Resistance Genes and Genetic Elements Associated with Antibiotic Resistance in Clinical and Commensal Isolates of Streptococcus salivarius.
ABSTRACT: 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: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 Φm46.1, designated the Φm46.1-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: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:Transferable genetic elements conferring macrolide resistance in Streptococcus pneumoniae can encode the efflux pump and ribosomal protection protein, mef(E)/mel, in an operon of the macrolide efflux genetic assembly (Mega) element- or induce ribosomal methylation through a methyltransferase encoded by erm(B). During the past 30 years, strains that contain Mega or erm(B) or both elements on Tn2010 and other Tn916-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 ?g/ml) due to the presence of the Mega element [mef(E)/mel] alone. High-level resistance due to mef(E)/mel 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 erm(B) or known ribosomal mutations. Deletion of mef(E)/mel in these isolates eliminated macrolide resistance. We also found that Mef(E) and Mel of Tn2010-containing pneumococci were functional but the high-level of macrolide resistance was due to Erm(B). Using in vitro competition experiments in the presence of macrolides, high-level macrolide-resistant S. pneumoniae conferred by either Mega-2.IVa or erm(B), had a growth fitness advantage over the lower-level, mef(E)/mel-mediated macrolide-resistant S. pneumoniae phenotypes. These data indicate the ability of S. pneumoniae 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 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:In recent years mef genes, encoding efflux pumps responsible for M-type macrolide resistance, have been investigated extensively for streptococci. mef(I) is a recently described mef variant detected in particular isolates of Streptococcus pneumoniae instead of the more common mef(E) and mef(A). This study shows that mef(I) is located in a new composite genetic element, whose sequence was completely analyzed and the left and right junctions determined, demonstrating a unique genetic organization. The new composite structure (30,505 bp), designated the 5216IQ complex, consists of two halves: a left one (15,316 bp) formed by parts of the known transposons Tn5252 and Tn916, and a right one (15,115 bp) formed by a new fragment, designated the IQ element. While the defective Tn916 contained a silent tet(M) gene, the IQ element, ending with identical transposase genes on both sides and containing the mef(I) gene with an adjacent new msr(D) gene variant and a catQ chloramphenicol acetyltransferase gene, was completely different from the genetic elements carrying other mef genes in pneumococci. This is the first report demonstrating catQ in S. pneumoniae and showing its linkage with a mef gene. Analysis of the chromosomal region beyond the left junction revealed an organization more similar to that of S. pneumoniae strain TIGR4 than to that of strain R6. The 5216IQ complex was apparently nonmobile, with no detectable transfer of erythromycin resistance being obtained in repeated transformation and conjugation assays.
Project description: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: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:Antimicrobial resistance among pneumococci has greatly increased over the past two to three decades. Resistance to tetracycline (tet(M)), chloramphenicol (cat) and macrolides (erm(B) and/or mef(A/E)) is generally conferred by acquisition of specific genes that are associated with mobile genetic elements, including those of the Tn916 and Tn5252 families. The first tetracycline-, chloramphenicol- and macrolide-resistant pneumococci were detected between 1962 and 1970; however, until now the oldest pneumococcus shown to harbour Tn916 and/or Tn5252 was isolated in 1974. In this study the genomes of 38 pneumococci isolated prior to 1974 were probed for the presence of tet(M), cat, erm(B), mef(A/E) and int (integrase) to indicate the presence of Tn916/Tn5252-like elements.Two Tn916-like, tet(M)-containing, elements were identified among pneumococci dated 1967 and 1968. The former element was highly similar to that of the PMEN1 multidrug-resistant, globally-distributed pneumococcal reference strain, which was isolated in 1984. The latter element was associated with a streptococcal phage. A third, novel genetic element, designated ICESpPN1, was identified in the genome of an isolate dated 1972. ICESpPN1 contained a region of similarity to Tn5252, a region of similarity to a pneumococcal pathogenicity island and novel lantibiotic synthesis/export-associated genes.These data confirm the existence of pneumococcal Tn916 elements in the first decade within which pneumococcal tetracycline resistance was described. Furthermore, the discovery of ICESpPN1 demonstrates the dynamic variability of pneumococcal genetic elements and is contrasted with the evidence for Tn916 stability.
