Abstract
Analysis of penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) isolates in Hong Kong by use of a combination of antibiogram typing, serotyping, multilocus sequence typing, and pulsed-field gel electrophoresis indicated that the dissemination of PNSP was the result of the spread of international clones: variants of the Spain23F-1 or Spain6B-2 clones were the predominant PNSP isolates from 1994 to 1997 and remained so, but Taiwan19F-14 and Taiwan serotype 6B clones were disseminated in Hong Kong in 1999 and 2000. Concomitant changes in antibiotic susceptibility profiles, with the rate of susceptibility to chloramphenicol rising from 10% in the period from 1994 to 1997 to 31% (P < 0.001) in 1999 and 2000, were noted to accompany the shift of clones.
The prevalence of penicillin-nonsusceptible Streptococcus pneumoniae (PNSP) in Hong Kong has risen rapidly since 1993 and reached 50% by 1997 (4). A concurrent rise in the prevalence of PNSP was also reported in other regions of Asia, with the proportion of PNSP isolates reported to be 71% in Taiwan in 1998 (1) and 79.7% in South Korea in 1999 (10). This is in part due to the result of widespread global dissemination of the Spain23F-1 pneumococcal clone (7, 11). Two prevalent Taiwanese clones were identified by multilocus enzyme sequence typing (MLST), the Taiwan19F-14 clone and the Taiwan23F-15 clone (9), and it was postulated that these Taiwanese clones might have originated in East Asia.
We investigated the contributions of these international and Taiwanese clones to penicillin resistance in S. pneumoniae isolates in Hong Kong. Our previous study that used a combination of antibiograms, capsular types, pbp fingerprintings, and pulsed-field gel electrophoresis (PFGE) profiles showed that 92% (97 of 105) of PNSP isolates recovered from 1994 to 1997 in Hong Kong were indistinguishable from the Spanish serotype 6B clone or the Spanish serotype 23F clone but expressed three serotypes, serotypes 23F, 19F, and 14 (5). MLST was performed with representative isolates from that collection to elucidate further the relatedness of the predominant strains of S. pneumoniae in Hong Kong and their relation to the Taiwanese clones. In addition, clinical pneumococcal isolates from the Prince of Wales Hospital (PWH), Hong Kong, saved during 1999 and 2000 were examined to study the changes in the distribution of these international clones over time.
A total of 118 nonduplicate, consecutive isolates of PNSP isolated from PWH from January 1999 to March 2000 were studied. PWH is a 1,350-bed teaching hospital that serves the eastern section of the New Territories in Hong Kong and approximately one-sixth of Hong Kong's population. Ninety percent (106 of 118 isolates) of the isolates were from sputum; the remaining isolates were from blood, cerebrospinal fluid, and pus swabs. Only isolates for which the penicillin MIC was ≥0.12 μg/ml were analyzed. Eleven representative isolates of PNSP from the previous collection (5) obtained during the period from 1994 to 1997 were also examined. These included nine isolates with a PFGE pattern (group A) that was indistinguishable from that for the Spanish 23F clone but that expressed serotype 23F, 19F, or 14 and two isolates whose PFGE pattern (PFGE group B) was indistinguishable from that of the Spanish 6B clone. These two PFGE groups represented 92% of 105 isolates studied in that period from PWH and Pamela Youde Nethersole Eastern Hospital, Hong Kong. Representatives of well-defined international clones Spain23F-1 and Spain6B-2 (strains Sp267 and Sp681, respectively; gifts from K. P. Klugman) were included for comparison.
