Antimicrobial resistance of Streptococcus pneumoniae isolated in Taiwan: an island-wide surveillance study between 1996 and 1997

Chang-Phone Funga,*, Bor-Shen Hub, Sai-Cheong Leec, Peter Yuk-Fong Liub, Tsrang-Neng Jangd, Hsieh-Shong Leue, Benjamin I. Kuoa, Muh-Yung Yenf, Cheng-Yi Liua, Yung-Chin Liuf, Yeu-Jun Laub and Kwok-Woon Yua

a Section of Infectious Diseases, Department of Medicine, Veterans General Hospital-Taipei and National Yang-Ming University; b Veterans General Hospital-Taichung; c Chang-Gung Memorial Hospital-Keelung, Keelung; d Hsin-Kung Wu Ho-Su Memorial Hospital, Taipei; e Chang-Gung Memorial Hospital-Linkou Medical Center, Taoyuan; f Veterans General Hospital-Kaohsiung, Kaohsiung, Taiwan, Republic of China


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Between August 1996 and July 1997, 550 clinically significant Streptococcus pneumoniae isolates were collected from 14 geographically separate laboratories in Taiwan. These isolates were serotyped and MICs were determined by agar dilution. Among serotypes covered by the 23-valent vaccine, types 19F, 19A, 23F, 23A and 6B dominated, comprising 255 isolates; among non-vaccine serotypes, types 35, 39, 34, 13 and 31 dominated, comprising 118 isolates. Of the 550 isolates, 310 (56.4%) were resistant to penicillin G (MIC 0.12 mg/L), 238 (43.3%) with intermediate resistance (MIC 0.12–1 mg/L) and 72 (13.1%) with high-level resistance (MIC 2 mg/L). Most non-susceptible pneumococci were of serotypes 19F and 23F; non-susceptible isolates of these serotypes were distributed across all of Taiwan. Fourteen other antibiotics were tested; 83% of the isolates were resistant to tetracycline, 78% to azithromycin, 74% to erythromycin, 54% to clindamycin and 23% to chloramphenicol. Thus, macrolides can no longer be used as first line agents to treat pneumococcal infections in Taiwan. Multi-resistance (isolates resistant to three or more chemically unrelated antibiotics) was found in each serotype or group, but mostly in types 19F and 23F. The emergence of such strains complicates antibiotic selection, but both types are covered by the 23-valent vaccine, as were 82% of the isolates from blood and eight of the nine from cerebrospinal fluid. Good antibiotic control and appropriate use of this vaccine may improve the current problem in Taiwan, especially for the elderly.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pneumoniae is one of the leading causes of pneumonia, bacteraemia, meningitis, and upper respiratory tract infections worldwide.1–4 Penicillin has been the drug of choice for nearly half a century, but its value is being eroded by resistance. A few penicillin non-susceptible S. pneumoniae (PNSSP) strains were reported from Boston in 1965, Australia in 1967 and South Africa in 1977.5–7 From the late 1980s onwards, PNSSP have been encountered with increasing frequency from many countries.8–10

In recent Asian studies, PNSSP comprised 9.2% of pneumococci in Pakistan, 11.8% in Bangladesh, 41% in Japan, 70% in Korea and 55.8% in Hong Kong.11–13 Huang et al.14 reported the first two cases of PNSSP meningitis in Taiwan in 1991. Since then, there has been an apparent increase in the prevalence of PNSSP, with local reports indicating rates from 12.2 to 45%,15–17 but there has been no island-wide surveillance.

This study aimed to evaluate the prevalence of antimicrobial resistance among clinical isolates of S. pneumoniae from 14 teaching hospitals in Taiwan in relation to age, region and strain serotype, and to compare the results with those for other Asian countries.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

From July 1996 to June 1997, 14 geographically scattered laboratories in Taiwan collected consecutive clinically significant isolates of S. pneumoniae. Only one isolate was accepted per episode of infection. Isolates from normally sterile body sites were included without further proof of significance; lower respiratory tract isolates were included if the Gram's stain showed >25 white blood cells per high-power field, or if phagocytosis of diplococci was apparent; ear, nose and throat isolates were included if judged to be of clinical significance by the physician responsible for the patient. The isolates were sent to the Veterans General Hospital-Taipei and stored in skimmed milk at –70°C. Speciation was confirmed by Gram-staining, colony morphology on blood agar, susceptibility to optochin discs and bile solubility.

