The mef(E)-carrying genetic element (mega) of Streptococcus pneumoniae: insertion sites and association with other genetic elements.
ABSTRACT: The structure of the macrolide efflux genetic assembly (mega) element, its genomic locations, and its association with other resistance determinants and genetic elements were investigated in 16 Streptococcus pneumoniae isolates carrying mef(E), of which 1 isolate also carried tet(M) and 4 isolates also carried tet(M) and erm(B). All isolates carried a mega element of similar size and structure that included the operon mef(E)-msr(D) encoding the efflux transport system. Among tetracycline-susceptible isolates, six different integration sites were identified, five of which were recognized inside open reading frames present in the R6 genome. In the five isolates also carrying tet(M), mega was inserted in different genetic contexts. In one isolate, it was part of previously described Tn916-like element Tn2009. In another isolate, mega was inserted in a transposon similar to Tn2009 that also included an erm(B) element. This new composite transposon was designated Tn2010. Neither Tn2009 nor Tn2010 could be transferred by conjugation to pneumococcal or enterococcal recipients. In the three isolates in which mega was not physically linked with tet(M), this gene was associated with erm(B) in transposon Tn3872, a Tn916-like element. Homologies between the chromosomal insertions of these composite transposons and sequences of multidrug-resistant pneumococcal genomes in the databases indicate the presence of preferential sites for the integration of composite Tn916-like elements carrying multiple resistance determinants in S. pneumoniae.
Project description:The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan(19F)-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.
Project description:The molecular genetics of macrolide resistance were analyzed in 49 clinical pneumococci (including an "atypical" bile-insoluble strain currently assigned to the new species Streptococcus pseudopneumoniae) with efflux-mediated erythromycin resistance (M phenotype). All test strains had the mef gene, identified as mef(A) in 30 isolates and mef(E) in 19 isolates (including the S. pseudopneumoniae strain) on the basis of PCR-restriction fragment length polymorphism analysis. Twenty-eight of the 30 mef(A) isolates shared a pulsed-field gel electrophoresis (PFGE) type corresponding to the England14-9 clone. Of those isolates, 27 (20 belonging to serotype 14) yielded multilocus sequence type ST9, and one isolate yielded a new sequence type. The remaining two mef(A) isolates had different PFGE types and yielded an ST9 type and a new sequence type. Far greater heterogeneity was displayed by the 19 mef(E) isolates, which fell into 11 PFGE types, 12 serotypes (though not serotype 14), and 12 sequence types (including two new ones and an undetermined type for the S. pseudopneumoniae strain). In all mef(A) pneumococci, the mef element was a regular Tn1207.1 transposon, whereas of the mef(E) isolates, 17 carried the mega element and 2 exhibited a previously unreported organization, with no PCR evidence of the other open reading frames of mega. The mef gene of these two isolates, which did not match with the mef(E) gene of the mega element (93.6% homology) and which exhibited comparable homology (91.4%) to the mef(A) gene of the Tn1207.1 transposon, was identified as a novel mef gene variant and was designated mef(I). While penicillin-nonsusceptible isolates (three resistant isolates and one intermediate isolate) were all mef(E) strains, tetracycline resistance was also detected in three mef(A) isolates, due to the tet(M) gene carried by a Tn916-like transposon. A similar mechanism accounted for resistance in four of the five tetracycline-resistant isolates carrying mef(E), in three of which mega was inserted in the Tn916-like transposon, giving rise to the composite element Tn2009. In the fifth mef(E)-positive tetracycline-resistant isolate (the S. pseudopneumoniae strain), tetracycline resistance was due to the presence of the tet(O) gene, apparently unlinked to mef(E).
Project description:The association between the macrolide efflux gene mef(E) and the tet(M) gene was studied in two clinical strains of Streptococcus pneumoniae that belonged to serotypes 19F and 6A, respectively, and that were resistant to both tetracycline and erythromycin. The mef(E)-carrying element mega (macrolide efflux genetic assembly; 5,511 bp) was found to be inserted into a Tn916-like genetic element present in the chromosomes of the two pneumococcal strains. In both strains, mega was integrated at the same site, an open reading frame identical to orf6 of Tn916. The new composite element, Tn2009, was about 23.5 kb and, with the exception of the tet(M)-coding sequence, appeared to be identical in both strains. By sequencing of the junction fragments of Tn2009 at the site of insertion into the chromosome, it was possible to show that (i) the insertion site was identical in the two clinical strains and (ii) the integration of Tn2009 caused a 9.5 kb-deletion in the pneumococcal chromosome. It was not possible to detect the conjugal transfer of Tn2009 to a recipient pneumococcal strain; however, transfer of the whole element by transformation was shown to occur. It is possible to hypothesize that Tn2009 relies on transformation for its spread among clinical strains of S. pneumoniae.
