Abstract
Among a collection of 48 multidrug-resistant pneumococcal strains colonizing healthy children in a small municipality of Mérida, Venezuela, we identified sequence types (STs) related to a variety of internationally spreading drug-resistant clones, as well as ST135, thus far isolated only in Europe. The clones invariably harbored one or more of the Tn916-related transposons Tn3872, Tn5253, Tn6002, Tn2009, and Tn2010. Finally, our data suggest both structural rearrangements in certain transposons and occurrence of novel transposable elements.
TEXT
In the past decade, the emergence and spread of resistant Streptococcus pneumoniae strains has caused major concern, and elucidation of the genetic mechanisms involved in the dissemination of drug resistance has become an important subject for study (11). The non-beta-lactam multidrug resistance in S. pneumoniae is related to the Tn916 family of transposons; however, not much is known about their prevalence in different geographical regions.
The city of Mérida is one of the main centers for education and tourism in Western Venezuela, and the Spinetti Dini municipality is located in this metropolitan area. In 2007, its population consisted of 32,630 inhabitants, of which 2,871 were children under the age of 5 years. We investigated S. pneumoniae colonization in the noses and nasopharynges of 475 healthy children, aged 0 to 5 years and living in this municipality, using a sample size well above the calculated size for representative sampling. The prevalence of S. pneumoniae colonization was 27% (n = 126 strains), of which 38% (n = 48 strains) were found to be multidrug-resistant (MDR) strains.
The MDR strains belonged to serotypes 6B (n = 23), 14 (n = 11), 23F (n = 6), 23A (n = 3), 19A (n = 1), and 19F (n = 1); 3 strains were nontypeable. Among all MDR strains (n = 48) we investigated the presence of the Tn916 family of transposons by PCR using the following genes as markers: tetM (14), ermB (13), mefA (15), mefE (15), catpC194 (12), aphA-3 (4), int-Tn916 (8), xis-Tn916 (1), tnpA-Tn917 (5), tnpR-Tn917 (5), and int-Tn5252 (15). According to their serotype and genetic profile, 21 representative strains were selected for multilocus sequence typing (MLST) (9) (Table 1).
Table 1.
Genetic characteristics, transposons, and STs among MDR strainsa
| Serotype | No. of strains with same characteristics | Presence of gene |
Transposon(s) | Code(s) of representative strain(s) selected for MLST | MLST typeb | Related international clonec | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ermB | mefA | mefE | tetM | cat | int-Tn916 | xis-Tn916 | tnpR-Tn917 | tnpA-Tn917 | int-Tn5252 | Aph3′-III | ||||||
| 6B | 7 | + | − | − | + | + | + | + | + | + | + | − | Tn5253 plus Tn917 | I-121 | 90 | Spain6B-2 |
| 6 | + | − | − | + | − | + | + | + | + | + | − | Tn5253 plus Tn917 | I-15 | 90 | Spain6B-2 | |
| 6B | 8 | + | − | − | + | + | + | + | + | + | − | − | Tn3872 | I-163 | 135 | None |
| I-13 | 4835 (SLV135) | |||||||||||||||
| 1 | + | − | − | + | − | + | + | + | + | − | − | Tn3872 | H-183 | 135 | None | |
| 1 | − | − | − | − | + | − | − | − | − | − | − | None | H-222 | 135 | None | |
| 14 | 7 | + | − | − | − | − | + | + | + | + | − | − | Tn3872-related | I-206 | 15 | SLV England14-9 |
| 1 | + | − | − | − | − | + | + | + | + | − | + | SpnRi3erm(B) or SpnAp3erm(B) | H-215 | 15 | SLV England14-9 | |
| 2 | + | − | − | + | − | + | + | − | − | − | − | Tn6002 | H-12 | 2563 | DLV England14-9 | |
| 14 | 1 | − | − | + | − | − | − | − | − | − | − | − | None | I-177 | 156 | Spain9V-3 |
| 23F | 1 | + | − | − | + | + | + | + | + | + | − | − | Tn3872 | I-196 | 5165 (SLV81) | SLV Spain23F-1 |
| 1 | − | − | + | + | + | + | + | − | − | − | − | Tn2009 | I-246 | 81 | Spain23F-1 | |
| 23F | 2 | + | − | − | + | − | + | + | + | + | − | − | Tn3872 | I-176 | 242 | Taiwan23F-15 |
| 1 | − | − | + | + | − | + | + | − | − | − | − | Tn2009 | H-59 | 242 | Taiwan23F-15 | |
| 23F | 1 | + | − | − | + | − | + | + | + | + | − | − | Tn3872 | I-210 | 5166 | DLV Colombia23F-26 |
| 23A | 3 | + | − | − | + | − | + | + | − | − | − | − | Tn6002 | I-120 | 42 | DLV Tennessee23F-4 |
| 19F | 1 | + | − | + | + | − | + | + | − | − | − | − | Tn2010 | I-248 | 5167 (SLV320) | DLV Taiwan19F-14 |
| 19A | 1 | + | − | + | + | − | + | + | − | − | − | − | Tn2010 | H-123 | 320 | DLV Taiwan19F-14 |
| NTd | 1 | − | − | + | + | − | + | + | − | − | − | − | Tn2009 | I-29 | 5168 (SLV1106) | None |
| 1 | − | − | + | + | − | + | + | − | − | − | − | Tn2009 | I-34 | 4837 (DLV1106) | None | |
| 1 | − | − | + | + | − | + | + | − | − | − | − | Tn2009 | H-22 | 4836 (SLV1106) | None | |
All genetic characteristics except for MLST were explored in all 48 MDR strains. MLST was performed in 21 representative strains.
