1 University of Tromsø and University Hospital of North Norway (UNN); 6 Haukeland University Hospital; 7 Reference Center for Detection of Antimicrobial Resistance, UNN, Norway; 2 Statens Serum Institut; 3 Aarhus University Hospital, Denmark; 4 Landspitali University Hospital, Reykjavik, Iceland; 5 Uppsala University Hospital, Sweden
Received 24 September 2002; accepted 19 October 2002
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Abstract |
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Keywords: enterococci, antimicrobial resistance, antimicrobial use
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Introduction |
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Materials and methods |
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Data were collected at five tertiary care university hospitals in Bergen (1100 beds) and Tromsø (500 beds) (Norway), Uppsala (1200 beds) (Sweden), Aarhus (1380 beds) (Denmark) and Reykjavik (800 beds) (Iceland).
Selection and identification of bacterial isolates
Enterococci were isolated from clinical specimens by conventional methods, as practiced at each participating laboratory. Three groups of enterococcal isolates were included in the study: (i) all blood culture isolates from 1999; (ii) consecutive clinically significant isolates from in-patients between 13 March and 12 May 2000, with a maximum of 40 isolates from each hospital; and (iii) consecutive clinically significant isolates from outpatients between 13 March and 12 May 2000, with a maximum 40 isolates from each hospital. Only the first isolate per patient was included. The strains were speciated by conventional assays for acidification of arabinose and methyl--D-glucopyranoside broths, as well as catalase, pyrrolidonyl-ß-naphthylamidase (PYR), tellurite reductase and pigment production.4 The results were verified by the ATB ID32 Strep system (bioMérieux, Marcy lÉtoile, France) and ddl-PCR when needed.9
Antimicrobial susceptibility testing
Susceptibility testing was performed at the Departments of Microbiology, University of Tromsø and University Hospital of North Norway. All strains were screened for resistance to ampicillin, gentamicin and vancomycin, by an agar dilution method. Briefly, 10 µL aliquots of a 0.5 McFarland standard bacterial suspension in 0.85% NaCl were inoculated on the following agar plates: brain heart infusion (BHI) agar (Oxoid, Basingstoke, UK) with 16 mg/L ampicillin (Bristol-Myers Squibb, Bromma, Sweden), BHI agar with 100 mg/L gentamicin (Gibco BLR, Life Technologies, Invitrogen, Oslo, Norway), BHI agar with 6 mg/L vancomycin (Abbott, Solna, Sweden) and BHI agar without antimicrobials as control. For practical purposes, we chose to use the same agar base and inoculum for all antimicrobials, even though this procedure would allow growth of some isolates that were susceptible to ampicillin, according to the NCCLS criteria. However, all isolates, growing on any of the antimicrobial screening agars, were subsequently examined using Etest for susceptibility to the antibiotics (AB Biodisk, Solna, Sweden), according to the recommendations of the manufacturer. Results were categorized according to the NCCLS breakpoints: susceptible (S), intermediate (I) and resistant (R), i.e. ampicillin S 8 mg/L, R
16 mg/L; gentamicin high-level resistance S < 500 mg/L, R
500 mg/L; and vancomycin S
4 mg/L, I 816 mg/L, R
32 mg/L.10 The following strains were used for quality control: E. faecium BM4147 (VanA), E. faecalis V583 (VanB), Enterococcus gallinarum ATCC 49608 (VanC), E. faecalis ATCC 29212, E. faecium ATCC 19434, E. faecium ATCC 51559 (VanA, resistant to ampicillin, high-level resistance to gentamicin) and E. faecalis ATCC 51575 (high-level resistance to gentamicin and streptomycin).
Genetic analysis
Isolates categorized as intermediate or resistant to, vancomycin by Etest were further analysed by PCR for the vanA, vanB and vanC genes.1113 Additionally, a group of 13 E. faecium isolates, with reduced susceptibility to ampicillin and/or gentamicin, from Aarhus University Hospital, Denmark, were examined by PFGE of SmaI total DNA digests, as described previously.12 Banding patterns were compared visually and by the GelCompare software package (Applied Maths, Kortrijk, Belgium), with Dice analysis of peak positions using UPGMA, 0% optimization and band-width tolerance set critically at 1.0%.
