Erythromycin resistance and genetic elements carrying macrolide efflux genes in Streptococcus agalactiae.
ABSTRACT: The macrolide resistance determinants and genetic elements carrying the mef(A) and mef(E) subclasses of the mef gene were studied with Streptococcus agalactiae isolated in 2003 and 2004 from 7,084 vaginorectal cultures performed to detect carrier pregnant women. The prevalence of carriage was 18% (1,276 isolates), and that of erythromycin resistance 11.0% (129 of the 1,171 isolates studied). erm(B), erm(A) subclass erm(TR), and the mef gene, either subclass mef(A) or mef(E), were found in 72 (55.8%), 41 (31.8%), and 12 (9.3%) erythromycin-resistant isolates, while 4 isolates had more than 1 erythromycin resistance gene. Of the 13 M-phenotype mef-containing erythromycin-resistant S. agalactiae isolates, 11 had the mef(E) subclass gene alone, one had both the mef(E) and the erm(TR) subclass genes, and one had the mef(A) subclass gene. mef(E) subclass genes were associated with the carrying element mega in 10 of the 12 mef(E)-containing strains, while the single mef(A) subclass gene found was associated with the genetic element Tn1207.3. The nonconjugative nature of the mega element and the clonal diversity of mef(E)-containing strains determined by pulsed-field gel electrophoresis suggest that transformation is the main mechanism through which this resistance gene is acquired.
Project description:Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.
Project description:Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal and maternal sepsis. Penicillin is recommended for intrapartum prophylaxis, but erythromycin or clindamycin is used for penicillin-allergic carriers. Antibiotic resistance (AR) has increased recently and needs to be monitored. We have developed a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to detect, simultaneously, seven genes encoding AR--erm(A/TR), erm(B), mef(A/E), tet(M), tet(O), aphA-3, and aad-6--and two AR-related genes, int-Tn and mreA. We tested 512 GBS isolates from Asia and Australasia and compared mPCR/RLB with antibiotic susceptibility phenotype or single-gene PCR. Phenotypic resistance to tetracycline was identified in 450 (88%) isolates, of which 442 had tet(M) (93%) and/or tet(O) (6%). Of 67 (13%) erythromycin-resistant isolates, 18 were susceptible to clindamycin, i.e., had the M phenotype, encoded by mef(A/E); 39 had constitutive (cMLS(B)) and 10 inducible clindamycin resistance, and of these, 34 contained erm(B) and 12 erm(A/TR). Of four additional isolates with mef(A/E), three contained erm(B) with cMLS(B) and one was erythromycin susceptible. Of 61 (12%) clindamycin-resistant isolates, 20 were susceptible to erythromycin and two had intermediate resistance. Based on sequencing, 21 of 22 isolates with mef had mef(E), and 8 of 353 with int-Tn had an atypical sequence. Several AR genes, erm(B), tet(O), aphA-3, aad-6, and mef(A/E), were significantly more common among Asian than Australasian isolates, and there were significant differences in distribution of AR genes between GBS serotypes. Our mPCR/RLB assay is simple, rapid, and suitable for surveillance of antibiotic resistance in GBS.
Project description:We assessed the mechanisms of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics and related antibiotics in erythromycin-resistant viridans group streptococci (n = 164) and Gemella spp. (n = 28). The macrolide resistance phenotype was predominant (59.38%); all isolates with this phenotype carried the mef(A) or mef(E) gene, with mef(E) being predominant (95.36%). The erm(B) gene was always detected in strains with constitutive and inducible MLS(B) resistance and was combined with the mef(A/E) gene in 47.44% of isolates. None of the isolates carried the erm(A) subclass erm(TR), erm(A), or erm(C) genes. The mel gene was detected in all but four strains carrying the mef(A/E) gene. The tet(M) gene was found in 86.90% of tetracycline-resistant isolates and was strongly associated with the presence of the erm(B) gene. The cat(pC194) gene was detected in seven chloramphenicol-resistant Streptococcus mitis isolates, and the aph(3')-III gene was detected in four viridans group streptococcal isolates with high-level kanamycin resistance. The intTn gene was found in all isolates with the erm(B), tet(M), aph(3')-III, and cat(pC194) gene. The mef(E) and mel genes were successfully transferred from both groups of bacteria to Streptococcus pneumoniae R6 by transformation. Viridans group streptococci and Gemella spp. seem to be important reservoirs of resistance genes.
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:Transferable genetic elements conferring macrolide resistance in <i>Streptococcus pneumoniae</i> can encode the efflux pump and ribosomal protection protein, <i>mef</i>(E)/<i>mel</i>, in an operon of the macrolide efflux genetic assembly (Mega) element- or induce ribosomal methylation through a methyltransferase encoded by <i>erm</i>(B). During the past 30 years, strains that contain Mega or <i>erm</i>(B) or both elements on Tn<i>2010</i> and other Tn<i>916</i>-like composite mobile genetic elements have emerged and expanded globally. In this study, we identify and define pneumococcal isolates with unusually high-level macrolide resistance (MICs > 16 μg/ml) due to the presence of the Mega element [<i>mef</i>(E)/<i>mel</i>] alone. High-level resistance due to <i>mef</i>(E)/<i>mel</i> was associated with at least two specific genomic insertions of the Mega element, designated Mega-2.IVa and Mega-2.IVc. Genome analyses revealed that these strains do not possess <i>erm</i>(B) or known ribosomal mutations. Deletion of <i>mef</i>(E)/<i>mel</i> in these isolates eliminated macrolide resistance. We also found that Mef(E) and Mel of Tn<i>2010</i>-containing pneumococci were functional but the high-level of macrolide resistance was due to Erm(B). Using <i>in vitro</i> competition experiments in the presence of macrolides, high-level macrolide-resistant <i>S. pneumoniae</i> conferred by either Mega-2.IVa or <i>erm</i>(B), had a growth fitness advantage over the lower-level, <i>mef</i>(E)/<i>mel</i>-mediated macrolide-resistant <i>S. pneumoniae</i> phenotypes. These data indicate the ability of <i>S. pneumoniae</i> to generate high-level macrolide resistance by macrolide efflux/ribosomal protection [Mef(E)/Mel] and that high-level resistance regardless of mechanism provides a fitness advantage in the presence of macrolides.
Project description: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 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: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.
Project description:Macrolide (including erythromycin and azithromycin) and lincosamide (including clindamycin) antibiotics are recommended for treatment of penicillin-allergic patients with Streptococcus pyogenes pharyngitis. Resistance to erythromycin in S. pyogenes can be as high as 48% in specific populations in the United States. Macrolide and lincosamide resistance in S. pyogenes is mediated by several different genes. Expression of the erm(A) or erm(B) genes causes resistance to erythromycin and inducible or constitutive resistance to clindamycin, respectively, whereas expression of the mef(A) gene leads to resistance to erythromycin but not clindamycin. We studied the resistance of S. pyogenes to erythromycin and clindamycin at an urban tertiary-care hospital. Of 196 sequential isolates from throat cultures, 15 (7.7%) were resistant to erythromycin. Three of these were also constitutively resistant to clindamycin and had the erm(B) gene. Five of the erythromycin-resistant isolates were resistant to clindamycin upon induction with erythromycin and had the erm(A) gene. The remaining seven erythromycin-resistant isolates were susceptible to clindamycin even upon induction with erythromycin and had the mef(A) gene. Pulsed-field gel electrophoresis analysis and emm typing demonstrated that the erythromycin-resistant S. pyogenes comprised multiple strains. These results demonstrate that multiple mechanisms of resistance to macrolide and lincosamide antibiotics are present in S. pyogenes strains in the United States.