New STs are depicted in bold numbers.
SLV, single-locus variant; DLV, double-locus variant.
NT, nontypeable strains.
We identified nine previously described STs, as well as seven novel STs (4835, 4836, 4837, 5165, 5166, 5167, and 5168). We found strains displaying STs identical to those of Spain6B-2 (ST90), Spain9V-3 (ST156), Spain23F-1 (ST81), and Taiwan23F-15 (ST242) clones, as well as strains having single-locus variant (SLV) or double-locus variant (DLV) types of England14-9 (ST15 and ST2563), Spain23F-1 (ST5165), Tennessee23F-4 (ST42), Taiwan19F-14 (ST320 and ST5167), and Colombia23F-26 clones (ST5166).
In addition to the presence of several international clones, we observed a group of serotype 6B MDR strains showing ST135/SLV135 displaying the same genetic profile (Table 1). Interestingly, at least 20 other ST135/SLV135 strains have been isolated in several European countries, such as Spain, Germany, Austria, United Kingdom, Poland, and Turkey, from healthy carriers as well as from patients suffering from both invasive and noninvasive pneumococcal diseases. These European strains display serotype 6B or 23F, and some of them are MDR (www.mlst.net). Whether these strains have the same genetic characteristics as the serotype 6B ST135/SLV135 clonal group we describe here and, consequently, constitute a pandemic clone remains to be investigated.
Different Tn916-related elements, including Tn1545, Tn3872, Tn6002, and Tn6003, have previously been detected in S. pneumoniae isolates with ermB-mediated erythromycin resistance, with geographical as well as temporal variations in their presence (4). Of these, the first ermB-carrying transposon described in S. pneumoniae (4), Tn1545, has been detected among Denmark14-32, England14-9, Spain14-5, Spain9V-3, and Poland6B-20 clones (3). However, it was not found among our MDR strains.
On the other hand, 10% of the strains in our study carried the combination of ermB, tetM, int-Tn916, and xis-Tn916 genes but not the tnpA-Tn917 and tnpR-Tn917 genes, which suggests the presence of the Tn6002 element. This transposon was detected among strains related to the England14-9 clone as previously described (3), as well as in strains related to the Tennessee23F-4 clone. The Tn6002 element was ranked first among ermB-carrying strains in two previous studies as well (2, 4).
Tn3872 is a composite mobile genetic element resulting from the insertion of the ermB-containing Tn917 transposon into Tn916 (4). In our collection, Tn3872-related elements were the most frequent elements associated with ermB-carrying MDR S. pneumoniae strains (44%) and were distributed across different serotypes, i.e., 6B, 23F, and 14. Further, Tn3872-related elements were found in strains related to different international clones, i.e., England14-9, Spain23F-1, and Taiwan23F-15, as previously described (3), as well as in one strain related to the Colombia23F-26 clone. In strains showing serotype 6B or 23F this transposon contained the expected tetM gene. However, a Tn3872-related element was observed in a group of serotype 14 strains in which the tetM gene was not detected. Interestingly, one of those strains carried the aphA-3 gene. Similarly, Cochetti and coworkers in 2007 described two new Tn3872-related elements, SpnRi3erm(B) and SpnAp3erm(B), which carried the aphA-3 gene but lacked the tetM gene (5).
Another transposon frequently observed among our MDR S. pneumoniae strains, particularly among serotype 6B strains, was Tn5253 (13% of strains). Tn5253 consists of a Tn916-like tetM-carrying element designated Tn5251, inserted within the Tn5252 element, which carries the cat gene (10); this transposon has been described in a variety of international clones (10). In our study, the cat gene could not be detected in some Spain6B-2 strains, suggestive of modifications of the transposon. As hypothesized previously (10), the high degree of variability described within the Tn5253 transposon might explain the absence of the cat gene in those strains. Interestingly, in our strains the tnpA and tnpR genes, which are usually not present on Tn5253 elements, were detected. We hypothesize that an additional Tn917 transposon or a new composite element might be carried by the Spain6B-2 strains circulating in our community.
Among the macrolide-resistant mefE-carrying strains, the presence of genetic elements carrying a mega-element such as Tn2009 and Tn2010 was expected (6). Indeed, the Tn2009 transposon was found among mefE-carrying strains related to the Taiwan23F-15 clone, as previously described (3), as well as in one strain of the Spain23F-1 clone. However, the Tn2010 transposon was detected only in the two ermB+ mefE-carrying strains showing DLV types of the Taiwan19F-14 clone. This Tn2010 transposon has been described in MDR strains of ST320 and other STs belonging to clonal complex 271 that are currently emerging (7).
