The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals

Franz-Josef Schmitza,b,*, Ad C. Fluitb, Mechthild Gondolfa, Ralf Beyraua, Elke Lindenlaufa, Jan Verhoefb, Hans-Peter Heinza and Mark E. Jonesb

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aminoglycosides still play an important role in antistaphylococcal therapies, although emerging resistance amongst staphylococci is widespread. To further our understanding of the prevalence of aminoglycoside resistance in Europe, we tested 699 and 249 consecutive unrelated clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS), respectively, from the SENTRY Antimicrobial Surveillance Program, for susceptibility to gentamicin, tobramycin, kanamycin and streptomycin, and examined the relationship between susceptibility to these antimicrobials and susceptibility to methicillin. Three hundred and sixty-three staphylococcal isolates demonstrated resistance to at least one of the aminoglycosides tested; all of these isolates were screened for the presence of aac(6')-Ie + aph(2''),ant(4')-Ia and aph(3')-IIIa, the genes encoding the most clinically relevant aminoglycoside-modifying enzymes. S. aureus isolates derived from hospital-acquired pneumonia tended to be more resistant to aminoglycosides and methicillin than isolates from blood or wound infections. In S. aureus, resistance to aminoglycosides was closely associated with methicillin resistance. Susceptibility of S. aureus to gentamicin has decreased by 9% from previous European studies to a current level of 77%, while susceptibility of CNS, currently at 67%, has increased by 21%. Geographical variation occurred, correlating with methicillin resistance, although intra-country variation was considerable. aac(6')-Ie + aph(2''),ant(4')-Ia and aph(3')-IIIa were found throughout Europe in 68%, 48% and 14% respectively of staphylococci resistant to at least one aminoglycoside. aph(3')-IIIa was considerably more common in methicillin-susceptible S. aureus and CNS isolates; the reverse was true for the other two resistance genes. The prevalence of ant(4')-Ia and aph(3')-IIIa genes in aminoglycoside-resistant staphylococci was significantly greater than that reported in previous European studies.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aminoglycoside antibiotics play an important role in the therapy of serious staphylococcal infections despite reports of increased resistance to these drugs in Europe. 1 ,2 ,3 Aminoglycosides are potent bactericidal agents, inhibiting protein synthesis by binding to the 30S ribosomal subunit. Gentamicin and tobramycin are the most active against staphylococci and are often used in combination with either a ß-lactam or a glycopeptide, especially in the treatment of staphylococcal endocarditis, as these drugs act synergically. 4

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Collection of bacterial isolates

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the S. aureus isolates, 23% were resistant to gentamicin, 29% to tobramycin, 31% to kanamycin and 21% to streptomycin (Table I). The proportion of MRSA that showed resistance to the aminoglycosides tested was 15-18 times higher than that of the MSSA isolates. For example, 75% of MRSA were fully resistant to gentamicin, compared with only 4% of MSSA (Table II). No significant differences in the prevalence of resistance to any of the aminoglycosides tested were apparent between sites 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, demonstrating the close relationship between methicillin and aminoglycoside resistance.


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Table I. Distribution of aminoglycoside resistance in S. aureus isolates from 19 European hospitals
 

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Table II. The distribution of aminoglycoside resistance in isolates of S. aureus in relation to methicillin resistance and sites of isolation
 
Of the CNS isolates, 33% of the strains were resistant to gentamicin, 35% to tobramycin, 49% to kanamycin and 7% to streptomycin (Table III). The differences in the percentage of aminoglycoside resistance between MRCNS and MSCNS were less pronounced than those between MRSA and MSSA. The percentages of MRCNS showing resistance to the aminoglycosides tested were equal (for streptomycin) or seven to eight times higher (for gentamicin and tobramycin) compared with MSCNS isolates. For example, 48%of MRCNS were fully resistant to gentamicin, as compared with only 7% of MSCNS ( Table IV).


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Table III. Distribution of aminoglycoside-resistance in coagulase-negative staphylococci from 19 different hospitals
 

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Table IV. The distribution of aminoglycoside resistance in isolates of coagulase-negative staphylococci (CNS) in relation to methicillin resistance and sites of isolation
 
Overall, MSCNS were more resistant to aminoglycosides than MSSA, whilst MRCNS showed less resistance than MRSA isolates (Tables II and IV). In CNS the association between methicillin resistance and aminoglycoside resistance was less pronounced than that in S. aureus.

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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Table V. The distribution of aminoglycoside resistance genes in isolates of S. aureus and coagulase-negative staphylococci (CNS) in relation to methicillin resistance
 
None of the tested genes encoding aminoglycoside resistance were detectable in any of the 50 fully aminoglycoside-sensitive staphylococci tested.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two Europe-wide surveys investigating aminoglycoside resistance and the presence of aminoglycoside-modifying enzymes have been published previously. 1 ,2 In 1984-85 and in 1987-88 the European Study Group on Antibiotic Resistance (ESGAR), which included 29 laboratories in 12 countries (1984-85) and 37 laboratories in 14 European countries (1987-88), studied aminoglycoside resistance in consecutively collected blood and urine culture isolates. Resistant isolates were tested for the presence of aminoglycoside-modifying enzymes. Higher aminoglycoside resistance rates were reported in the second ESGAR study than in the previous one. In 1987-88, 818 S. aureus and 460 CNS isolates were tested, of which 74% of all staphylococci were susceptible to gentamicin (86% of S. aureus and 46% of CNS tested). Resistance to each of the aminoglycosides tested was lower in northern Europe than in central or southern Europe. CNS were more often aminoglycoside-resistant than S. aureus, although no correlation of aminoglycoside-resistance to methicillin resistance was demonstrated in either study.

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.


    Acknowledgments
 
This work was funded in part by Bristol-Myers Squibb Pharmaceuticals via the SENTRY Antimicrobial Surveillance Program and via European grant number ERBCHRCT940554 for the European Network for Antimicrobial Resistance and Epidemiology (ENARE). We thank the following persons for referring isolates from their institutes for use in this study: Professor Helmut Mittermayer, Professor Marc Struelens, Professor Jacques Acar, Professor Vincent Jarlier, Professor Jerome Etienne, Professor Rene Courcol, Professor Franz Daschner, Professor Ulrich Hadding, Professor Nikos Legakis, Professor Gian-Carlo Schito, Professor Carlo Mancini, Professor Piotr Heczko, Professor Dario Costa, Professor Evilio Perea, Professor Fernando Baquero, Dr Rogelio Martin Alvarez, Professor Jacques Bille and Professor Gary French.


    Notes
 
* Tel/Fax: +49-2132-72040. Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 23 June 1998; returned 7 August 1998; revised 9 September 1998; accepted 17 September 1998