a Institute for Medical Microbiology and Virology, Heinrich-Heine University Düsseldorf, Universitätsstrasse 1, Geb. 22.21, D-40225 Düsseldorf, Germany; b Eijkman-Winkler Institute for Clinical Microbiology, University Hospital Utrecht, The Netherlands
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Abstract |
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Introduction |
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The main mechanism of aminoglycoside resistance in staphylococci is drug inactivation by cellular aminoglycoside-modifying enzymes. Several distinct gene loci encoding such modifying enzymes have been characterized in staphylococci. Clinically, the most important of these encode acetyltransferase (AAC), adenylyltransferase (ANT) or phosphotransferase (APH) activity. Aminoglycosides modified at amino groups by AAC enzymes or at hydroxyl groups by ANT or APH enzymes, lose their ribosome-binding ability and thus no longer inhibit protein synthesis. 5
Resistance to gentamicin and concomitant resistance to tobramycin and kanamycin in staphylococci are mediated by a bifunctional enzyme displaying AAC(6') and APH(2'') activity. 6 ,7 The aac(6')-Ie +aph(2'') gene encodes this bifunctional enzyme and is encoded on composite transposon Tn4001. Tn4001-like elements are widely distributed in both Staphylococcus aureus and coagulase- negative staphylococci (CNS). Tn4001 has been found on pSK1 family plasmids, conjugative plasmids, such as pSK41, occasionally on ß-lactamase/heavy metal resistance plasmids, such as pSK23, and also in various chromosomal locations. 5
Resistance to neomycin, kanamycin, tobramycin and amikacin in staphylococci is mediated by an ANT(4')-I enzyme encoded by ant(4')-Ia. This gene is often carried on small plasmids, and then integrated into larger conjugative plasmids, such as pSK41, and subsequently into the mec region of the chromosome of some S. aureus isolates, probably as a result of IS257-mediated recombination events. 8 ,9 ,10 A variety of other plasmids encoding ANT(4')-I activity have also been detected. 5
Resistance to neomycin and kanamycin conferred by an APH(3')-III enzyme has also been described for staphylococci. The aph(3')-IIIa gene responsible for this phenotype is carried on the transposon Tn5405, which may be located on both the chromosome and plasmids. 11
The genetics of streptomycin resistance is somewhat more complex, being associated with an ant(6)-Ia gene, a resistance gene called str, chromosomal mutations (strA), an aph(3')-III gene and an ant(4')-Ia gene. 12 ,13 ,14
To our knowledge no Europe-wide surveillance studies have investigated the status of aminoglycoside resistance in staphylococci since two previous comprehensive studies in 1987 and 1990. 1 ,2 In an attempt to determine the current status of aminoglycoside resistance we investigated the prevalence of the most clinically important aminoglycoside resistance genes, aac(6')-Ie +aph(2''), aph(3')-IIIa and ant(4')-Ia, and the prevalence of resistance to gentamicin, tobramycin, kanamycin, streptomycin and methicillin, in the first 948 unrelated clinical staphylococcal isolates derived from 19 different hospitals as part of the European SENTRY Antimicrobial Surveillance Program.
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Materials and methods |
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All staphylococcal isolates used in this study were isolated between April and November 1997 from 19 hospitals in 12 different European countries as part of the SENTRY Antimicrobial Surveillance Program, as described previously. 15
Susceptibility testing
All isolates were tested, using a disc diffusion method, for resistance to gentamicin, tobramycin, kanamycin, streptomycin and oxacillin. NCCLS guidelines for susceptibility testing and qualitative interpretation were used throughout. 16 Methicillin susceptibility was further confirmed by detection of mecA using a multiplex PCR protocol as described previously. 17
PCR for detection of genes encoding aminoglycoside-modifying enzymes
Every staphylococcal isolate demonstrating resistance to at least one of the aminoglycosides tested was screened for the presence of the three aminoglycoside-modifying enzyme genes of interest. In total 363 staphylococci were analysed, comprising 38 methicillin-susceptible S. aureus (MSSA), 191 methicillin-resistant S. aureus (MRSA), 22 methicillin-susceptible CNS (MSCNS) and 112 methicillin-resistant CNS (MRCNS). In addition, 50 isolates fully susceptible to all of the aminoglycosides tested, comprising 25 S. aureus and 25 CNS, randomly selected, were screened for the presence of aminoglycoside genes as described below.
