Rapid increase in macrolide resistance among penicillin non-susceptible pneumococci in Finland, 1996–2000

Marja Pihlajamäki1,*, Tarja Kaijalainen2, Pentti Huovinen1, Jari Jalava and the Finnish Study Group for Antimicrobial Resistance1,§

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.


    Abstract
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 Materials and methods
 Results
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The aims of this study were to evaluate the resistance patterns and serotypes/groups of penicillin non-susceptible pneumococci (PNSP) in Finland, and to determine phenotypes and resistance mechanisms of the erythromycin-resistant isolates. A total of 1190 PNSP were collected during 1996–2000 in Finland. The MICs of 18 antimicrobials were determined by the agar plate dilution method, and PCR was used to study the resistance mechanisms of the macrolide-resistant isolates. For serotyping, counterimmunoelectrophoresis and latex agglutination were used. Erythromycin resistance increased from 32% in 1996 to 62% in 2000 among PNSP in Finland. Multiresistance (co-resistance to erythromycin, tetracycline and co-trimoxazole) was present in 22% of the isolates in 1996 and in 40% in 2000. The most common macrolide resistance phenotype was the MLSB phenotype (72%), 25% had the M phenotype and 3% the MS phenotype. The MLSB and M phenotypes increased in the same proportion during the study period. All the MLSB isolates had the erm(B) gene, the M isolates the mef(A) gene, and in 11 MS isolates, ribosomal mutations were the cause of resistance. The most common serotypes/groups were 14, 19 and 6. We found a significant increase in multiresistance among PNSP within a short period of time in Finland. Although pneumococcal resistance to erythromycin was 11% in 2000, the same figure was 50% among the PNSP. The rise in erythromycin resistance is worrying, as macrolides are commonly used as first- and second-line drugs in pneumococcal infections.


    Introduction
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 Introduction
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Penicillin resistance in Streptococcus pneumoniae (pneumococcus) has increased remarkably during recent decades. Macrolide resistance is also increasing, as well as the number of strains resistant to more than one antimicrobial agent.1 In macrolide-resistant pneumococci, there are two well-characterized resistance mechanisms: target site modification by methylation of the 23S rRNA [caused by the erm(B) gene] and macrolide efflux caused by the mef(A) gene.25 Recently, two more mechanisms have been described; mutations in the 23S rRNA or ribosomal proteins, and another methylase gene, erm(TR), previously described to cause erythromycin resistance in Streptococcus pyogenes.610 The bacteria carrying the erm(B) gene usually have the MLSB phenotype, which means that they are resistant to the 14-, 15- and 16-membered ring macrolides, as well as to clindamycin and streptogramin B. The strains carrying the mef(A) gene are resistant to the 14- and 15-membered ring macrolides, and have the phenotype M. The mutations in pneumococcal 23S rRNA or ribosomal proteins often cause high-level resistance to 14-, 15- and 16-membered ring macrolides and streptogramin B, but the level of clindamycin susceptibility varies from susceptible to low-level resistant: the MS phenotype. The erm(TR) gene has been described in pneumococcus once, and it caused resistance to 14- and 15-membered ring macrolides and inducible resistance to clindamycin.8

We studied the resistance patterns and serotype/group distribution of penicillin non-susceptible pneumococci (PNSP) isolated from clinical samples in Finland during 1996–2000. 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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Bacterial strains

A total of 1190 PNSP (MIC >= 0.125 mg/L) were collected during 1996–2000 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 l’Étoile, 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 Mueller–Hinton 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 manufacturer’s 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 {chi}2 test was used for statistical analyses.


    Results
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Of the 1190 pneumococci, 490 were penicillin resistant and 700 were penicillin intermediate. Ninety-six were invasive isolates (isolates from blood or cerebrospinal fluid samples) and 1094 were non-invasive isolates (mostly from respiratory tract and eye samples). Penicillin MICs varied between 0.125 and 8 mg/L. Of the penicillin-resistant pneumococci, 55% (268 of 490), and of the penicillin-intermediate pneumococci, 47% (330 of 700), were also resistant to erythromycin (Table 1).


