These comprised S. aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619 and Enterococcus faecalis ATCC 29212 together with one multi-resistant isolate of each of these species. The sentinel laboratories were asked to test these strains according to the study protocol and to return the results. The identity of the control strains was not known by the sentinel laboratories. Testing of clinical isolates was commenced only after a centre was obtaining MICs within one dilution of the MIC range designated for the control strains by the ARMRL.
Further quality control (QC) was achieved by the sentinel laboratories' sending every 10th isolate collected to the ARMRL for retesting. Isolates with unusual resistances were also collected for retesting. These comprised any isolates with vancomycin or linezolid MICs > 4 mg/L, any S. aureus with teicoplanin MICs > 4 mg/L, any enterococci identified as a species other than E. faecalis, and any staphylococci where routine and Etest results for oxacillin disagreed.
Re-identification at the ARMRL
Identification of enterococcal species other than E. faecalis was undertaken using published methods.12,13
Susceptibility testing at the ARMRL
MIC determinations at the ARMRL were undertaken using an agar incorporation method, except for trovafloxacin, where the Etest was used. MuellerHinton agar (Oxoid) was employed, supplemented with 5% lysed horse blood (Tissue Culture Services, Buckingham, UK) for pneumococci. Inocula of 104 cfu/spot were delivered with a multipoint inoculator. Incubation was as described above for the Etest. MICs were defined as the lowest drug concentrations to prevent growth completely and isolates were defined as sensitive or resistant based on NCCLS criteria.14
All antimicrobial powders for determining MICs were obtained from Sigma (Poole, UK), with the exception of ciprofloxacin (Bayer, Newbury, UK) and linezolid (Pharmacia Corporation, Milton Keynes, UK).
Molecular studies
All enterococci with vancomycin MICs > 4 mg/L were collected by the ARMRL and the presence of vanA and vanB genes was determined by PCR.15 Sentinel laboratories were also asked to send in erythromycin-resistant pneumococci for the detection of the mefE16,17 and ermB18 genes by PCR. An Escherichia coli control strain with the cloned ermB gene was kindly supplied by Dr Marilyn Roberts from the University of Washington (Seattle, WA, USA) and an S. pneumoniae control strain with mefE by Dr Virginia Shortridge from Abbott Laboratories (Abbott Park, IL, USA).
Statistical analyses
Statistical analysis was performed using the chi-squared test with Yates' correction, and with a P value <0.05 indicating significance.
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Results |
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The sentinel laboratories collected and tested 3770 isolates. These comprised 1000 S. pneumoniae; 1005 S. aureus; 769 CNS and 996 enterococci. The enterococci comprised 875 E. faecalis, 108 E. faecium and 13 isolates belonging to other species including Enterococcus gallinarum, Enterococcus avium and Enterococcus raffinosus. Among the S. aureus strains, 755 were consecutive isolates and 250 were collected specifically as MRSA. A total of 940 isolates were retested at the ARMRL.
Quality assurance
Two types of QC were built into the study. In the first instance, centres were asked to test six reference strains before commencing the study. These organisms comprised three susceptible ATCC strains and three resistant ARMRL strains. The sentinel laboratories' results fell within the ARMRL QC ranges for 90.5% and 86.7% of tests with the ATCC and ARMRL strains, respectively (Table I). Even when a sentinel laboratory's MIC result fell outside the ARMRL MIC ranges, the deviation was commonly only one dilution. There was 98% agreement between the ARMRL and the sentinel laboratories with regard to the susceptibility categorization. Categorization agreement was maintained for S. aureus strain ARU 30300 with gentamicin, penicillin and oxacillin and for S. aureus ATCC 29213 tested with erythromycin, although many sentinel laboratories obtained MIC values outside the ARMRL's control range in these cases. These differences were not confined to a single laboratory (Table I
). In the case of S. pneumoniae ARU 3489, where the QC ranges spanned the intermediate/resistant border, both intermediate and resistant categories were accepted as correct.
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Isolates received by the ARMRL as unusually resistant
Sentinel laboratories were also asked to refer in all isolates with atypical resistances. Two MRSA isolates were reported initially as resistant to vancomycin with MICs of 8 mg/L, but were found susceptible on retesting (MIC 4 mg/L). Two centres initially reported linezolid resistance in enterococci (a total of eight isolates) and S. aureus (a total of 12 isolates). On retesting at the ARMRL, the linezolid MIC for all these 20 isolates was 4 mg/L. All enterococci (n = 38) received as vancomycin resistant were confirmed as such.