Oligonucleotide primers for use in a multiplex PCR were selected using published DNA sequences for aac(6')-Ie + aph(2''),ant(4')-Ia and aph(3')-IIIa.18 Primer sequences used were as follows: aac(6')-Ie + aph(2''), 5'-primer, 2022-CCAAGAGCAATAAGGGCATACC; 3'-primer, 2369-CACACTATCATAACCATCACCG; ant(4')-Ia, 5'-primer: 605-CTGCTAAATCGGTAGAAGC; 3'-primer, 777-CAGACCAATCAACATGGCACC; aph(3')-IIIa, 5'-primer: 329-CTGATCGAAAAATACCGCTGC; 3'-primer, 597-TCATACTCTTCCGAGCAAAGG. The specificity of the different sets of primers was tested with DNA extracts of staphylococcal reference strains containing, respectively, aac(6')-Ie + aph(2''), ant(4')-Ia and aph(3')-IIIa genes (kindly supplied by R. Vanhoof, Pasteur Institute of Brabant, Brussels, Belgium). 18
Primers specific for conserved regions of the staphylococcal 16S rRNA gene (5'-primer: 294-GCCGGTGGAGTAACCTTTTAGGAGC; 3'-primer: 1522- AGGAGGTGATCCAACCGCA), were used as additional internal controls. 16 When amplification failed to produce a product corresponding to the target sequence, this was taken as an indication that there was an error in the experimental conditions and hence that the procedure needed to be repeated.
Staphylococcal DNA was extracted by incubating it with lysostaphin followed by purification with a commercially available purification kit (Qiagen, Hilden, Germany) before PCR amplification. Multiplex PCR amplifications were carried out using a GeneAmp PCR System 2400 (Perkin Elmer, Weiterstadt, Germany), and all reagents (GeneAmp dNTPs, High Fidelity Taq DNA polymerase and 10x PCR buffer) were purchased from Perkin Elmer or Boehringer Mannheim (Mannheim, Germany). Ten microlitres of purified DNA solution was added to the PCR mixture, which consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 100 µM dNTPs, 3 U High Fidelity Taq DNA polymerase and 0.4 µM of the respective primers in a final volume of 50 µL. Samples were denatured at 94°C for 10 min followed by 35 amplification cycles using the following parameters: 94°C for 20 s, 55°C for 60 s and 72°C for 50 s. A final extension cycle of 72°C for 10 min was used. After amplification PCR products were resolved using 1.5% (w/v) agarose gels. All isolates were tested at least twice before being considered as positive.
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Results |
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Geographical differences in aminoglycoside resistance in isolates of S. aureus strongly reflected geographical variations in susceptibility to methicillin, as described previously. 15 Thus those countries with a higher prevalence of MRSA, such as Belgium, France, Italy, Portugal and Spain, showed higher levels of aminoglycoside resistance, although there was variation within countries in terms of the prevalence of such resistance phenotypes. Nevertheless, the relationship between the prevalence of methicillin resistance and aminoglycoside resistance remains (Table II).
In CNS the relationship between methicillin resistance and aminoglycoside resistance was less apparent, although approximately one half of all MRCNS showed resistance to gentamicin, tobramycin and kanamycin (Tables III and IV). Whilst in S. aureus the percentage of isolates resistant to streptomycin was comparable to resistance to other aminoglycoside compounds and was clearly associated with methicillin resistance, resistance to streptomycin in CNS was considerably less common and not associated with methicillin resistance (Tables I,II, III, IV).
The prevalence of aminoglycoside resistance genes studied in staphylococci resistant to at least one of the aminoglycoside drugs tested is shown in Table V. Amongst isolates of S. aureus the most prevalent resistance gene was aac(6')-Ie + aph(2''), found in 76% of MRSA and 50% of MSSA. The least common was aph(3')-IIIa, occurring in 7% of MRSA and 13% of MSSA. Similarly in MRCNS, 67% of isolates carried aac(6')-Ie + aph(2''), whilst this gene was detected in only 32% of MSCNS. As in S. aureus, the least common aminoglycoside resistance gene, aph(3')-IIIa, was found more frequently in methicillin-susceptible CNS (50% of isolates) than in methicillin-resistant CNS (20% of isolates). The reverse was true for the other two genes. aph(3')-IIIa was considerably more prevalent amongst aminoglycoside-resistant isolates of CNS than S. aureus (25% versus 8%).