Serotyping

Capsular typing was performed by the Quellung reaction and chessboard methods18 using pool (A to I), group or type (P to T) and major-factor sera, all obtained from Statens Seruminstitut, Copenhagen, Denmark.

Determination of MICs

MICs were determined on Mueller–Hinton agar (Difco, Detroit, MI, USA) supplemented with 5% sheep blood. Ampicillin, co-amoxiclav (2:1), azithromycin, cefotaxime, cefpodoxime, ceftriaxone, clindamycin, erythromycin, imipenem, teicoplanin, tetracycline and vancomycin were obtained as laboratory standard powders from manufacturers; chloramphenicol, penicillin G and rifampicin were purchased from Sigma (St Louis, MO, USA). Inocula were prepared from growth in brain–heart infusion broth (Oxoid, Basingstoke, UK) and comprised 104 cfu/spot, applied with a multipoint inoculator (Denley, Billings-hurst, UK). All plates were incubated in air at 37°C for 18 h. The MIC was defined as the lowest concentration to inhibit visible growth, but a single colony or a fine haze was disregarded when reading the plates. Interpretative criteria for penicillin were: <=0.06 mg/L, susceptible; 0.12–1.0 mg/L, intermediately resistant and >=2.0 mg/L highly resistant.19 Other breakpoints are listed in Table IGo and follow the recommendation of the NCCLS.19 S. pneumoniae ATCC49619 and Staphylococcus aureus ATCC29213 were used as controls.


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Table I. Serotype distribution of S. pneumoniae studied in relation to penicillin susceptibility
 
Statistical analysis

The data were entered into a dBASE III program on a personal computer. Descriptive statistics were generated and correlations between serotypes and other factors were examined by Chi-squared or Fisher's exact test, using the SPSS package. A P value of <0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sources of S. pneumoniae isolates

Five hundred and fifty isolates of S. pneumoniae were included. These comprised 396 isolates from northern Taiwan, 77 from central Taiwan, 66 from southern Taiwan and 11 isolates from the east coast. The patients' ages varied from 4 months to 92 years: 185 isolates (33.6%) were from patients older than 65 years, 123 isolates (22.4%) from children less than 2 years old; 111 isolates (20%) from adults aged between 31 and 65. The 15–29 year age group provided the fewest isolates (3.3%).

Two hundred and two isolates (36.7%) were from sputa, 155 (28.2%) from blood, 55 (10%) from ascitic fluid, 31 (5.6%) from eye swabs, 15 (2.7%) from pleural fluid and nine (1.6%) from cerebrospinal fluid (CSF). Discharges from middle ears, noses and throats yielded 42 (7.6%), 17 (3.1%) and 24 (4.4%) isolates, respectively. Most isolates from patients over 65 years of age were from sputum whereas most from patients aged <15 years were from discharges of the middle ear, nose, throat and eyes. Most isolates were obtained between November and February, with a peak in January.

Serotype distribution

Based on the Danish serotyping system, 348 (63.3%) of the isolates belonged to or were closely related to serotypes covered by the 23-valent vaccine, whereas 162 (29.5%) belonged to non-vaccine types and 40 (7.3%) were non-typeable (Table IIGo). Isolates of types 19F, 23F, 6B, 23A, 35, 39 and 34 comprised 316 organisms, or 57.5% of the total. Among vaccine-or vaccine-related types, 19F, 19A, 23F, 23A and 6B were dominant; among non-vaccine types, 35, 39, 34, 13 and 31 were dominant. Among isolates from blood, 82% (127/155 isolates) belonged to a vaccine- or vaccine- related type, as did eight of nine CSF isolates. Isolates from sputa and swabs or discharges from the middle ear, nose and throat also belonged to either vaccine- or vaccine-related types in over 60% of cases. Type 19F, 23F and 6B isolates were most commonly seen in sputum, blood, middle ear and ophthalmic specimens. There was no relationship between the serotype of isolates and the patients' ages (P > 0.05) except that types 4 and 9V were not seen in those aged under 14 years.


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Table II. MICs of 15 antimicrobial agents for the S. pneumoniae isolates (n = 550)
 
Antimicrobial susceptibility tests

Of the 550 isolates, 310 (56.4%) were resistant to penicillin G: 238 (43.3%) being intermediately resistant and 72 (13.1%) highly resistant (Table IIGo). The great majority of PNSSP belonged to serotypes 23F and 19F; within these types 93% (53/57 isolates) and 90% (85/94), respectively, were resistant to penicillin. Of 14 isolates of type 7, seven exhibited high-level resistance. PNSSP were distributed across all of Taiwan, reaching 65%, 57% and 46% in the central, northern and southern regions, respectively.