Antimicrobial use
National consumption data were collected from public sources, and hospital consumption data were based on sales from hospital pharmacies to the hospitals. The 1999 version of WHO Anatomical Chemical Therapeutics (ATC) classification index and defined daily doses (DDDs) were used.14 Yearly overall national antimicrobial use was reported as the mean yearly number of DDDs per 1000 inhabitants and per day (DDD/1000 inhabitant-days) for the years 19971999. Yearly hospital antimicrobial use was reported as the mean yearly number of DDDs per 1000 occupied beds and per day (DDD/1000 bed-days) and as the mean proportion of total use in DDDs for the period 19971999.
Statistical analysis
Groups of isolates were compared by 2 and Fisher exact tests (Epi Info 2000, CDC, Atlanta, GA, USA and WHO, Geneva, Switzerland). P values <0.05 were considered significant.
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Results |
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A total of 509 enterococcal isolates were included in the study, comprising 420 E. faecalis (82.5%), 82 E. faecium (16.1%), four E. gallinarum (0.8%), two Enterococcus casseliflavus (0.4%) and one Enterococcus hirae (0.2%). The strains were isolated from 249 men (48.9%) and 260 women (51.1%). Among 156 blood culture isolates, there were 104 E. faecalis (66.7%), 49 E. faecium (31.4%), two E. gallinarum (1.3%) and one E. casseliflavus (0.6%). Both the 184 consecutive non-systemic isolates from in-patients (group ii) and the 169 consecutive non-systemic isolates from outpatients (group iii) were predominantly E. faecalis (84.2% and 95.3%) from urinary tract specimens. The overall proportion of E. faecium among isolates from in-patients was 22.1%, in contrast to 4.1% among isolates from outpatients. E. faecium represented 31.4% (range 5.943.1%) of blood culture isolates, and only 14.1% (range 037.1%) of non-systemic in-patient isolates. The proportion of E. faecium among blood culture enterococcal isolates was significantly lower in Reykjavik (1/17) than in the other hospitals (48/139) (P = 0.016). The detailed distribution of enterococcal isolates by species, hospital, patient type and specimen type is presented in Table 1.
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Reduced susceptibility to ampicillin was not detected in E. faecalis isolates, whereas 33.361.3% of E. faecium isolates from Bergen, Tromsø, Uppsala and Aarhus were resistant to this agent. The 40 isolates with this phenotype had ampicillin MICs of 16 (n = 16), 32 (n = 21), 64 (n = 2) and 128 mg/L (n = 1). Only one E. faecium isolate was recovered in Reykjavik. High-level gentamicin resistance (HLGR) was detected in 1.127.6% of E. faecalis isolates, with a low prevalence in Reykjavik (1.1%), Aarhus (1.2%) and Tromsø (3.6%), and a relatively high prevalence in Bergen (15.7%) and Uppsala (27.6%). There were no significant differences in HLGR prevalences between in-patient and outpatient E. faecalis isolates in any hospital, but the HLGR prevalence in E. faecalis isolates was significantly higher in Uppsala and Bergen than in the other three hospitals (P < 0.01 for both). HLGR in E. faecium isolates was only detected in Aarhus, where nine out of 17 (52.9%) E. faecium isolates from in-patients displayed this trait. Among these nine isolates, six (five from blood culture and one from an intravenous catheter) showed co-resistance to ampicillin. No isolates with acquired vancomycin resistance were detected. The presence of the intrinsic vanC1 and vanC2/3 genes was confirmed by PCR in four E. gallinarum and two E. casseliflavus isolates. The E. gallinarum, E. casseliflavus and E. hirae isolates were all susceptible to ampicillin and gentamicin. The distribution of antimicrobial susceptibility testing results among species, hospitals, patient types and specimen types is presented in Table 1.
