a Department of Microbiology, Division of Infectious Diseases, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Pokfulam, Hong Kong SAR; h School of Professional and Continuing Education, The University of Hong Kong; b Department of Clinical Pathology, Pamela Youde Nethersole Eastern Hospital; c Department of Clinical Pathology, Queen Elizabeth Hospital; d Department of Clinical Pathology, Tuen Mun Hospital; e Department of Clinical Pathology, Kwong Wah Hospital; f Department of Microbiology, Queen Mary Hospital; g Department of Clinical Pathology, Princess Margaret Hospital, Hong Kong SAR, China
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Abstract |
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Introduction |
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Materials and methods |
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During a 4 month period from January to April 2000, isolates of S. pneumoniae were collected from six participant hospital laboratories located in widely separated areas of Hong Kong. These hospitals cover over half of the population in this region. Each laboratory submitted 30 consecutive, non-duplicate isolates of S. pneumoniae derived from clinical samples. Isolates obtained from the same patient during the same episode of infection were only included once. From each laboratory only one isolate per patient accompanied by relevant strain-specific patient data (date of isolation, site of bacterial isolation, age, gender, patient identifying number and co-morbidity) was referred for study. Co-morbidity was defined as any underlying illness that might increase the risk of infection. All isolates were subcultured and re-identified using colony morphology, Gram's stain, optochin susceptibility and bile solubility. Isolates were stored at 20°C until tested in batches. Only pure culture isolates were included in the final study. During the study period, the infection control service in the hospitals did not identify any clustering of pneumococcal diseases.
Antimicrobial agents
Etest strips of penicillin, amoxicillin (as co-amoxiclav 2:1), erythromycin, cefuroxime, cefotaxime, quinupristin/dalfopristin, ciprofloxacin, levofloxacin, sparfloxacin, grepafloxacin, gatifloxacin, moxifloxacin and clinafloxacin were purchased from AB Biodisk, Solna, Sweden. Moxifloxacin and levofloxacin powders with known potencies were kindly provided by Bayer China Company, Ltd, Hong Kong, China and the R. W. Johnson Pharmaceutical Research Institute, Raritan, NJ, USA, respectively.
Determination of MICs and interpretation
Etest MICs were determined according to the manufacturer's instructions. All susceptibility testing was carried out in a single laboratory at the University of Hong Kong. Test inocula were prepared from pneumococcal colonies grown on sheep blood agar that had been incubated for 2024 h in 5% CO2. Colonies were suspended in 0.9% saline to obtain a suspension equivalent to the turbidity of a 0.5 McFarland standard. From this suspension, Etests were performed on MuellerHinton agar with 5% sheep blood (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). The plates were incubated at 35°C in 5% CO2 for 2024 h. MICs falling between two marks on the Etest strip were rounded up to the next highest two-fold dilution, as recommended in the instructions. Broth microdilution tests were performed using the procedures described by the NCCLS. Cation-adjusted MuellerHinton broth (Difco, Detroit, MI, USA) supplemented with 5% horse blood was used as the test medium. Test inocula were prepared as above and further diluted within 15 min to provide a final inoculum density of 5 x 105 cfu/mL in the wells of the microdilution panels. For all MIC determinations, the bacterial inocula were validated by back titration in 10% of the tests to ensure the desired inoculum density. Quality control strains (S. pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922) were included with each run. Interpretation of results was according to published breakpoints of the NCCLS.6 For ciprofloxacin, the criteria were: susceptible, 2 mg/L; resistant,
4 mg/L.2 Fluoroquinolone non-susceptible pneumococci were defined as those for which the MIC of levofloxacin was at least 4 mg/L.
Typing of isolates
The subset of 24 isolates with reduced susceptibility to levofloxacin was examined further by serotyping, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Serotyping of the isolates was determined by the Quellung reaction7 using sera with various reactivities from the Statens Seruminstitut (Copenhagen, Denmark). Protocols for PFGE analysis and MLST have been described previously.8,9 SmaI was used for digestion of DNA in the PFGE analysis and the results were interpreted according to a scheme suggested by Tenover et al.10 Strain SP264 (kindly provided by K. P. Klugman), representative of the Spain23F-1 clone was used as a control in the PFGE and MLST analysis.
Statistical analysis
The 2, Fisher's exact or the KruskalWallis test was used for statistical analysis. A P value of <0.05 was considered significant.