Project description:The diversity of clinical (n = 92) and oral and digestive commensal (n = 120) isolates of Streptococcus salivarius was analyzed by multilocus sequence typing (MLST). No clustering of clinical or commensal strains can be observed in the phylogenetic tree. Selected strains (92 clinical and 46 commensal strains) were then examined for their susceptibilities to tetracyclines, macrolides, lincosamides, aminoglycosides, and phenicol antibiotics. The presence of resistance genes tet(M), tet(O), erm(A), erm(B), mef(A/E), and catQ and associated genetic elements was investigated by PCR, as was the genetic linkage of resistance genes. High rates of erythromycin and tetracycline resistance were observed among the strains. Clinical strains displayed either the erm(B) (macrolide-lincosamide-streptogramin B [MLSB] phenotype) or mef(A/E) (M phenotype) resistance determinant, whereas almost all the commensal strains harbored the mef(A/E) resistance gene, carried by a macrolide efflux genetic assembly (MEGA) element. A genetic linkage between a macrolide resistance gene and genes of Tn916 was detected in 23 clinical strains and 5 commensal strains, with a predominance of Tn3872 elements (n = 13), followed by Tn6002 (n = 11) and Tn2009 (n = 4) elements. Four strains harboring a mef(A/E) gene were also resistant to chloramphenicol and carried a catQ gene. Sequencing of the genome of one of these strains revealed that these genes colocalized on an IQ-like element, as already described for other viridans group streptococci. ICESt3-related elements were also detected in half of the isolates. This work highlights the potential role of S. salivarius in the spread of antibiotic resistance genes both in the oral sphere and in the gut.
Project description:Efflux-mediated macrolide resistance due to mef(E) and mel, carried by the mega element, is common in Streptococcus pneumoniae, for which it was originally characterized, but it is rare in Streptococcus pyogenes In S. pyogenes, mega was previously found to be enclosed in Tn2009, a composite genetic element of the Tn916 family containing tet(M) and conferring erythromycin and tetracycline resistance. In this study, S. pyogenes isolates containing mef(E), apparently not associated with other resistance determinants, were examined to characterize the genetic context of mega. By whole-genome sequencing of one isolate, MB56Spyo009, we identified a novel composite integrative and conjugative element (ICE) carrying mega, designated ICESpy009, belonging to the ICESa2603 family. ICESpy009 was 55 kb long, contained 61 putative open reading frames (ORFs), and was found to be integrated into hylA, a novel integration site for the ICESa2603 family. The modular organization of the ICE was similar to that of members of the ICESa2603 family carried by different streptococcal species. In addition, a novel cluster of accessory resistance genes was found inside a region that encloses mega. PCR mapping targeting ICESpy009 revealed the presence of a similar ICE in five other isolates under study. While in three isolates the integration site was the same as that of ICESpy009, in two isolates the ICE was integrated into rplL, the typical integration site of the ICESa2603 family. ICESpy009 was able to transfer macrolide resistance by conjugation to both S. pyogenes and S. pneumoniae, showing the first evidence of the transferability of mega from S. pyogenes.