Prevalence of resistance
S. aureus.
A total of 755 S. aureus isolates were tested by the sentinel laboratories as consecutive isolates, and a further 250 as confirmed MRSA. Skin and wound infections were the most frequent sources (63.2%), followed by respiratory tract infections (10.2%) and blood (7.6%). The prevalence of MRSA amongst the consecutive isolates was 19.2%, but this rose to 29% (114/392) if out-patient isolates were excluded. MRSA rates for individual hospitals varied from 0 to 43%. Most methicillin-susceptible S. aureus (MSSA) isolates were resistant to penicillin (84.2%), but only 8.2% were resistant to erythromycin and 3.4% to ciprofloxacin. In contrast, 87.9% of MRSA isolates were resistant to erythromycin and 94.7% were resistant to ciprofloxacin. Resistance to gentamicin was also more prevalent among MRSA than MSSA but remained infrequent (8.8%).
No glycopeptide-intermediate S. aureus isolates were reported, but vancomycin MICs for 5% of MRSA isolates were on the breakpoint at 4 mg/L. Linezolid MIC ranges were similar for MSSA and MRSA, with most MIC values between 0.5 and 4 mg/L, although one MSSA (not retested at the ARMRL) was reported with a linezolid MIC of 0.064 mg/L. Modal MICs of linezolid for MRSA and MSSA were both 2 mg/L. The MIC90 for MRSA was 4 mg/L, compared with 2 mg/L for MSSA. This difference, however, reflected only a slightly higher proportion of MRSA for which the MIC was 4 mg/L and the MIC95 values for both MRSA and MSSA were 4 mg/L.
Coagulase-negative staphylococci.
MIC distributions and resistance rates for CNS are shown in Table II. Isolates from blood or line infections accounted for 52.7% of the total infections. Based on the NCCLS breakpoint of 0.25 mg/L, 68.9% of the CNS were oxacillin resistant, whereas only 39% were resistant with respect to the BSAC breakpoint of 2 mg/L.
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Enterococci.
Resistance rates and MIC distributions for the enterococci are shown in Table III. E. faecalis and E. faecium accounted for 87.9 and 10.8% of the enterococcal isolates, respectively, after re-identification at the ARMRL. E. faecalis isolates were mostly from urine samples (70.5%), and were divided equally between those from hospital patients and those from community patients. By contrast, virtually all the E. faecium isolates (99/108) were from hospitalized patients and many (36%) were from blood. About 91% of the confirmed E. faecium isolates were resistant to ampicillin (MIC > 8 mg/L), as were single isolates of E. raffinosus, E. avium and E. gallinarum. Eight E. faecium isolates were sensitive to ampicillin, with MICs
8 mg/L as confirmed at the ARMRL. One of these was vancomycin resistant.
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PCR-based identification of Van determinants was undertaken on all 38 VRE. Twenty-six, including the four resistant E. faecalis isolates, carried the vanA gene. These characteristically had high-level resistance to vancomycin (MIC 256 mg/L). The vanB gene was present in five isolates and was characteristically associated with lower-level vancomycin resistance (MIC
64 mg/L) and susceptibility to teicoplanin (MIC
4 mg/L). The seven remaining VRE were E. gallinarum isolates with low-level resistance to vancomycin (MICs 816 mg/L) but were susceptible to teicoplanin. This behaviour corresponds to the VanC phenotype, which is characteristic of this species. These isolates did not give PCR products with primers for vanA or vanB.
Pneumococci.
A total of 1000 S. pneumoniae isolates were tested by the sentinel laboratories, but two duplicates were identified from the case record forms received and were excluded. Pneumococci were most often isolated from the respiratory tract (53%), eyes (16.5%), blood (14.7%) and ears (10.8%). Overall, 8.9% of the pneumococcal isolates were resistant to penicillin. Most resistance was intermediate (MIC 0.121 mg/L), but 1% of the isolates had high-level resistance (MIC 2 mg/L). Resistance rates to cefotaxime (MIC > 0.5 mg/L) and erythromycin (MIC > 0.5 mg/L) were 2.0 and 12.3%, respectively. No resistance to linezolid or vancomycin was found and linezolid was active over a narrow range of MICs (0.1254 mg/L), with a mode of 1 mg/L.
To analyse the susceptibility profiles of pneumococci more fully, the isolates were categorized according to their resistance to penicillin (Table IV). Of the 911 isolates susceptible to penicillin, 10.5% were resistant to erythromycin and none was resistant to any other drug tested. Of 78 isolates with intermediate resistance to penicillin, 15.4% had intermediate resistance to cefotaxime (MIC > 0.5 mg/L) and 29.8% were resistant to erythromycin. One penicillin-intermediate isolate from a respiratory tract infection in a community patient was resistant to trovafloxacin, with an MIC of 4 mg/L. The nine isolates resistant to penicillin were all resistant or intermediately resistant to cefotaxime (MIC > 0.5 mg/L), and five were resistant to erythromycin. These nine isolates were received from six hospitals and were isolated from sputa (six), eyes (two) and an ear (one). No highly resistant pneumococci came from blood, but 8.2% (12/147) of the bacteraemia isolates had intermediate resistance to penicillin and one had intermediate resistance to cefotaxime.