Project description:In streptococci mef(I) and catQ, two relatively uncommon macrolide and chloramphenicol resistance genes, respectively, are typically linked in a genetic module designated IQ module. Though variable, the module consistently encompasses, and is sometimes reduced to, a conserved ∼5.8-kb mef(I)-catQ fragment. The prototype IQ module was described in Streptococcus pneumoniae. IQ-like modules have subsequently been detected in Streptococcus pyogenes and in different species of viridans group streptococci, where mef(E) may be found instead of mef(I). Three genetic elements, one carrying the prototype IQ module from S. pneumoniae and two carrying different, defective IQ modules from S. pyogenes, have recently been characterized. All are integrative and conjugative elements (ICEs) belonging to the Tn5253 family, and have been designated ICESpn529IQ, ICESpy029IQ and ICESpy005IQ, respectively. ICESpy029IQ and ICESpy005IQ were the first Tn5253 family ICEs to be described in S. pyogenes. A wealth of new information has been obtained by comparing their genetic organization, chromosomal integration, and transferability. The origin of the IQ module is unknown. The mechanism by which it spreads in streptococci is discussed.
Project description:Long-term macrolide therapy reduces rates of pulmonary exacerbation in bronchiectasis. However, little is known about the potential for macrolide therapy to alter the composition and function of the oropharyngeal commensal microbiota or to increase the carriage of transmissible antimicrobial resistance. We assessed the effect of long-term erythromycin on oropharyngeal microbiota composition and the carriage of transmissible macrolide resistance genes in 84 adults with bronchiectasis, enrolled in the Bronchiectasis and Low-dose Erythromycin Study (BLESS) 48-week placebo-controlled trial of twice-daily erythromycin ethylsuccinate (400 mg). Oropharyngeal microbiota composition and macrolide resistance gene carriage were determined by 16S rRNA gene amplicon sequencing and quantitative PCR, respectively. Long-term erythromycin treatment was associated with a significant increase in the relative abundance of oropharyngeal Haemophilus parainfluenzae (P = 0.041) and with significant decreases in the relative abundances of Streptococcus pseudopneumoniae (P = 0.024) and Actinomyces odontolyticus (P = 0.027). Validation of the sequencing results by quantitative PCR confirmed a significant decrease in the abundance of Actinomyces spp. (P = 0.046). Erythromycin treatment did not result in a significant increase in the number of subjects who carried erm(A), erm(B), erm(C), erm(F), mef(A/E), and msrA macrolide resistance genes. However, the abundance of erm(B) and mef(A/E) gene copies within carriers who had received erythromycin increased significantly (P < 0.05). Our findings indicate that changes in oropharyngeal microbiota composition resulting from long-term erythromycin treatment are modest and are limited to a discrete group of taxa. Associated increases in levels of transmissible antibiotic resistance genes within the oropharyngeal microbiota highlight the potential for this microbial system to act as a reservoir for resistance.IMPORTANCE Recent demonstrations that long-term macrolide therapy can prevent exacerbations in chronic airways diseases have led to a dramatic increase in their use. However, little is known about the wider, potentially adverse impacts of these treatments. Substantial disruption of the upper airway commensal microbiota might reduce its contribution to host defense and local immune regulation, while increases in macrolide resistance carriage would represent a serious public health concern. Using samples from a randomized controlled trial, we show that low-dose erythromycin given over 48 weeks influences the composition of the oropharyngeal commensal microbiota. We report that macrolide therapy is associated with significant changes in the relative abundances of members of the Actinomyces genus and with significant increases in the carriage of transmissible macrolide resistance. Determining the clinical significance of these changes, relative to treatment benefit, now represents a research priority.