Prevalence of resistance to ampicillin, gentamicin and vancomycin in Enterococcus faecalis and Enterococcus faecium isolates from clinical specimens and use of antimicrobials in five Nordic hospitals

G. S. Simonsen1, L. Småbrekke1, D. L. Monnet2, T. L. Sørensen2, J. K. Møller3, K. G. Kristinsson4, A. Lagerqvist-Widh5, E. Torell5, A. Digranes6, S. Harthug6 and A. Sundsfjord1,7,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We determined the species distribution and prevalence of ampicillin resistance, high-level gentamicin resistance (HLGR) and vancomycin resistance among clinical enterococcal isolates from five Nordic laboratories (Bergen, Tromsø, Uppsala, Aarhus and Reykjavik). Isolates represented three different groups: (i) all blood culture isolates from 1999; (ii) consecutive in-patient isolates (maximum 40); and (iii) consecutive outpatient isolates (maximum 40) collected during March to May 2000. Antimicrobial use data were collected at the national and hospital level. A high proportion (31.4%) of Enterococcus faecium was detected among blood culture isolates, in contrast to only 4.2% among isolates from outpatients. Ampicillin resistance was not found in Enterococcus faecalis, in contrast to 48.8% in E. faecium isolates. HLGR rates varied considerably between laboratories (1.1–27.6%). Acquired vancomycin resistance was not detected. There were no significant differences in the prevalences of HLGR between in-patient and outpatient isolates at individual hospitals. A cluster of clonally related ampicillin-resistant and HLGR E. faecium isolates was demonstrated in one of the hospitals. The lowest level of hospital antimicrobial use, the lowest proportion of E. faecium and the lowest prevalence of resistance were observed in Reykjavik.The study showed a relatively low level of resistance in enterococci, as compared with most European countries and the USA. However, there were large differences between hospitals with regard to the relative proportion of E. faecium isolates, their susceptibility to ampicillin and gentamicin, as well as the prevalence of HLGR in E. faecalis isolates. This indicates a potential for further improvement of antibiotic policies, and possibly hospital infection control, to maintain the low resistance levels observed in these countries.

Keywords: enterococci, antimicrobial resistance, antimicrobial use


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Enterococci are commensals of the gastrointestinal tract of most human beings. They have gained increasing clinical importance through the 1990s due to changes in hospital patient populations and antimicrobial use patterns.1,2 The most frequent clinical enterococcal infections are urinary tract infections and endocarditis.3 Enterococci are also isolated from polymicrobial intra-abdominal abscesses and wound infections, but their clinical significance in these cases has been questioned.3 Enterococcus faecalis and Enterococcus faecium have traditionally been estimated to account for ~90% and 5–10% of enterococcal infections.4 Enterococci are considered important difficult-to-treat pathogens, due to their intrinsic resistance to several antimicrobial agents and their propensity to acquire resistance. Several trends have been identified in the epidemiology of enterococcal infections: (i) an increasing incidence of enterococcal infections, particularly among severely ill patients;5 (ii) an increasing proportion of nosocomial enterococcal infections caused by E. faecium;6,7 and (iii) an increasing level of resistance to ampicillin, aminoglycosides and glycopeptides.1,8 The aims of the present study were to determine the relative proportions of enterococcal species among clinical enterococcal isolates from five Nordic hospitals, and their susceptibility to ampicillin, gentamicin and vancomycin. Additionally, since the level of resistance in a hospital can be the result of cross-transmission, antimicrobial pressure or both, we typed some of the isolates by pulsed-field gel electrophoresis (PFGE) and measured antimicrobial use overall in the four countries, and specifically in the five participating hospitals.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hospitals

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-{alpha}-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 l’Étoile, 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 8–16 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 1997–1999. 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 1997–1999.

Statistical analysis

Groups of isolates were compared by {chi}2 and Fisher exact tests (Epi Info 2000, CDC, Atlanta, GA, USA and WHO, Geneva, Switzerland). P values <0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

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 5.9–43.1%) of blood culture isolates, and only 14.1% (range 0–37.1%) 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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Table 1. Ampicillin and high-level gentamicin resistance in E. faecalis and E. faecium isolates from Haukeland Hospital (Bergen), Tromsø University Hospital (Tromsø), Uppsala University Hospital (Uppsala), Aarhus University Hospital (Aarhus) and Landspitali University Hospital (Reykjavik) by patient and specimen type, 1999–2000
 
Antimicrobial susceptibility testing

Reduced susceptibility to ampicillin was not detected in E. faecalis isolates, whereas 33.3–61.3% 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 1.1–27.6% 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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Figure 1. PFGE and UPGMA dendrogram of 13 E. faecium strains from Aarhus, Denmark, with reduced susceptibility to ampicillin and/or high-level gentamicin resistance. L, low-range PFG marker (New England Biolabs). Strains D-6 and D-23 are indistinguishable, whereas strains D-24 and D-38 are defined as closely, or possibly, related. Strains D-2, D-10, D-31 and D-37 are defined as closely, or possibly, related. Strains D-43 and D-84 are indistinguishable. Strains D-34, D-65 and D-91 are unrelated to each other, or to the other strains.

 
Antimicrobial use

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 (0.05–0.08 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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Table 2. Average yearly consumption of antibacterials for systemic use at Haukeland Hospital (Bergen), Tromsø University Hospital (Tromsø), Uppsala University Hospital (Uppsala), Aarhus University Hospital (Aarhus) and Landspitali University Hospital (Reykjavik), 1997–1999
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we determined the species distribution and antimicrobial susceptibility of clinical enterococcal isolates from five Nordic hospitals, and calculated local and national data on antimicrobial use. Longitudinal studies have indicated an increasing incidence of enterococcal infections in tertiary care teaching hospitals, often accompanied by a high proportion of E. faecium isolates.1618 Our cross-sectional design did not allow us to determine trends in the incidence of enterococcal infections, but confirms the high proportion of E. faecium among hospital enterococcal isolates. E. faecium is reported to account for 5–10% of enterococcal infections.4 Although we found a similar percentage among outpatient isolates in our study, E. faecium represented 31.4% (range: 5.9–43.1%) of blood culture isolates and 14.1% (range: 0–37.1%) of non-systemic in-patient isolates. The exception was Landspitali University Hospital, Reykjavik, where only one of 45 in-patient isolates (a blood culture isolate) was identified as E. faecium. The relatively high proportion of E. faecium among hospital isolates in our study is consistent with those reported in other studies.7,19 Thus, the traditional outnumbering of E. faecium by E. faecalis by 10:1 in clinical specimens only seems to be valid for outpatient isolates. It has been suggested that the emergence of E. faecium bloodstream infections is the result of changes in the hospital patient populations and antimicrobial use patterns.4 In this context, it is of interest that the hospital in Reykjavik had the lowest yearly overall antimicrobial use of the five hospitals.

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 33.3–61.3%) 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 16–32 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 1997–1999. 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.


    Acknowledgements
 
We thank Bjørg C. Haldorsen for excellent laboratory assistance, and the laboratory staff in all the participating laboratories for valuable collaboration and help in the collection of samples. The study was supported by a grant from the Scandinavian Society for Antimicrobial Chemotherapy.

This study was presented in part at the Eleventh European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), Istanbul, Turkey, April 1–4 2001.


    Footnotes
 
* Corresponding author. Tel: +47-7-64-62-02; Fax: +47-77-62-70-15; E-mail: arnfinns{at}fagmed.uit.no Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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