1Antimicrobial Research Laboratory, National Public Health Institute, Kiinamyllynkatu 13, 20520 Turku; 2National Reference Laboratory for Pneumococcus, National Public Health Institute, Oulu, Finland
Received 24 September 2001; returned 2 January 2002; revised 8 February 2002; accepted 15 February 2002.
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Abstract |
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Introduction |
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We studied the resistance patterns and serotype/group distribution of penicillin non-susceptible pneumococci (PNSP) isolated from clinical samples in Finland during 19962000. Macrolide resistance, as well as multiresistance, nearly doubled among the PNSP during the study period. For the macrolide-resistant isolates, the phenotypes and resistance mechanisms were determined.
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Materials and methods |
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A total of 1190 PNSP (MIC 0.125 mg/L) were collected during 19962000 from clinical microbiology laboratories in Finland. Duplicate isolates were excluded from the study by removing repeat isolates from the same patient within a time period of 3 months from the first isolate.
Each laboratory identified pneumococci using their own standard microbiology techniques. In the Antimicrobial Research Laboratory, the identification of the strains was further confirmed by typical colony morphology and haemolysis on blood agar plates (Oxoid Ltd, Basingstoke, UK) supplemented with 5% (v/v) sheep blood. The strains were further tested for optochin sensitivity (Optochin Disc; Oxoid Ltd) and, to confirm unclear results, they were tested with the Slidex Pneumo-Kit (bioMérieux, Marcy lÉtoile, France) agglutination test. All the strains were also serotyped.
MIC testing
The MICs were determined by the agar plate dilution technique. The bacteria were cultured, and incubated for 20 h in 5% CO2 at 35°C on MuellerHinton II (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) agar plates supplemented with 5% (v/v) sheep blood. When testing co-trimoxazole and trimethoprim, lysed horse blood was used. The antibiotics tested were: erythromycin, azithromycin, spiramycin, levofloxacin, clindamycin, cefalothin, cefaclor, cefuroxime, ceftriaxone, tetracycline, ampicillin, chloramphenicol, penicillin, co-trimoxazole, vancomycin, trimethoprim, ciprofloxacin and RP59500 (quinupristin/dalfopristin). Isolates that were simultaneously resistant to erythromycin, tetracycline and co-trimoxazole were considered multiresistant. The NCCLS MIC breakpoints were used.11 A control strain, S. pneumoniae ATCC 49619, was tested together with the isolates.
Macrolide resistance phenotypes
The phenotypes were determined for macrolide-resistant isolates by the double disc method with erythromycin and clindamycin discs (Rosco Neo-sensitabs; A/S Rosco, Taastrup, Denmark), as well as with the MIC data. The double disc test was used for differentiating constitutive and inducible resistance.12
Resistance gene detection
Isolation of DNA for PCR was carried out using the High Pure DNA isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturers instructions. Macrolide resistance genes were detected by PCR.13 They were studied in a random sample of 324 erythromycin-resistant pneumococci with phenotypes MLSB and M, all isolates with the MS phenotype, all erythomycin-intermediately resistant isolates and 50 erythromycin-susceptible isolates. The genes tested were erm(B) and mef(A); erm(TR) was tested in isolates where the two previous tests were negative, in a sample of 50 isolates with the erm(B) or mef(A) gene to look for double mechanisms, and in a sample of 50 erythromycin-susceptible isolates.
The primers for detection of the genes erm(B) and mef(A) have been described previously.14,15 For erm(TR), primers 5'-CTTGTGGAAATGAGTCAACGG-3' [erm(TR) 1] and 5'-TTGTTCATTGGATAATTTATC-3' [erm(TR) 2] were used. S. pyogenes A200 [erm(TR)], S. pyogenes A569 [mef(A)] and Escherichia coli with plasmid pJIR229 [erm(B)] were used as positive controls.
Serotyping
Serotyping of the pneumococci was performed by counterimmunoelectrophoresis, and, for the electrically neutral serotypes/groups 7 and 14, by latex agglutination. The capsular swelling test was used when an uncertain result needed to be confirmed. All antiserum pools, group- or type-specific antisera, as well as factor antisera for subtyping within groups containing the 7-valent vaccine serotypes (6B, 9V, 18C, 19F and 23F) were purchased from Statens Serum Institut, Copenhagen, Denmark.