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Table 1.  Antimicrobial resistance in penicillin-resistant and penicillin-intermediate pneumococci
 
Among the PNSP, erythromycin resistance increased from 32% in 1996 to 62% in 2000 (P < 0.01). This trend was from 30% to 78% (P < 0.01) in penicillin-resistant isolates, and from 35% to 54% (P < 0.01) in penicillin-intermediate isolates (Figure 1). Multiresistance occurred in 22% of the isolates in 1996 and in 40% in 2000 (P < 0.01). One isolate was resistant to levofloxacin (MIC 16 mg/L). It was isolated from an elderly patient (61 years old) from a blood sample, and was of serogroup 9.



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Figure 1. Antimicrobial resistance (%) of penicillin-intermediate (a) and -resistant (b) pneumococci in Finland in 1996–2000. Triangles, erythromycin; large squares, tetracycline; diamonds, co-trimoxazole; small squares, erythromycin/tetracycline/co-trimoxazole; n, the total number of isolates during the year.

 
Co-resistance to erythromycin or tetracycline was more common among the pneumococci isolated from the respiratory tract than among the invasive isolates, while co-trimoxazole resistance was more frequent in the invasive isolates (Figure 2). Co-trimoxazole-resistant invasive isolates decreased in number from 26 in 1996 to eight in 2000. When comparing three age groups, 0–1 years (n = 488 isolates), 2–7 years (n = 263) and >7 years (n = 384), no remarkable differences were seen in the prevalence of erythromycin, co-trimoxazole or tetracycline resistance, or in multiresistance.



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Figure 2. Resistance properties of PNSP according to the specimen type. Black bars, erythromycin; dark grey bars, tetracycline; light grey bars, co-trimoxazole; white bars, erythromycin/tetracycline/co-trimoxazole.

 
Of the macrolide-resistant pneumococci, 72% (429 of 598) had the MLSB phenotype, 25% (151 of 598) the M phenotype and 3% (20 of 598) the MS phenotype. There were no statistically significant differences in the proportions of the phenotypes between the years 1996 and 2000. Of the invasive macrolide-resistant isolates, 71% (22 of 31) had the MLSB, 27% (eight of 31) the M and 3% (one of 31) the MS phenotype. No erythromycin–clindamycin inducibly resistant strains were found.

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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Figure 3. Co-resistance to chloramphenicol (Chlor), co-trimoxazole (Sut) and tetracycline (Tetra) in erm(B)-positive (white bars) and mef(A)-positive (black bars), erythromycin-resistant pneumococci.

 
Serotypes/groups were determined for all isolates. The most common serotypes/groups were 14, 19, 6 and 23, accounting for 30, 27, 16 and 12% of the isolates tested, respectively (Table 2). These four serotypes/groups were the most common in all age groups. The most common serogroups were serogroup 19 among the isolates resistant to erythromycin, serogroup 23 among chloramphenicol-resistant isolates, serotype 14 among co-trimoxazole-resistant isolates and serogroup 19 among tetracycline-resistant isolates. In multiresistant isolates, 38% of the isolates were serogroup 6 and 31% were serogroup 19. Among both the invasive and non-invasive isolates the three most common serotypes/groups were 14, 19 and 6. Among the 82 invasive co-trimoxazole-resistant pneumococci, 47 were serotype 14.


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Table 2.  The most common serotypes/groups among PNSP, among the isolates simultaneously resistant to erythromycin, tetracycline, co-trimoxazole or chloramphenicol, and among multiresistant isolates
 
The pneumococci were isolated throughout the year. The highest frequency of the isolations was in March, May and April (in that order), and the fewest isolations were made in July and other summer months.

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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Figure 4. Antimicrobial consumption of outpatients in Finland during 1993–2000. Diamonds, penicillin and amoxicillin; squares, macrolides; triangles, co-trimoxazole; crosses, tetracyclines; asterisks, first-generation cephalosporins; circles, fluoroquinolones (hospital use included). DDD/1000 inh/day, defined daily dose per 1000 inhabitants per day.