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Discussion |
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Although the data presented here cannot be compared directly with those from the previous ESGAR studies, as a result of different participating hospitals and criteria for including bacterial isolates, our data are at least suggestive of a decrease in susceptibility to gentamicin in S. aureus of nearly 10% over the last decade, whilst susceptibility to gentamicin in CNS has increased by nearly 20%.
CNS remain more resistant than isolates of S. aureus (with the exception of streptomycin) and, as before, southern European countries retain a higher prevalence of aminoglycoside-resistant staphylococci. In common with the ESGAR studies we did not detect significant differences between resistance patterns and site of infection, although S. aureus isolates implicated as the causative agent in hospital-acquired pneumonia tended to be more resistant to methicillin and aminoglycosides, especially gentamicin.
In the 1987-88 ESGAR study 280 aminoglycoside-resistant staphylococci were tested for the presence of aminoglycoside-modifying enzymes. Ten percent of the staphylococci produced ANT(4')-I. The presence of APH(2'') and AAC(6'), alone and also in combination with ANT(4')-I, was found in 51% and 36%of cases, respectively. Three strains produced APH(3')-III alone, while four strains of S. aureus did so in combination with APH(2'') and AAC(6'). As seen in earlier studies, the large majority of resistant staphylococci produced APH(2'') and AAC(6'), alone or in combination with ANT(4')-I. 2
Since PCR is a reliable tool for the identification of aminoglycoside-modifying enzyme genes in staphylococci, 18 it was used in this study to detect the aac(6')-Ie + aph(2''), aph(3')-IIIa and ant(4')-Ia genes in the staphylococci tested, and, hence, the enzymes they encode. The data were used to assess their distribution in S. aureus and CNS isolates. Overall 48% of 363 staphylococci tested carried ANT(4')-I, four to five times more than recorded 10 years ago. 2 As reported in previous ESGAR studies, the bifunctional enzyme was the most common, occurring in 87% of all staphylococci tested. In our study 68% of all staphylococci carried the gene encoding this enzyme, the reduced percentage occurring as a result of reduced prevalence of aminoglycoside resistance in CNS from our study. In contrast to the seven strains (2.5%) detected that carried aph(3')-IIIa in the ESGAR study, we detected 51 isolates (14%) showing an increase in prevalence of this gene amongst aminoglycoside-resistant staphylococci in Europe. All three genes tested occurred Europe-wide with a higher prevalence in southern Europe. Similar observations concerning the presence of aminoglycoside-modifying enzymes were reported in the ESGAR studies. 1 ,2
Resistance to streptomycin occurs at very low frequencies in CNS, unlike S. aureus in which resistance occurs at levels similar to other aminoglycosides tested.
Changes in the prevalence of genes encoding aminoglycoside-modifying enzymes could be caused by changes in antibiotic policies or by the introduction and/or consequent inter-hospital spread of resistant strains, especially MRSA and MRCNS. Additionally, the possibility cannot be excluded that these resistance genes originated from an environmental source. Indeed, saphrophytic staphylococci have frequently been reported to carry resistance determinants, including genes encoding aminoglycoside-modifying enzymes and as such have the capacity to function as a genetic reservoir. 19 Conjugal transfer of resistance determinants between S. aureus and S. epidermidis, leading to rapid dissemination of these determinants in the hospital environment, has been demonstrated. 5 ,20 The higher prevalences of aminoglycoside-resistant CNS compared with S. aureus observed in our study and in the two ESGAR studies are concordant.
In every isolate demonstrating phenotypic resistance to any one of the compounds tested, we detected a known gene encoding an aminoglycoside-modifying enzyme which could account for the phenotype. This suggests that no new aminoglycoside resistance genes are circulating within the staphylococcal population.
In summary, we have demonstrated an increase in the number of aminoglycoside-resistant S. aureus and an upward trend in the proportions of ant(4')-Ia and aph(3')-IIIa genes in aminoglycoside-resistant staphylococci in Europe between 1983 and 1997 which gives cause for concern. Continued surveillance at both the genotypic and phenotypic levels as well as adherence to well-designed antibiotic and infection control policies are necessary to understand and limit further increases.
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Acknowledgments |
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Notes |
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References |
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Received 23 June 1998; returned 7 August 1998; revised 9 September 1998; accepted 17 September 1998