Antimicrobial consumption rates
Data on the consumption of antibiotics were obtained from the National Agency of Medicines.16 The consumption is expressed as the number of defined daily doses (DDD) per 1000 inhabitants per day.17
Statistical analysis
The 2 test was used for statistical analyses.
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Results |
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The erythromycin MICs of the isolates carrying the mef(A) gene were between 0.5 (intermediate) and 256 mg/L (resistant), while in the isolates with the erm(B) gene, they were between 0.5 and >256 mg/L. The erythromycin MIC50 and MIC90 for mef(A)-positive isolates were 4 and 16 mg/L, respectively, and for erm(B)-positive isolates both values were >256 mg/L. The MIC for one mef(A)-positive isolate was 256 mg/L, while among the erm(B)-positive isolates, 98% had MICs 256 mg/L. All the strains with the erm(B) gene had the phenotype MLSB. All but one of the isolates with the mef(A) gene had the M phenotype, and the one that did not had the MLSB phenotype. Five isolates of the MLSB phenotype carried both mef(A) and erm(B) genes. Co-resistance to chloramphenicol, co-trimoxazole and tetracycline was more common among the strains with the erm(B) than with the mef(A) gene (Figure 3). Of the six erythromycin-intermediate (MIC 0.5mg/L) strains, one had the erm(B) gene and two the mef(A) gene. Resistance was not inducible. The erm(TR) gene was not found. In the 50 erythromycin-susceptible pneumococci tested, neither erm(B) nor mef(A) was found. In addition, there was a total of 20 macrolide-resistant strains with no known acquired resistance gene detected. These isolates had the MS phenotype. In eleven of these strains, mutations in 23S ribosomal RNA or ribosomal protein L4 were the cause of the macrolide resistance.18
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In Finland, the consumption of antimicrobial agents in outpatient care was in decline during the late 1990s (Figure 4). However, the use of the first-generation cephalosporins, macrolides and fluoroquinolones increased from 1993 to 2000. In Figure 4, hospital use of fluoroquinolones (c. 40% of the total use) was included, as the consumption was so low. The use of penicillin and amoxicillin increased until 1995, after which it decreased until 1999. The use of co-trimoxazole and tetracyclines decreased during the 1990s.
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Discussion |
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One could argue that as the isolates were all penicillin non-susceptible, they were more likely to be isolated from patients with chronic infections. This would explain the high level of resistance to the other drugs.
Fluoroquinolone-resistant pneumococci were rare in our study. Only one isolate was resistant to levofloxacin. In Canada, the prevalence of pneumococci with reduced susceptibility to fluoroquinolones was as high as 1.7% in 1997, following a rise in the prescriptions of the fluoroquinolones in that country.19 This may also be seen in Finland, as the consumption of fluoroquinolones is increasing.
The pneumococci isolated from invasive infections were more often susceptible to erythromycin and tetracycline than the isolates of non-invasive origin. Nevertheless, co-trimoxazole resistance was higher in the invasive isolates (Figure 2), which may be due to a clonal spread of pneumococci with certain virulence factors (ability to cause invasive infections) combined with co-trimoxazole resistance determinants. Dominant serotype 14 covered c. 50% of these isolates.
