Eijkman-Winkler Center, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
Received 15 December 2004; returned 27 January 2005; revised 19 April 2005; accepted 19 April 2005
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Abstract |
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Methods: MICs of antibiotics were determined using broth microdilution. For ß-lactamase-negative isolates with reduced susceptibility to amoxicillin, the nucleotide sequence of the penicillin-binding domain of PBP3 was determined.
Results: The prevalence of ß-lactamase-positive isolates in certain countries has reached 38%. During the period 1997/1998, 8.8% of the isolates were ß-lactamase-negative and non-susceptible to amoxicillin (BLNAR). During the period 2002/2003, 9.6% of the isolates were BLNAR. The emergence of the BLNAR phenotype of H. influenzae was demonstrated in all countries with a prevalence ranging from 2% to 20%. The penicillin-binding domain of PBP3 from 30 sequenced isolates showed known amino acid substitutions, although no amino acid changes were observed in two BLNAR isolates. Clonal spread of BLNAR strains was limited or absent in our study. Both ß-lactamase-producing and BLNAR strains of H. influenzae were fully susceptible to cefixime. However, neither ß-lactamase-producing nor BLNAR isolates were fully susceptible to the other cephalosporins tested. All isolates were also fully susceptible to levofloxacin, moxifloxacin, azithromycin and telithromycin.
Conclusions: The prevalence of the BLNAR phenotype in Europe is increasing, but no new amino acid substitutions were detected in the penicillin-binding domain of PBP3. Cefixime remains a useful treatment option for respiratory tract infections, including in areas with increasing resistance problems.
Keywords: PBP3 , surveillance , cefixime
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Introduction |
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The objective of this study was to test cefixime and comparators against H. influenzae from various European countries prospectively and to compare the susceptibilities of isolates from 1997/1998 with isolates from 2002/2003 paying special attention to the epidemiology of ampicillin resistance mediated by ß-lactamase-production and altered penicillin-binding proteins.
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Materials and methods |
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A total of 915 H. influenzae isolates were tested, comprising 499 isolates from the period 1997/1998 and 416 isolates from the period 2002/2003. The isolates derived from various university hospitals in Austria, France, Germany, The Netherlands, Italy, Spain, Portugal, Poland, UK, Ireland and Turkey and were isolated from the respiratory tract of patients with respiratory tract infections (Table 1). Only one isolate per patient was included. Identification of all isolates was confirmed at the Eijkman-Winkler Institute by colony morphology on chocolate agar and by X and V factor requirements. ß-Lactamase production was determined by the nitrocefin test (Oxoid, Basingstoke, UK). Table 1 shows the number of isolates tested per country broken down by the year of isolation.
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The antimicrobial agents and the dilution ranges tested are listed in Table 2. Microtitre plates containing freeze-dried serial dilutions of the antibiotics were prepared by Trek Diagnostics (East Grinstead, West Sussex, UK). MICs were determined by a microdilution method according to NCCLS guidelines using Haemophilus Test Medium and an inoculum of 5 x 105 cfu/mL. Plates were read after 2024 h incubation at 35°C in ambient air. MICs were recorded as the lowest concentration that inhibited visible growth. H. influenzae ATCC 49247 and ATCC 49766 were used for quality control.14 ß-Lactamase-negative isolates, which were non-susceptible to amoxicillin (MIC 2 mg/L), were categorized as BLNAR. BLNAR isolates were further subdivided into high-BLNAR (amoxicillin MIC
4 mg/L; resistant) and low-BLNAR (amoxicillin MIC 2 mg/L; intermediate resistant).
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Mutations relevant for ß-lactam resistance in H. influenzae occur in the transpeptidase domain of PBP 3B,12,13 which is located between amino acids 320 and 540 of this protein. The coding sequence for this domain was amplified by PCR and sequenced.
A loopful of H. influenzae bacteria was suspended in 200 µL of water. A total of 50 µg of lysozyme was added and the suspension was incubated for 30 min at 37°C. The bacteria were heated for 7 min at 96°C and stored at 20°C until use.