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Results |
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The susceptibilities of the 180 pneumococcal isolates to 13 antimicrobial agents are summarized in Table 1. The MICs of amoxicillin were generally identical or within a two-fold dilution of that of penicillin. High MICs of penicillin (4 mg/L), cefotaxime (4 mg/L) and erythromycin (32
256 mg/L) were found in 11.1% (20/180), 3.9% (7/180) and 25.6% (46/180) of the isolates, respectively. Rates of penicillin non-susceptibility were not significantly different between the various age groups. Penicillin MICs (geomean ± s.d.) were highest in hospital A (0.66 ± 1.1 mg/L), followed by hospitals C (0.51 ± 1.1 mg/L), E (0.5 ± 1.1 mg/L), B (0.23 ± 1.1 mg/L), F (0.21 ± 1.1 mg/L) and D (0.14 ± 0.7 mg/L) (KruskalWallis test, P = 0.001). Between 12.2 and 17.8% of the strains were intermediately resistant or resistant to the seven fluoroquinolones. All fluoroquinolone non-susceptible strains were non-susceptible to penicillin (MIC range 24 mg/L), cefotaxime (MIC range 14 mg/L) and erythromycin (MIC range 4
256 mg/L).
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Discussion |
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This investigation, in agreement with our earlier study,14 showed that fluoroquinolone non-susceptibility in pneumococci is associated with COPD and old age. Of the 24 resistant isolates, 20 (83.3%) were isolated from patients aged 65 years and 14 (58.3%) from patients with COPD. COPD patients are a group that are commonly colonized or infected with S. pneumoniae and when an exacerbation occurs the colony counts increase,15 therefore increasing the likelihood of first-step mutants being present. If such mutants are not killed or eradicated, as might happen when patients are treated with a fluoroquinolone with borderline activity, when an inadequate dose is used or in the case of poor absorption, then a higher level of resistance will emerge. Indeed, a history of suboptimal use of fluoroquinolone was common among our patients with fluoroquinolone-resistant pneumococci.14 Such practice might be related in part to confusion regarding the relative potency, dosage and indications of ofloxacin and levofloxacin. For both ofloxacin and levofloxacin, the 100 mg tablet is the only oral formulation available in Hong Kong, and in MIMS Hong Kong16 (an index of essential prescribing information similar to the British National Formulary in the UK), the recommended doses of both ofloxacin and levofloxacin for various infections, including respiratory tract infections, were 200600 mg/day in one to three divided doses. Finally, a drug efflux mechanism is known to be common in local strains of S. pneumoniae,17 which might also facilitate mutational resistance by permitting short-term bacterial survival. In the study by Chodosh et al.,18 the pneumococcal isolates that persisted in two chronic bronchitis patients treated with ciprofloxacin were found to change from susceptible to resistant. Weiss et al.19 also reported a similar finding recently.
In agreement with reports by other investigators,2,3,5 this and our previous study14 showed that fluoroquinolone non-susceptibility is associated with resistance to penicillin, cephalosporins, co-trimoxazole and macrolides. Molecular analyses confirmed that this finding is due to the dissemination of a multiply resistant clone that shares an identical MLST allelic profile (4-4-2-4-4-1-1) with the globally distributed strain Spain23F-1. This fluoroquinolone-resistant variant, designated Hong Kong23F-1, was found to have serotype 14 and 19F variants. In contrast, fluoroquinolone non-susceptible strains in Spain and Canada were not clonally related.2,5
Dissemination of resistant clones of S. pneumoniae has often been associated with increasing antimicrobial usage.20 In Hong Kong, the most frequently prescribed fluoroquinolones are ofloxacin, ciprofloxacin and levofloxacin (these three fluoroquinolones together constitute >90% of the total usage of this antibiotic class locally). Ofloxacin was licensed for use in this area in 1985, followed by ciprofloxacin in 1988 and levofloxacin in 1994. Data obtained on the inpatient and outpatient use of these three agents from 1994 to 2000 in the Hospital Authority (HA) are shown in the Figure. The HA manages all the public hospitals and specialist outpatient services in this area. Serving a population of 7 million, there are approximately 1 million inpatient discharges, 8 million specialist outpatient attendances and 2 million emergency room attendances each year. Between 1994 and 2000, fluoroquinolone prescriptions increased from 66.8 to 91.8 daily defined doses (DDDs)/1000 persons/year. A breakdown of the usage in terms of clinical indication was not available but we believe that most of the increase is related to increased use for empirical therapy of respiratory tract infections.
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In conclusion, fluoroquinolone resistance in pneumococci has reached disturbingly high rates in Hong Kong both quantitatively and qualitatively. Our data showed that this is related to the emergence and widespread dissemination of a fluoroquinolone-resistant, and multiply-resistant clone. Typing studies suggests that this clone (Hong Kong23F-1) is genetically related to the globally distributed strain Spain23F-1. These findings are of major significance for local patient care, public health and the pharmaceutical industry.
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Acknowledgements |
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Notes |
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References |
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2
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Received 16 January 2001; returned 9 April 2001; revised 30 July 2001; accepted 14 August 2001