The most common macrolide resistance mechanism was the ribosomal methylation caused by the erm(B) gene (72% of the macrolide-resistant isolates). In most of those isolates, erythromycin MICs were 256 mg/L, indicating that they are clinically very important. Nevertheless, erythromycin resistance in the isolates with either the erm(B) or mef(A) gene varied from erythromycin intermediate resistance to high-level resistance. This indicates a great variation in the expression of the genes. The mef(A) gene was seen in 25% of the macrolide-resistant isolates. Among these, one isolate had the MLSB phenotype. It is possible that this isolate has a mutation as a second resistance mechanism. More multiresistance among the isolates with the erm(B) gene than in those with the mef(A) gene (Figure 3) was seen in our study. It is likely that the genes causing resistance to the other antimicrobials are more often in the same transposon with the erm(B) gene than with the mef(A) gene. According to Seral et al.20 there is an association between erm(B), tet(M) (mediating tetracycline resistance) and catpC194 (mediating chloramphenicol resistance) genes, and our findings are in concordance with those results. Three per cent of the macrolide-resistant isolates were of the MS phenotype. This is a novel phenotype linked to mutations in the ribosomal RNA and proteins. The mutations have been found in several bacterial species and recently also in pneumococci.6,9,10 Mutations were also detected in 11 isolates of our material.18
There are differences in the prevalence of the macrolide-resistant pneumococci, and the distribution of the macrolide resistance genes among different countries and continents. In Finland, macrolide resistance among pneumococci increased from 0.6% in 19871990 to 5.3% in 1997 and 11% in 2000 (Finnish Study Group for Antimicrobial Resistance data21,22 and unpublished results). In the present study, erythromycin resistance was high among the penicillin-resistant and -intermediate pneumococci (55% and 47%), and the erm(B) gene was the most common cause of macrolide resistance. In an Italian study with material collected between 1994 and 1998, 60% of the penicillin-intermediate and 70% of the penicillin-resistant pneumococci were erythromycin resistant.23 Also in Italy, the most common erythromycin resistance mechanism was ribosomal methylation, found in 90% of macrolide-resistant pneumococci.24 In two studies from France, all erythromycin-resistant pneumococci carried the erm(B) gene.25,26 It was hypothesized that this could be explained by the high consumption of the 16-membered ring macrolides in France. In Greece, the proportions of the erm(B) and mef(A) genes among the macrolide-resistant pneumococci were 67.9% and 29.2%, respectively.27 As appears to be typical for Europe, in Belgium the MLSB phenotype was the most common in the 1990s; over 90% of macrolide-resistant pneumococci had the MLSB phenotype and carried the erm(B) gene.28,29 In material collected between 1988 and 1995 in Northern Ireland, 19% of penicillin-resistant pneumococci were also resistant to erythromycin.30 A comparison based on the penicillin MICs was made with clinical isolates of pneumococcus collected during 19971998 in the USA.31 Similar to the present study, it was shown that the macrolide and co-trimoxazole resistance increased as the penicillin MIC increased. In North America, the M phenotype was the most common, covering 55.8% of the macrolide-resistant pneumococci in Canada and 65% in the USA.32,33 In Hong Kong, 92% of PNSP were found to be resistant to erythromycin;34 the strains were collected during 19941998, and the M phenotype was the most common, covering 73% of the macrolide-resistant isolates.
There were certain serotypes/groups that accounted for the majority of the PNSP in our material. The three most common serotypes/groups covered three-quarters of the isolates (Table 2). They were the most common in all age groups, and in both the invasive and non-invasive isolates, while in the isolates resistant to at least one additional antimicrobial drug, the most common serotypes were different.
Outpatient antimicrobial consumption decreased in Finland during the 1990s (Figure 4). Generally speaking, the use of the older antimicrobials decreased, while the use of the newer drugs, such as macrolides, increased. It was previously shown in Finland that regional use of macrolides correlates with the macrolide resistance in pneumococci.22 Concerning the increasing co-resistance to and the increasing use of the macrolides, these results are in concordance with the earlier results.
In conclusion, multiresistance is very common among PNSP in Finland. Of the penicillin-resistant pneumococci, 78% were also resistant to erythromycin in 2000. The most common macrolide resistance mechanism was ribosomal methylation, which commonly causes high-level resistance. In Finland, penicillin non-susceptibility remains low (c. 7%), and amoxicillin and penicillin are drugs of choice for infections caused by pneumococci.22 Our results indicate that wide susceptibility testing and local resistance surveillance of pneumococci, especially PNSP, is essential to determine the optimal antimicrobial therapy.
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Acknowledgements |
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Finnish Study Group for Antimicrobial Resistance |
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Footnotes |
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Members of the Finnish Study Group for Antimicrobial Resistance are listed after the Acknowledgements.
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References |
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