Capsular typing of the isolates was by the chessboard agglutination method with Pneumotest antisera (Statens Seruminstitut, Copenhagen, Denmark). Briefly, the pneumococci were typed by using two panels of pooled antisera (12). A positive capsular reaction test was obtained with an antiserum from each panel, and the combination of the two positive reactions corresponded to a particular capsular type. The bacteria agglutinated when a positive capsular reaction was obtained. The MICs of penicillin, cefotaxime, trimethoprim, chloramphenicol, tetracycline, erythromycin, and lincomycin were determined by the NCCLS agar dilution method (8). Strains S. pneumoniae ATCC 6305 and ATCC 49619 were included as controls. MLST was carried out as described previously (3). Internal fragments of seven housekeeping genes, aroE, gdh, gki, recP, spi, xpt, and ddl, were amplified by PCR (3). Sequencing was performed with an ABI 310 sequencer and an ABI Prism d-Rhodamine terminator cycle sequencing kit (Applied Biosystems, Foster City, Calif.), according to the instructions of the manufacturer. The forward and reverse sequences were obtained for each locus. The sequence of each locus was compared with all those of the known alleles from the pneumococcal MLST database, and an appropriate allele number was assigned by use of the software at the pneumococcal MLST website (http://www.mlst.net). PFGE with restriction enzyme SmaI was performed as described previously (5). The detection of macrolide resistance determinants was done by PCR with published primers chosen to be specific for the mef and erm genes (6).
A total of 81% (95 of 118) of the isolates had intermediate susceptibilities to penicillin, and 19% were resistant to penicillin, with the penicillin MICs being 2 μg/ml. Among the 108 strains of PNSP serotyped, only five serotypes were identified, namely, serotypes 23F (46%; 50 of 108 isolates), 19F (28%; 30 of 108 isolates), 6B (16%; 17 of 108 isolates), 14 (9%; 10 of 108 isolates), and 9V (1%; 1 of 108 isolates). Five isolates were nontypeable, and five died and were not available for typing. The distribution of serotypes in this study did not differ from that of the collection of isolates recovered from 1994 to 1997 (5).
By use of antibiotic resistance profiles, two major groups of PNSP from 1999 and 2000 were identified (Table 1). A total of 67% (79 of 118) of the isolates had the resistance profile to penicillin, tetracycline (erythromycin), chloramphenicol, and trimethoprim, and these isolates consisted of four serotypes, namely, serotypes 23F, 19F, 6B, and 14. Representatives from the predominant capsular types were subjected to MLST. All except 1 of the 13 isolates subjected to MLST had multilocus sequence types identical to that of the Spain23F-1 or Spain6B-2 clone; one isolate had a multilocus sequence type identical to that of the England14-9 clone. Twenty-five percent (30 of 118) of the PNSP isolates had the resistance profile to penicillin, tetracycline (erythromycin), and trimethoprim. The multilocus sequence types of all 12 of these isolates indicated that they were identical to that of the Taiwan19F-14 clone or Taiwanese 6B clones. The characteristics of the PNSP isolates and their PFGE and MLST allelic profiles are listed in Table 2. The results for representative isolates recovered from 1994 to 1997 are also shown. MLST performed on representative isolates recovered from 1994 to 1997 indicated that they all belonged to the Spanish clones. Isolates belonging to PFGE group A gave an identical allelic profile, 44-2-4-4-1-1, to that which belonged to the Spain23F-1 clone. Strains that expressed serotype 19F or 14 were variants of the Spain23F-1 clone, likely due to recombinational exchanges of the capsular biosynthetic genes (2). PFGE group B isolates obtained from 1994 to 1997 had allelic profiles identical to that of the Spain6B-2 clone.
TABLE 1.