PFGE typing of antimicrobial-resistant E. faecium isolates from Aarhus
A total of 13 E. faecium isolates, with reduced susceptibility to ampicillin and/or HLGR, were detected in Aarhus University Hospital (nine blood culture isolates, one urinary tract isolate and three isolates from other specimens). PFGE typing revealed two pairs with indistinguishable patterns: D-6/D-23 and D-43/D-84 (Figure 1). The D-6/D-23 pair clustered with two additional isolates (D-24 and D-38), whereas a second group was formed by strains D-2, D-10, D-31 and D-37, thus forming two groups of four isolates, each with >80% identity. There were from one to six band differences within each group, thus indicating closely or possibly related isolates, as defined by Tenover.15 Isolates D-34, D-65 and D-91 were unrelated to each other, or to any other isolate.
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The mean yearly national consumption of antimicrobials varied from 13.5 DDD/1000 inhabitant-days in Denmark, 14.5 in Norway and 15.5 in Sweden, to 21.3 in Iceland. There were large differences in the mean yearly consumption of penicillins, with extended spectrum: 1.6 DDD/1000 inhabitant-days in Sweden, 1.92 in Norway, 2.73 in Denmark and 5.72 in Iceland. The mean yearly consumption of glycopeptides was the highest in Iceland (0.017 DDD/1000 inhabitant-days) and lowest in Norway (0.007 DDD/1000 inhabitant-days). Sweden had a lower mean yearly consumption of aminoglycosides (0.01 DDD/1000 inhabitant-days) than the other Nordic countries (0.050.08 DDD/1000 inhabitant-days).
In the participating hospitals, the mean yearly consumption of antimicrobials varied from 295 DDD/1000 bed-days in Reykjavik to 483 in Tromsø. The mean yearly consumption of penicillins with extended spectrum varied from 28 DDD/1000 bed-days in Uppsala to 134 in Aarhus. In Uppsala, this class accounted for 7.3% of the total number of DDD, whereas in Aarhus it accounted for 29.7% of the total. The mean yearly consumption of cephalosporins varied from 46 DDD/1000 bed-days in Aarhus to 90 in Tromsø. In Aarhus, this class accounted for 10.2% of the total number of DDD, whereas in Tromsø it accounted for 18.6% of the total. The mean yearly consumption of aminoglycosides varied from 7 DDD/1000 bed-days in Reykjavik to 24 in Aarhus. In Reykjavik, this class accounted for 2.4% of the total number of DDD, whereas in Aarhus it accounted for 5.1% of the total. In Aarhus, the mean yearly consumption of glycopeptides was 11 DDD/1000 bed-days, which was almost three times the consumption in Tromsø, Bergen and Reykjavik. Detailed hospital antimicrobial use data are presented in Table 2.
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Discussion |
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Treatment of enterococcal endocarditis should comprise a bactericidal synergic combination of an aminoglycoside and a cell-wall active agent, such as ampicillin or vancomycin.20,21 Ampicillin is usually preferred to glycopeptides, unless the strain is ampicillin resistant; gentamicin is the aminoglycoside of choice, due to the synergic effect against E. faecium, which harbours chromosomally encoded low-level resistance against all other aminoglycosides except streptomycin.22 Thus, emergence of resistance to aminoglycosides, ampicillin and glycopeptides is a cause for great concern, as clinicians may have to use new and less established alternatives, such as quinupristin/dalfopristin or linezolid to treat enterococcal endocarditis. In the present study, the overall prevalence of resistance was relatively low. Acquired high-level glycopeptide resistance was not detected, which is in accordance with previous studies in Nordic countries.2327 All E. faecalis isolates were susceptible to ampicillin. The prevalence of HLGR was low in Reykjavik (1.1%) and Tromsø (3.6%), and in E. faecalis isolates from Aarhus (1.2%). However, the prevalence of HLGR in E. faecalis in Uppsala (27.6%) and Bergen (15.7%), as well as in E. faecium in Aarhus (58.8%), was relatively high and comparable to the situation reported in other countries.28,29 Similar results have been reported previously from Uppsala and Bergen.24,30,31 Although Nordic countries generally report low prevalence of resistance in enterococci, there are apparently large differences in gentamicin susceptibility among hospitals.