PCR amplification of the sequence encoding the transpeptidase domain of PBP3 was performed with the primer set F1: 5'-TAA TGC GTA ACC GTG CAA TTA C and F2: 5'-ACC ACT AAT GCA TAA CGA GGA TC12 or with the primer set PBP3-F1alt2: AAT CCA AAC CGT GTC GG and PBP3-F2ALT2: CGG AAT AGC ATT TGC ACG TA. Amplifications were performed for 35 cycles of 1 min at 94°C, 1 min at 54°C and 1 min at 72°C using a PE 9600 Thermocycler (Perkin-Elmer, Gouda, The Netherlands). Part of the amplification products was analysed on 1% agarose gel. The remainder was purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany) and recovered in 30 µL of water.
The purified products were sequenced using the Big Dye Terminator Sequence Reaction system and an ABI Prism 377 DNA sequencer (both Applied Biosystems, Warrington, UK). Sequencing was performed with four primers. Besides primers F1 and F2 described above, the primers PBP3.seqexf: 5'-CTG GGC AGA TAT TGA GCG TG and PBP3.seqexr: 5'-CAC GCT CAA TAT CTG CCC AG were used to sequence products obtained with primer set F1 and F2. To sequence the products of the second primer set, the amplification primers were used in combination with primers PBP2.seqF2: TAA ACG CTG GGC AGA TAT TG and PBP2.seqR2: CAATAT CTG CCC AGC GTT TA. Sequence reactions were performed during 25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C using a PE 9600 Thermocycler.
Sequence amplification products were purified by ethanol precipitation and dissolved in 6 µL of formamide loading buffer directly before analysis. Analysis of 2 µL of sequence amplification product solution was performed on a 4% acrylamide gel prepared with Longranger gel solution (Bio Whittaker, Rockland, ME, USA). Electrophoresis was performed for 4 h on an ABI Prism 377 DNA sequencer. DNA sequence data were analysed using Align Plus 2.0 (Scientific Educational Software, Durham, NC, USA) and Chromas 2.0 beta 3 (Technelysium Pty Ltd, Helensvale, Australia). Mutations were determined by comparing the sequence data with the PBP3B DNA sequence from GenBank accession no. L42023.
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Results |
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The results of the susceptibility testing of H. influenzae isolates isolated in 1997/1998 are shown in Table 2. Of the 499 isolates tested, 99 (19.8%) were non-susceptible to amoxicillin. In 55 isolates (11.0%), resistance was due to ß-lactamase production. Forty-four (8.8%) ß-lactamase-negative isolates non-susceptible to amoxicillin (MIC 2 mg/L) were categorized as BLNAR. Of these, 14 (2.8%) were high-BLNAR isolates resistant to amoxicillin (MIC
4 mg/L) and 30 (6.0%) low-BLNAR isolates intermediately resistant to amoxicillin (MIC = 2 mg/L). According to the NCCLS breakpoints, all 499 isolates were susceptible to cefixime, azithromycin, levofloxacin and moxifloxacin, whereas clarithromycin, cefaclor and cefuroxime exhibited reduced susceptibility rates (75.6%, 88.4% and 97.4% susceptibility, respectively). With the exception of the quinolones, cefixime was the most active agent tested with MIC50/90 values of 0.03/0.06 mg/L. It was 32 times more active than cefuroxime and 256 times more active than cefaclor. The activity of telithromycin was comparable to that of azithromycin (MIC50/90 1/2 mg/L) and these two agents were eight times more active than clarithromycin (MIC50/908/16 mg/L). Levofloxacin and moxifloxacin were the most active agents tested (MIC90s
0.03 mg/L). Comparing the activity of the antibiotics against ß-lactamase-positive and ß-lactamase-negative isolates, and excluding amoxicillin, no difference in the MIC90 values was observed. Cefixime was highly active against BLNAR isolates, all being inhibited at 0.5 mg/L. In contrast, only 40.9% of these isolates were susceptible to cefaclor and 81.8% to cefuroxime.
In Table 3, the susceptibility data are presented for the 416 H. influenzae isolates isolated in 2002/2003. Compared with the results for isolates from 1997/1998, the percentage of isolates that were non-susceptible to amoxicillin, cefaclor, cefuroxime and clarithromycin increased slightly from 19.8% to 23.3%, from 11.6% to 15.1%, from 2.6% to 5.0%, and from 24.4% to 28.8%, respectively. Also, the proportion of ß-lactamase-producing isolates increased (11.0% versus 13.7%). Of the 416 isolates, 40 (9.6%) were categorized as BLNAR comprising 13 (3.1%) high-BLNAR (resistant to amoxicillin) and 27 (6.3%) low-BLNAR (intermediate resistant to amoxicillin). The high activity of cefixime against H. influenzae including ß-lactamase-positive and BLNAR isolates was again confirmed: all isolates were inhibited at 0.5 mg/L.