Project description:This study was directed at characterizing the genetic elements carrying the methylase gene erm(B), encoding ribosome modification-mediated resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics, in Streptococcus pyogenes. In this species, erm(B) is responsible for MLS resistance in constitutively resistant isolates (cMLS phenotype) and in a subset (iMLS-A) of inducibly resistant isolates. A total of 125 erm(B)-positive strains were investigated, 81 iMLS-A (uniformly tetracycline susceptible) and 44 cMLS (29 tetracycline resistant and 15 tetracycline susceptible). Whereas all tetracycline-resistant isolates carried the tet(M) gene, tet(M) sequences were also detected in most tetracycline-susceptible isolates (81/81 iMLS-A and 7/15 cMLS). In 2 of the 8 tet(M)-negative cMLS isolates, erm(B) was carried by a plasmid-located Tn917-like transposon. erm(B)- and tet(M)-positive isolates were tested by PCR for the presence of genes int (integrase), xis (excisase), and tndX (resolvase), associated with conjugative transposons of the Tn916 family. In mating experiments using representatives of different combinations of phenotypic and genotypic characteristics as donors, erm(B) and tet(M) were consistently cotransferred, suggesting their linkage in individual genetic elements. The linkage was confirmed by pulsed-field gel electrophoresis and hybridization studies, and different elements, variably associated with the different phenotypes/genotypes, were detected and characterized by amplification and sequencing experiments. A previously unreported genetic organization, observed in all iMLS-A and some cMLS isolates, featured an erm(B)-containing DNA insertion into the tet(M) gene of a defective Tn5397, a Tn916-related transposon. This new element was designated Tn1116. Genetic elements not previously described in S. pyogenes also included Tn6002, an unpublished transposon whose complete sequence is available in GenBank, and Tn3872, a composite element resulting from the insertion of the Tn917 transposon into Tn916 [associated with a tet(M) gene expressed in some cMLS isolates and silent in others]. The high frequency of association between a tetracycline-susceptible phenotype and tet(M) genes suggests that transposons of the Tn916 family, so far typically associated solely with a tetracycline-resistant phenotype, may be more widespread in S. pyogenes than currently believed.
Project description:Amongst 100 Streptococcus pneumoniae isolated from clinical cases and nasopharynx of healthy individuals, 60 erythromycin resistant strains were isolated and characterized using MLST, PFGE, transposon analysis and Quellung reaction. Most of the S. pneumoniae erythromycin resistant (80%) were found to be attributable to the ermB-edncoded ribosome methylase activity which differs from the dominant mechanism of macrolide resistance seen in North America. The most predominant transposons were; Tn1545/6003 (27%), Tn6002 (22%), Tn2009 (20%), Tn2010 (17%). Number of the clinical isolates carrying Tn2010 was more significant than the normal flora. The serotypes found were; 14 (33%), 3 (22%), 23F (15%), 19F (15%), 19A (7%), 6A (3%), 9V (3%) and 6B (2%). The most prevalent serotypes among the clinical (n = 28) and normal flora (n = 32) isolates were serotypes 14 (46%) and 3 (31%), respectively. The most prevalent vaccine serotypes amongst the clinical isolates and the healthy individuals were pneumococcal conjugate vaccines (PCV) 13 and PCV10, respectively. PFGE revealed 34 pulsotypes with 9 common and 25 single types. Significant number of the normal isolates belonged to CT5 and CT6. On the other hand, significant number of clinical isolates belonged to CT8 as compared to the normal flora isolates. MLST showed 2 dominant sequence types. ST3130 (23%) and ST180 (22%) were the most predominant sequence types in the clinical and normal isolates, respectively. There was no significant difference in other sequence types between clinical and normal flora isolates. Three polyclonal complexes including Sweden15A -25, Spain23F-1 and Spain9V-3 constituted 58% of the isolates. Our results suggest that the genetic diversity and transposon distribution were high among S. pneumoniae, particularly in the isolates containing erm(B) and double antibiotic resistant genes (erm/mef). The results presented here could influence the change in the current vaccination practices in Iran which currently calls for vaccination with PCV7 or PCV10.