E. faecalis isolates were uniformly susceptible to ampicillin, whereas almost half (range 33.361.3%) of E. faecium isolates were resistant to ampicillin, according to the NCCLS breakpoints. These results are in accordance with previous reports from Norway and Sweden, where a high proportion (6.9 and 21.5%) of hospitalized patients were faecal carriers of ampicillin-resistant enterococci, predominantly E. faecium.24,30 The situation in Aarhus was more complex, with the occurrence of a cluster of HLGR E. faecium isolates. Many of the Aarhus isolates were resistant to both gentamicin and ampicillin, thus further complicating the choice of treatment. However, treatment of infections due to isolates with ampicillin MICs of 1632 mg/L could, in some cases, be achieved by adequate dose adjustments. Molecular analysis indicated that at least some of these isolates were clonally related. Gentamicin resistance determinants in enterococci have been linked to mobile genetic elements.1,32 HLGR isolates that appear unrelated to PFGE in our study may thus represent an outbreak of a mobile genetic element carrying gentamicin resistance determinants.
The number of isolates from each laboratory is limited, and one should be careful when drawing conclusions. However, the prevalence of resistance varied widely among laboratories, underlining the need for local surveillance of resistance, and implementation of appropriate infection control measures. The apparent clonal outbreaks of antimicrobial-resistant E. faecium in Aarhus were first recognized through the present study, and the finding led to further investigations in this hospital. The value of antimicrobial resistance monitoring, in combination with molecular investigation of suspected outbreaks, is obvious for both infection control and the development of antimicrobial treatment guidelines.
Yearly hospital antimicrobial use was given as the mean for the period 19971999. There were striking differences between hospitals, with overall antimicrobial use in Tromsø being 1.6 times greater than in Reykjavik. There were also differences among hospitals with regard to the patterns of antimicrobial agents being used. Aarhus had the highest consumption of extended-spectrum penicillins, aminoglycosides, glycopeptides and macrolides, and the lowest consumption of tetracyclines and cephalosporins. Uppsala had the lowest consumption of penicillins, and the highest consumption of quinolones and carbapenems. Tromsø had the highest consumption of cephalosporins and, together with Bergen, the highest consumption of ß-lactamase-sensitive penicillins. The observation of the high consumption of extended-spectrum penicillins and aminoglycosides in Aarhus is interesting, but not sufficient to establish a causal relationship with the cluster of ampicillin-resistant/HLGR E. faecium found at this hospital. There are several reasons why the putative link between antimicrobial use and the occurrence of resistance was difficult to demonstrate. Hospitals may differ in other aspects than their antimicrobial use patterns. The level of resistance may also be too low to discern underlying differences. Antimicrobial resistance is not a phenomenon restricted to a specific class of antimicrobials, because of cross-resistance due to overlapping targets of different antimicrobials, or co-selection related to genetic linkage between resistance genes or other loci. Thus there is rarely a monospecific association between the use of one single class of antimicrobials and the occurrence of a specific resistance mechanism. The dynamic relationship between antimicrobial use, and occurrence of resistant bacteria isolated from humans, should probably be analysed by time series analysis and transfer function models, or other suitable statistical tools using large longitudinal data sets.3335 The present study did not contain sufficient data for such analyses.
Despite these shortcomings, several interesting observations can be made. Reykjavik had the lowest level of hospital antimicrobial use and the lowest prevalence of resistance, in spite of the highest level of community antimicrobial use being observed in Iceland. In both Bergen and Uppsala, similarly high prevalences of HLGR were seen among E. faecalis isolates from in-patients and outpatients. This is in accordance with the mathematical model presented by Lipsitch et al.36 in which the successful control of resistance in hospitals is highly dependent on the influx of susceptible clones from the community. Because of the continuous admission of patients, a resistance problem that is prevalent both within and outside hospitals will obviously be difficult to control. Further studies are warranted to elucidate these relationships further.
In conclusion, the present study documents low levels of antimicrobial use, and occurrence of resistance in E. faecalis and E. faecium, as compared with other areas in Europe and the USA. However, large differences among hospitals in both antimicrobial use and prevalence of resistance indicate a potential for further improvement of antibiotic policies, and possibly hospital infection control, to maintain the low resistance levels observed in the Nordic countries.
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Acknowledgements |
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This study was presented in part at the Eleventh European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Istanbul, Turkey, April 14 2001.
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Footnotes |
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References |
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