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Amino acid changes in the penicillin binding domain of PBP3
The DNA encoding the transpeptidase domain of PBP3 was sequenced from 30 isolates obtained from 12 centres. DNA sequences diverged between isolates by as much as 7% (data not shown). Two groups of isolates showed identical DNA sequences (18L015/58L085/58L092 and 20C050 and 20C053) and in both groups, two isolates came from the same hospital (58 and 20). Multiple isolates selected from five other centres all differed.
The amino acid substitutions deduced for the isolates are listed in Table 4. Fifteen different amino acid sequences were obtained. For two isolates (05B006 and 26K103), no amino acid substitutions were found when compared with the reference sequence suggesting a different mechanism of resistance. Most isolates had amino acid substitutions at Ala-502 and Asn-526. Only one isolate had a single amino acid substitution (26K105). The isolate that was amoxicillin-susceptible also showed an amino acid change Ala-437Ser.
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Discussion |
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The results of our surveillance study on resistance of H. influenzae in Europe show that the prevalence of ß-lactamase-positive isolates in certain countries like the UK, France, and Spain is high and has reached 38% in Portugal. There was a striking increase in ß-lactamase-positive isolates obtained from Portugal and BLNAR isolates obtained from Poland in the two collection periods (from 7.338.2% and 420%, respectively) (Table 1).
BLNAR isolates were first reported in 1980,1,2 and have generally been reported at low frequencies.4,5 A surveillance study in the USA conducted between December 1997 and May 1998 did not detect any BLNAR.7 Also, the PROTEKT study in the USA showed only 0.4% high-BLNAR for 20012002.10 Another US study conducted in 20002001 involving 1234 isolates from two hospitals reported 0.6% of the H. influenzae as BLNAR.9 However, at the end of the 1990 s, higher frequencies of 4.3% and 3.4% for high-BLNAR were reported from Spain and Japan, respectively.15,16 Here we have shown that during 1997/1998, the frequency of BLNAR was 8.8% with 2.8% of the isolates fully resistant to amoxicillin. During 2002/2003, these percentages increased to 9.6 and 3.1, respectively. It should be noted that a large geographic variation is present with prevalences ranging from 2% in Germany to 20% in Poland. So the prevalence of BLNAR in Europe seems much higher than in the USA.
A large number of silent mutations occurred in the DNA encoding the transpeptidase domain of PBP3, and DNA sequences could diverge as much as 7%. Only two groups of isolates (with three and two representatives) showed identical sequences. In the cluster of three isolates, two were from the same hospital, while the third was from elsewhere. The cluster of two isolates again came from the same hospital. These results indicate that clonal spread of BLNAR isolates was limited or absent in our study population.
Most of the amino acid changes observed have also been described for French and Japanese isolates,12,13,17 and also among 30 BLNAR isolates from the Alexander Project.18 However, amino acid changes for Leu-519 were not observed in our study. Not only amino acid changes in PBP3 may contribute to resistance to ß-lactam antibiotics. Additional mutations in other PBP genes appear to be involved, especially when MICs of amoxicillin for BLNAR isolates are 4 mg/L.19 It has been suggested that changes in PBP2, PBP4 and PBP5 may play a role as well.1921 The involvement of mechanisms other than changes in PBP3 is consistent with our finding of two isolates with no amino acid changes.
In summary, the results of our surveillance study on resistance of H. influenzae in Europe show that the prevalence of ß-lactamase-positive isolates in certain countries like the UK, France, Spain and Portugal is high, reaching up to 38%. The emergence of the BLNAR phenotype of H. influenzae was demonstrated in all countries with a prevalence ranging from 2% in Germany to 20% in Poland. The results of DNA sequencing indicate that clonal spread of BLNAR isolates was limited or absent in our study. In contrast to the other cephalosporins tested, cefixime showed excellent activity against both ß-lactamase-producing and BLNAR isolates of H. influenzae and thus, remains a useful treatment option for respiratory tract infection and in areas with increasing resistance problems.
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Acknowledgements |
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References |
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