Interestingly, our results are indicative of horizontal gene transfer processes involving transposable elements within some S. pneumoniae clones. For instance, in the strains related to Spain6B-2, Spain23F-1, Taiwan23F-15, and England14-9 clones, more than one transposon was detected.
Variability in the structure of the Tn5253 and Tn3872 elements was observed among strains belonging to the same clone. The presence of different transposons or of similar transposons with different structures within a clone supports at least two hypotheses. First, it is likely that those clones have entered the population more than once. Second, horizontal gene transfer processes might have occurred within these clones after they entered this community. Mérida is a tourist city, frequently visited by people from all over the world. Consequently, it is plausible that the spread of these clones and transposons has been facilitated by international travel (16).
In summary, our study describes a variety of international resistant clones and a potentially new pandemic clone among MDR S. pneumoniae strains colonizing healthy children in our community. In addition to the demonstration of the presence of a plethora of transposons, our PCR analyses suggest structural changes in certain transposons, as well as the occurrence of new transposable elements in some MDR clones. Our study further demonstrates that nasopharyngeal carriage in children is an important reservoir for perpetuation and dissemination of MDR pneumococcal clones in the community.
Footnotes
Published ahead of print on 25 July 2011.
REFERENCES
- 1. Amezaga M. R., Carter P. E., Cash P., McKenzie H. 2002. Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites. J. Clin. Microbiol. 40:3313–3318 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Calatayud L., et al. 2007. Serotypes, clones, and mechanisms of resistance of erythromycin-resistant Streptococcus pneumoniae isolates collected in Spain. Antimicrob. Agents Chemother. 51:3240–3246 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Calatayud L., et al. 2010. Serotype and genotype replacement among macrolide-resistant invasive pneumococci in adults: mechanisms of resistance and association with different transposons. J. Clin. Microbiol. 48:1310–1316 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Cochetti I., Tili E., Mingoia M., Varaldo P. E., Montanari M. P. 2008. erm(B)-carrying elements in tetracycline-resistant pneumococci and correspondence between Tn1545 and Tn6003. Antimicrob. Agents Chemother. 52:1285–1290 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Cochetti I., et al. 2007. New Tn916-related elements causing erm(B)-mediated erythromycin resistance in tetracycline-susceptible pneumococci. J. Antimicrob. Chemother. 60:127–131 [DOI] [PubMed] [Google Scholar]
- 6. Del Grosso M., Camilli R., Iannelli F., Pozzi G., Pantosti A. 2006. The mef(E)-carrying genetic element (mega) of Streptococcus pneumoniae: insertion sites and association with other genetic elements. Antimicrob. Agents Chemother. 50:3361–3366 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Del Grosso M., Northwood J. G., Farrell D. J., Pantosti A. 2007. The macrolide resistance genes erm(B) and mef(E) are carried by Tn2010 in dual-gene Streptococcus pneumoniae isolates belonging to clonal complex CC271. Antimicrob. Agents Chemother. 51:4184–4186 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Doherty N., Trzcinski K., Pickerill P., Zawadzki P., Dowson C. G. 2000. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44:2979–2984 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Enright M. C., Spratt B. G. 1998. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology 144:3049–3060 [DOI] [PubMed] [Google Scholar]
- 10. Henderson-Begg S. K., Roberts A. P., Hall L. M. 2009. Diversity of putative Tn5253-like elements in Streptococcus pneumoniae. Int. J. Antimicrob. Agents. 33:364–367 [DOI] [PubMed] [Google Scholar]
- 11. Lynch J. P., III, Zhanel G. G. 2009. Streptococcus pneumoniae: does antimicrobial resistance matter? Semin. Respir. Crit. Care Med. 30:210–238 [DOI] [PubMed] [Google Scholar]
- 12. Marchese A., Ramirez M., Schito G. C., Tomasz A. 1998. Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996. J. Clin. Microbiol. 36:2944–2949 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Nagai K., et al. 2001. Evaluation of PCR primers to screen for Streptococcus pneumoniae isolates and beta-lactam resistance, and to detect common macrolide resistance determinants. J. Antimicrob. Chemother. 48:915–918 [DOI] [PubMed] [Google Scholar]
- 14. Olsvik B., Olsen I., Tenover F. C. 1995. Detection of tet(M) and tet(O) using the PCR in bacteria isolated from patients with periodontal disease. Oral Microbiol. Immunol. 10:87–92 [DOI] [PubMed] [Google Scholar]
- 15. Shiojima T., Fujiki Y., Sagai H., Iyobe S., Morikawa A. 2005. Prevalence of Streptococcus pneumoniae isolates bearing macrolide resistance genes in association with integrase genes of conjugative transposons in Japan. Clin. Microbiol. Infect. 11:808–813 [DOI] [PubMed] [Google Scholar]
- 16. Tomasz A., et al. 1998. Molecular epidemiologic characterization of penicillin-resistant Streptococcus pneumoniae invasive pediatric isolates recovered in six Latin-American countries: an overview. PAHO/Rockefeller University Workshop. Pan American Health Organization. Microb. Drug Resist. 4:195–207 [DOI] [PubMed] [Google Scholar]