Project description:This study investigated the genetic organization of erm(B)-carrying transposons of Streptococcus pneumoniae and their distribution in tetracycline-resistant clinical isolates. By comparatively analyzing reference pneumococci carrying erm(B)/tet(M) transposon Tn1545, Tn6003, Tn6002, or Tn3872, we demonstrated a substantial correspondence between Tn1545 and Tn6003, which have the same resistance gene combination [tet(M) (tetracycline), erm(B) (erythromycin), and aphA-3 (kanamycin)]; share the macrolide-aminoglycoside-streptothricin element, containing a second erm(B); and only differ by a ca. 1.2-kb insertion (containing a putative IS1239 insertion sequence) detected in Tn1545 from S. pneumoniae reference strain BM4200. These results enabled elucidation of the structure of Tn1545, the first erm(B)-carrying transposon described in S. pneumoniae. A collection of 83 erythromycin- and tetracycline-resistant clinical pneumococci, representative of recent Italian isolates carrying erm(B) as the sole erythromycin resistance gene, was used to investigate the distribution of the different transposons. All 83 organisms were positive for tet(M) and bore an erm(B)/tet(M) transposon that could be characterized by using a specific set of primer pairs; Tn3872 was detected in 18 isolates, Tn6002 in 59 isolates, and Tn6003 in 6 (the sole kanamycin-resistant) isolates. The genetic organization of transposon Tn1545, with its specific insertion, was not detected in any of the isolates tested. The erm(B)-carrying elements of tetracycline-resistant pneumococci substantially corresponded to those [bearing a silent tet(M) gene] recently detected in tetracycline-susceptible pneumococci. Overall, in erm(B)-positive pneumococci, Tn6003 was the least common erm(B)-carrying Tn916-related element and Tn6002 the most common.
Project description:In Streptococcus pyogenes, efflux-mediated erythromycin resistance is associated with the mef gene, represented mostly by mef(A), although a small portion of strains carry different mef subclasses. We characterized the composite genetic elements, including mef subclasses other than mef(A), associated with other resistance genes in S. pyogenes isolates. Determination of the genetic elements was performed by PCR mapping. The strains carrying mosaic mef(A/E), in which the 5' region was identical to mef(A) and the 3' region was identical to mef(E), also carried tet(O). The two genes were found enclosed in an element similar to S. pyogenes prophage ?m46.1, designated the ?m46.1-like element. In S. pyogenes strains carrying mef(E) and tet(M), mef(E) was included in a typical mega element, and in some strains, it was physically associated with tet(M) in the composite element Tn2009. S. pyogenes strains carrying mef(I) also carried catQ; the two genes were linked in a fragment representing a portion of the 5216IQ complex of Streptococcus pneumoniae, designated the defective IQ element. In the only isolate carrying a novel mef gene, this was associated with catQ and tet(M) in a genetic element similar to the 5216IQ complex of S. pneumoniae (5216IQ-like complex), suggesting that the novel mef is in fact a variant of mef(I). This study demonstrates that the composite elements containing mef are shared between S. pyogenes and S. pneumoniae and suggests that it is important to distinguish the mef subclass on the basis of the genetic element containing it.
Project description:Macrolide resistance in Streptococcus pneumoniae has emerged as an important clinical problem worldwide over the past decade. The aim of this study was to analyze the phenotypes (serotype and antibiotic susceptibility), genotypes (multilocus sequence type [MLST] and antibiotic resistance gene/transposon profiles) among the 31% (102/328) of invasive isolates from children in New South Wales, Australia, in 2005 that were resistant to erythromycin. Three serotypes--19F (47 isolates [46%]), 14 (27 isolates [26%]), and 6B (12 isolates [12%])--accounted for 86 (84%) of these 102 isolates. Seventy four (73%) isolates had the macrolide-lincosamide-streptogramin B (MLS(B)) resistance phenotype and carried Tn916 transposons (most commonly Tn6002); of these, 73 (99%) contained the erythromycin ribosomal methylase gene [erm(B)], 34 (47%) also carried the macrolide efflux gene [mef(E)], and 41 (55%) belonged to serotype 19F. Of 28 (27%) isolates with the M phenotype, 22 (79%) carried mef(A), including 16 (57%) belonging to serotype 14, and only six (19%) carried Tn916 transposons. Most (84%) isolates which contained mef also contained one of the msr(A) homologues, mel or msr(D); 38 of 40 (95%) isolates with mef(E) (on mega) carried mel, and of 28 (39%) isolates with mef(A), 10 (39%) carried mel and another 11(39%) carried msr(D), on Tn1207.1. Two predominant macrolide-resistant S. pneumoniae clonal clusters (CCs) were identified in this population. CC-271 contained 44% of isolates, most of which belonged to serotype 19F, had the MLS(B) phenotype, were multidrug resistant, and carried transposons of the Tn916 family; CC-15 contained 23% of isolates, most of which were serotype 14, had the M phenotype, and carried mef(A) on Tn1207.1. Erythromycin resistance among S. pneumoniae isolates in New South Wales is mainly due to the dissemination of multidrug-resistant S. pneumoniae strains or horizontal spread of the Tn916 family of transposons.