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Discussion |
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The overall rate of resistance to methicillin among S. aureus isolates was 19.2%, but rose to 29% among isolates from hospitalized patients and 28% among isolates from bacteraemias. Routine laboratory reports for blood culture isolates in England and Wales, as sent to the Public Health Laboratory Service (PHLS),2 show the prevalence of methicillin resistance among S. aureus, increasing from 1.7% in 1990 to 34% in 1998. The latter figure is in good agreement with the present study, allowing that only 60 S. aureus bacteraemias were represented here. The MRSA prevalence rate in the UK remains lower than some southern European countries but is much higher than in Scandinavian countries.20
The prevalence of methicillin resistance amongst S. aureus varied between the sentinel hospitals: six hospitals had methicillin resistance rates <5%, whereas others had prevalence rates as high as 43%. Analogous surveys by Andrews et al.4,5 have revealed similar variation, and have also shown that rates in individual hospitals can vary widely between two consecutive years.
Higher resistance rates to other antibiotics were seen amongst MRSA than amongst MSSA. Specifically, most MRSA isolates were resistant to ciprofloxacin and erythromycin, though resistance to gentamicin remained rare (8.8% amongst MRSA). This profile is typical of the epidemic MRSA (EMRSA) 15 and 16 types currently prevalent in the UK.21 None of the S. aureus isolates was resistant to teicoplanin or vancomycin but MICs of these glycopeptides were on the breakpoint of 4 mg/L for c. 5% of the isolates. All the S. aureus isolates were susceptible to linezolid with MICs 4 mg/L.
The resistance rate to oxacillin among CNS was 68.9% based on the new NCCLS breakpoint14 (>0.25 mg/L) but this would decrease to 38.9% based upon the BSAC breakpoint (2 mg/L). PCR examination of CNS isolates with oxacillin MICs of 0.52 mg/L showed these isolates to lack mecA (results not shown), supporting the BSAC breakpoint.
E. faecalis was the predominant enterococcal species, with most isolates coming from urine specimens (70.5%), collected in almost equal proportions from hospital and community patients. On the other hand, the E. faecium isolates were predominantly from hospitalized patients, with 36% of the isolates from bacteraemias. Vancomycin resistance was seen mostly in E. faecium (24.1%) and was much rarer in E. faecalis (0.5%). VanA was the most prevalent phenotype. The prevalence rate for vancomycin resistance in E. faecium agreed well with data for bacteraemias in England and Wales in 1998 (24%), as reported to the PHLS,2 but the prevalence rate for this resistance in E. faecalis was lower. This discrepancy perhaps reflects the high proportion of isolates from urine samples from community patients in the present collection and the fact that 1520% of E. faecalis bacteraemias reported to the PHLS involve ampicillin-resistant organisms more likely to be E. faecium.2 The overall rates of VRE in this survey were similar to those reported by Andrews et al.5 in a 19971998 UK survey.
The resistance rate to penicillin amongst pneumococci was 8.9%. This shows an increase in resistance from that found in previous multi-centre PHLS surveys,3 which indicated rates of 1.5% in 1990 and 3.9% in 1995. Penicillin resistance was mostly intermediate level, with considerable variation in prevalence between hospitals. Past surveys in the UK have shown similar variations in resistance rates between regions,22 but an upward trend in intermediate resistance is apparent at many hospitals.5 In contrast to the USA,23,24 differences in penicillin resistance rates were not observed in relation to age groups or isolation sites. Resistance to erythromycin appears to have stabilized at around 813%, with no significant differences to the rates found in the 1995 UK survey3 (P > 0.05). This conclusion is also supported by the routine data for pneumococci from bacteraemia, as reported to the PHLS.2 Investigation of mechanisms of macrolide resistance amongst the S. pneumoniae isolates by PCR suggested that mefE was more prevalent than ermB, but the sample size was small.
Linezolid is being developed to treat serious infections caused by Gram-positive bacteria, including those resistant to the antibiotics currently available. Previous studies have shown linezolid to have promising laboratory2526 and clinical1011 activity. Its in vitro activity was confirmed here for UK isolates. Linezolid was equally active against methicillin-susceptible and -resistant staphylococci, vancomycin-susceptible and -resistant enterococci and against all pneumococci, irrespective of their penicillin or macrolide resistance. For each group of bacteria, linezolid MICs spanned a very narrow range of dilutions, with MICs never exceeding 4 mg/L. Together with promising clinical results,27 the present observations suggest that linezolid will become a valuable agent for the treatment of serious infections caused by Gram-positive cocci.
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Acknowledgments |
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Notes |
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Deceased; this paper is dedicated to his memory.
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References |
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Received 19 April 2000; returned 2 July 2000; revised 21 July 2000; accepted 15 August 2000