 

    Discussion
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 Materials and methods
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There was a rapid increase in the prevalence of multiresistance in PNSP in Finland during 1996–2000. The rates of erythromycin and tetracycline resistance more than doubled among penicillin-resistant pneumococci to 78% and 73%, respectively, in 2000. Co-trimoxazole resistance, instead, decreased from 97% in 1996 to 75% in 2000. This phenomenon may be due to the decrease in the consumption of co-trimoxazole during the 1990s, and can be partly explained by the decrease of invasive co-trimoxazole-resistant isolates.

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 1987–1990 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 1997–1998 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 1994–1998, 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.


    Acknowledgements
 
We thank Anna-Liisa Lumiaho, Saija Nylander, Hilkka Ohukainen and Tuula Randell for their excellent technical assistance, and Jari Ahvenainen for the statistical analyses. This work was supported by a grant from the Academy of Finland and by a Special Government Grant.


    Finnish Study Group for Antimicrobial Resistance
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The members of the Finnish Study Group for Antimicrobial Resistance are: Anja Kostiala-Thompson and Merja Rautio (Jorvi Hospital, Espoo); Risto Renkonen and Anna Muotiala (MedixDiacor Laboratory Service, Helsinki); Martti Vaara and Petteri Carlson (Helsinki University Central Hospital, Helsinki); Hannele Jousimies-Somer (Mehiläinen Hospital, Helsinki); Katariina Pekkanen (Yhtyneet Laboratoriot Oy, Helsinki); Jukka Korpela and Ritva Heikkilä (Central Hospital of Kanta-Häme, Hämeenlinna); Suvi-Sirkku Kaukoranta and Heikki Kaukoranta (Central Hospital of North-Karelia, Joensuu); Pirkko Hirvonen and Antti Nissinen (Central Hospital of Keski-Suomi, Jyväskylä); Pekka Ruuska (Central Hospital of Kainuu, Kajaani); Henrik Jägerroos (Central Hospital of Lapland, Rovaniemi); Martti Larikka (Central Hospital of Länsi-Pohja, Kemi); Simo Räisänen (Central Ostrobothnian Hospital District, Kokkola); Ulla Larinkari and Benita Forsblom (Central Hospital of Kymenlaakso, Kotka); Marja-Leena Katila and Ulla Kärkkäinen (Kuopio University Hospital, Kuopio); Hannu Sarkkinen and Pauliina Kärpänoja (Central Hospital of Päijät-Häme, Lahti); Maritta Kauppinen and Seppo Paltemaa (Central Hospital of South-Karelia, Lappeenranta); Päivi Kärkkäinen (Mikkeli Central Hospital, Mikkeli; Savonlinna Central Hospital, Savonlinna); Sylvi Silvennoinen-Kassinen (Deaconess Institution in Oulu, Oulu); Markku Koskela (Oulu University Central Hospital, Oulu); Marja-Liisa Klossner and Sini Pajarre (Central Hospital of Satakunta, Pori); Sinikka Oinonen and Virpi Ratia (Central Hospital of Seinäjoki, Seinäjoki); Paul Grönroos (Koskiklinikka, Tampere); Risto Vuento and Oili Liimatainen (Central Hospital of Tampere, Tampere); Maj-Rita Siro (Health Center Pulssi, Turku); Erkki Eerola and Raija Manninen (University of Turku, Turku); Olli Meurman (Turku University Central Hospital, Turku); Marko Luhtala (Central Hospital of Vaasa, Vaasa); and Katrina Lager (Antimicrobial Research Laboratory, National Public Health Institute, Turku).


    Footnotes
 
* Corresponding author. Tel: +358-2-2519255; Fax: +358-2-2519254; E-mail: mapihla{at}utu.fi Back

§ Members of the Finnish Study Group for Antimicrobial Resistance are listed after the Acknowledgements. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Finnish Study Group for...
 References
 
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