Resistance (combined intermediate and resistant strains) was also found to tetracycline (83%) (MIC90 = 32 mg/L); azithromycin (78%) (MIC90 >= 256 mg/L); erythromycin (74%) (MIC90 = 128 mg/L); clindamycin (54%) (MIC90 >= 256 mg/L); chloramphenicol (23%) (MIC90 = 8 mg/L); rifampicin (7%); imipenem (14.5%) (MIC90 = 0.5 mg/L). As for other ß-lactam antibiotics, 49% of the isolates were resistant to co-amoxiclav (MIC90 = 2 mg/L) but fewer than 14% to cefotaxime and ceftriaxone (MIC90 = 1 mg/L). The proportions of isolates resistant to ampicillin and cefpodoxime could not be determined owing to the lack of interpretative criteria; however, the MIC90s of these drugs were as high as for penicillin G. None of the isolates was resistant to teicoplanin or vancomycin (MIC90 = 0.06 and 0.25 mg/L, respectively). Even among isolates susceptible to penicillin, many were resistant to clindamycin (48.4%), tetracycline (40%), erythromycin (41%) and chloramphenicol (21%) (Table IIIGo). PNSSP isolates were more often resistant to other drugs than were penicillin-susceptible isolates. Multidrug-resistant strains (resistant to three or more chemically unrelated agents) were found widely in each serotype or group (FigureGo), but mostly in types 19F and 23F, where simultaneous resistance to penicillins, tetracycline, clindamycin and macrolides was especially frequent.


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Table III. Penicillin resistance and susceptibility among S. pneumoniae isolates resistant to other agents
 


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Figure. Serotype distribution of S. pneumoniae isolates studied ({blacksquare}, isolates resistant to three or more chemically unrelated drugs; {square}, isolates resistant to two or less unrelated drugs; NT, non-typeable).

 
Table IVGo shows the distribution of PNSSP by age. Most resistant isolates were from those aged under 2 years (63.5% resistant) or over 65 years (57.8% resistant). Strains with intermediate resistance were observed most frequently in patients aged under 2 years, whereas those with high-level resistance were mostly from patients aged over 65 years.


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Table IV. Penicillin susceptibility in relation to patient age
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study, covering the whole of Taiwan, showed that 56.4% of S. pneumoniae isolates were PNSSP, 13.1% with high-level resistance. The prevalence of PNSSP was lower than in South Korea (70%), similar to Hong Kong (55.8%), but higher than in Japan (41.8%).11–13 In a 1996 report from southern Taiwan, 12.2% of S. pneumoniae were found to be resistant to penicillin, but only 1.8% had high-level resistance;15 a recent report from Kaohsiung (southern Taiwan) found that 71% (415/584) of S. pneumoniae isolates from nasopharyngeal swab specimens of healthy children were non-susceptible to penicillin with 41% exhibiting high-level resistance.20

Of the present 550 isolates, 348 belonged to types (19F, 23F, 6B, 23A, 19A, 18, 9N, 12, 7, 14, 4, 6A and 9V in descending frequency) covered by the 23-valent vaccine or related to those covered by it. Types 19F, 23F and 6B comprised 58.3% of all vaccine-covered isolates, a proportion which resembles those for South Korea and Hong Kong.12,13

The finding that most strains with high-level resistance to penicillin belonged to serotypes 19F and 23F also resembles the results of studies in South Korea and Hong Kong. In Hong Kong, 20% of PNSSP strains were serotype 23F between 1993 and 1994, as were 62.2% in 1995.13 A South Korean report12 suggested that the rise in PNSSP might be related to excessive prescription of ampicillin and erythromycin to treat upper respiratory tract infections; a Hong Kong report emphasized the possible role of crowding in a small densely populated territory.13 In Taiwan, both crowding and profuse prescription are present; ampicillin and macrolides can be obtained without a prescription.