Antibiotic resistance profiles and serotypes of 118 penicillin-nonsusceptible S. pneumoniae isolatesa isolated at PWH during 1999 and 2000
| Antibiotic resistance profileb | No. (%) of isolates (n = 118) | Serotype(s) (no. of isolates) | MLST sequence type identity (no. of isolates tested) |
|---|---|---|---|
| PT(E)ChTm | 79 (67) | 23F (46) | Spain23F-1 (7) |
| 19F (13) | Spain23F-1-19F (3) | ||
| 6B (11) | Spain6B-2 (2) | ||
| 14 (4) | England14-9 (1) | ||
| NTc (5) | |||
| PT(E)Tm | 30 (25) | 19F (17) | Taiwan19F-14 (9) |
| 6B (6) | Taiwan 6B (3) | ||
| 23F (1) | |||
| 14 (2) | |||
| NT+ (4) | |||
| P(E)ChTm | 3 (2.5) | 23F, 14, NT+ | - |
| Others (PTE, PETm, PTm) | 6 (5) | 23F, 14, 9V | - |
Penicillin MICs, ≥0.12 μg/ml.
Resistance to penicillin (P; MIC, ≥0.1 μg/ml); cefotaxime (C; MIC, ≥0.5 μg/ml), tetracycline (T; MIC, ≥4.0 μg/ml), erythromycin (E; MIC, ≥0.5 μg/ml); chloramphenicol (Ch; MIC, ≥8.0 μg/ml); and trimethoprim (Tm; MIC, ≥1.0 μg/ml).
NT, not done or nontypeable.
TABLE 2.
Serotypes, antibiotic susceptibility patterns, MLST allelic profiles, and PFGE groups of penicillin-nonsusceptible S. pneumoniae isolates recovered from 1994 to 2000
| Yr of isolation | Isolate | PFGE group | Serotype | MLST allelic profile
|
Sequence type identity | Antibiograma | Presence of erm or mef gene | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| aroE | gdh | gki | recP | spi | xpt | ddl | |||||||
| 1994 | 31 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 1995 | 36 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 1996 | 548 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 1994 | 15 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 1995 | 62 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 1996 | 785 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 1996 | 746 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 1996 | 771 | A | 14 | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-14 | PCTEChTm | mef |
| 1997 | 234 | A | 14 | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-14 | PCTEChTm | mef |
| 1994 | 45 | B | 6B | 5 | 6 | 1 | 2 | 6 | 3 | 4 | Spain6B-2 | PCTEChTm | erm |
| 1997 | s2700 | B | 6B | 5 | 6 | 1 | 2 | 6 | 3 | 4 | Spain6B-2 | PCTChTm | |
| 2000 | 384 | 14 | 1 | 5 | 4 | 5 | 5 | 1 | 8 | England14-9 | PEChTm | erm | |
| 1999 | 610 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 1999 | 404 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PTChTm | |
| 1999 | 399 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PTChTm | |
| 2000 | 730 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 2000 | 865 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 2000 | 617 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTEChTm | mef |
| 2000 | 218 | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PTChTm | mef |
| 1999 | 14 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 1999 | 607 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 2000 | 385 | A | 19F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1-19F | PCTEChTm | mef |
| 1999 | 101 | B | 6B | 5 | 6 | 1 | 2 | 6 | 3 | 4 | Spain6B-2 | PTEChTm | erm |
| 2000 | 680 | B | 6B | 5 | 6 | 1 | 2 | 6 | 3 | 4 | Spain6B-2 | PCTEChTm | erm |
| 1999 | 107 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | mef |
| 1999 | 601 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | mef |
| 1999 | 112 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | mef |
| 1999 | 300 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | mef |
| 2000 | 074 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | mef |
| 2000 | 114 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | mef |
| 2000 | 133 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTETm | erm |
| 2000 | 496 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTTm | |
| 2000 | 49 | D | 19F | 15 | 16 | 19 | 15 | 6 | 20 | 1 | Taiwan19F-14 | PTTm | |
| 1999 | 684 | C | 6B | 2 | 13 | 9 | 15 | 6 | 19 | 42 | Taiwan6B | PTETm | mef |
| 1999 | 21 | C | 6B | 2 | 13 | 9 | 15 | 6 | 19 | 42 | Taiwan6B | PTETm | mef |
| 1999 | 205 | C | 6B | 2 | 13 | 9 | 15 | 6 | 19 | 42 | Taiwan6B | PTETm | mef |
| Sp267b | A | 23F | 4 | 4 | 2 | 4 | 4 | 1 | 1 | Spain23F-1 | PCTChTm | ||
| Sp681b | B | 6B | 5 | 6 | 1 | 2 | 6 | 3 | 4 | Spain6B-2 | PCTEChTm | erm | |
Resistance to penicillin (P; MIC, ≥0.1 μg/ml), cefotaxime (C; MIC, ≥0.5 μg/ml), tetracycline (T; MIC, ≥4.0 μg/ml); erythromycin (E; MIC, ≥0.5 μg/ml); chloramphenicol (Ch; MIC, ≥8.0 μg/ml), and trimethoprim (Tm; MIC, ≥1.0 μg/ml).
Reference strains.
The results indicate that there was a shift of the Spanish to the Taiwanese clones, in particular the Taiwan19F-14 clone, in 1999 and 2000 in Hong Kong. The Taiwanese clones differ from the Spanish clones by their susceptibilities to chloramphenicol (MICs, ≤4 μg/ml). These isolates were also resistant to erythromycin (MICs, 2 to 8 μg/ml) and expressed the M phenotype. Serotype 6B isolates with unique allelic profiles (profile 2-13-9-15-6-19-42) identical to that of a Taiwanese serotype 6B isolate from 1996, as described in the S. pneumoniae MLST database (isolate ID406 [Multilocus Sequence Typing website; http://mlst.zoo.ox.ac.uk]), are now also found among our isolates recovered in 1999 and 2000, suggesting their spread to Hong Kong.
This study showed that the high prevalence of PNSP during the early part of the 1990s was the result of the spread of variants of the Spain23F-1 and Spain6B-2 clones. These clones were characterized by multidrug resistance (resistance to erythromycin, tetracycline, and chloramphenicol) and thus led to the widespread presence of erythromycin resistance determinants among pneumococci, particularly the mef gene (6). Chloramphenicol-susceptible isolates accounted for only 10% of the PNSP isolates studied from 1994 to 1997. By 1999, Taiwan19F-14 and Taiwanese serotype 6B clones had also disseminated in Hong Kong. The Taiwan19F-14 clone was described to be the most prevalent clone in Taiwan; it has been present since 1993 and has spread intercontinentally (9). Taiwan19F-14 isolates are characterized by chloramphenicol susceptibility. Previously, loss of antibiotic resistance traits by deletion or inactivation of the resistance gene in the Icelandic serotype 6B clone (13) over a period of time has had a beneficial effect on the clone's susceptibilities to certain antibiotics. However, the impacts of the introduction and spread of new clones with unique antibiotic resistance phenotypes in our region were significant, such that an increase in the rates of chloramphenicol susceptibility, from 10% in 1994 to 1997 to 31% (P < 0.001 by the chi-square test) among PNSP isolates in 1999 and 2000, was observed.
Pneumococcal populations are dynamic, and factors such as population density, antimicrobial selection, and antimicrobial consumption influence the success of the arrival and spread of predominant clones. The loss of the chloramphenicol resistance phenotype due to the spread of new clonal lineages in a short time illustrates the importance of surveillance and the fact that molecular methods such as PFGE and MLST are requisites for the study of these PNSP isolates. New clones originating from Hong Kong have not yet been observed, although it is likely that new clones will originate in areas with high prevalences of PNSP. The Taiwanese serotype 6B clone has not yet been described to have spread sufficiently widely to be classified as one of the international pneumococcal clones, but it may be considered so as more of these isolates are seen in other countries.
Acknowledgments
This publication made use of the Multilocus Sequence Typing website (http://mlst.zoo.ox.ac.uk) developed by Man-Suen Chan at the Wellcome Trust Centre for the Epidemiology of Infectious Disease and funded by the Wellcome Trust. The work described in this paper was partially supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region (projects CUHK 4215/97M and 4431/99M).
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