A French study found that 65% of PNSSP were of serotype 23F;21 moreover, whereas penicillin-susceptible strains exhibited diverse DNA electrophoresis patterns, the resistant 23F isolates gave identical patterns. They suggested that many or all PNSSP 23F isolates might belong to a single clone that had spread widely. Tarasi et al.22 used pulsed-field gel electrophoresis to demonstrate close similarity among 23F isolates from Cleveland (OH), North Carolina, New York and California in the USA, Croatia, Portugal and South Korea, suggesting worldwide spread of a multidrug-resistant clone. People travel frequently between Hong Kong, South Korea and Taiwan, again potentially facilitating spread of the 23F strain. During the surveillance period, we sent several PNSSP strains to the University of Oxford, UK, for multilocus sequence typing: this revealed three major clones among the multidrug-resistant PNSSP from Taiwanese hospitals.23 Of those, two new clones (Taiwan-19F and Taiwan-23F) were not closely related to any previously described PNSSP clones. This finding suggested that new strains are emerging and spreading in Taiwan.

The PNSSP in this study were mostly resistant or had reduced susceptibility to ampicillin and co-amoxiclav, but resistance to third-generation cephalosporin was still rare (<14% for cefotaxime and ceftriaxone). Some investigators have suggested the use of some third-generation cephalosporins to treat patients with PNSSP pneumonia, but treatment failures have been on the rise,24,25 and the regimen can only be considered as a short-term expedient. Most (83%) S. pneumoniae isolates in this study were resistant to tetracycline. This figureGo is close to that found in Hong Kong (78.9%),26 and far higher than the 7.5% rate recorded in the USA.27 The resistance rate to erythromycin (74%) was higher than in Japan (56.7%), South Korea (52%) or Hong Kong (39.2%), and considerably higher than in the USA (10%).11,12,26,27 It is worth noting that 78% of S. pneumoniae isolates were resistant also to azithromycin, a drug only recently made available in Taiwan. Clearly macrolides can no longer be used as first-line drugs to treat pneumococcal infections in Taiwan.27 Resistance to clindamycin (54%) was as frequent as in Japan (58.1%);11 data for other Asian countries were not available. The resistance rate to chloramphenicol (23%) was higher than in the USA (4.3%), but lower than in South Korea (65%) and Hong Kong (37%).12,26,27

The high prevalence of PNSSP, many of them multidrug resistant, increases the need for prophylaxis of pneumococcal infections, especially for those aged over 65 years (57.8% PNSSP were found in this group, who represented 19.5% of the 550 patients). The current 23-valent capsular polysaccharide vaccine has been available since 1983, but was only licensed in Taiwan in 1998. In this study, 82% of blood isolates and eight of the nine from CSF belonged to types covered by this vaccine, or related to those covered by it. Types 19F and 23F—which comprised most of the isolates with high-level penicillin resistance and with multi-drug resistance in patients over 65 years old—are covered by the vaccine. Appropriate use of this vaccine, together with better control of antibiotic usage, may therefore improve the current situation for the elderly population in Taiwan. Unfortunately, the polysaccharide antigens of this vaccine are poorly immunogenic in children aged under 2 years. However, a new generation of protein-conjugate vaccine is under investigation, with encouraging results.28,29 Vaccination of young children with such a new vaccine may resolve the serious problem of pneumococcal resistance in the future.


    Acknowledgments
 
The authors wish to thank the medical and laboratory staff of the following participating laboratories: Chang-Gung Memorial Hospital-Keelung, Keelung; Veteran General Hospital-Taipei, Taipei; Tri-Service Hospital, Taipei; Air Force Hospital, Taipei; Shin-Kung Wu Ho-Su Memorial Hospital, Taipei; MacKay Memorial Hospital, Taipei; The Cathay General Hospital, Taipei; Provincial Hsin-Chu Hospital, Hsin-Chu; Chang-Gung Memorial Hospital– Linkuo Medical Center, Taoyuan; Veteran General Hospital-Taichung, Taichung; China Medical College Hospital, Taichung; Cheng-Tsin General Hospital, Taichung; Chang-Gung Memorial Hospital Kaoshiung, Kaoshiung; Veteran General Hospital-Kaoshiung, Kaoshiung; Lo-Tung Po-Ai Hospital, Lo-Tung. They also thank David M. Livermore for his thoughtful review of the manuscript. This research was supported by a grant from the Department of Health, Taiwan, ROC (DOH86-TD-036) and a grant from Merck Sharp & Dohme Ltd, Taiwan.


    Notes
 
* Correspondence address. Section of Infectious Diseases, Department of Medicine, Veterans General Hospital-Taipei, 201, Section 2, Shih-Pai Road, Taipei, 11217, Taiwan, ROC. Tel: +886-2-2875-7494; Fax: +886-2-2873-0052; E-mail: cpfung{at}vghtpe.gov.tw Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 8 April 1999; returned 12 July 1999; revised 9 August 1999; accepted 8 September 1999