Prophage association of mef(A) elements encoding efflux-mediated erythromycin resistance in Streptococcus pyogenes.

OBJECTIVES: To compare different mef(A) elements of Streptococcus pyogenes for a possible chimeric genetic nature, i.e. a transposon inserted into a prophage. METHODS: Eleven S. pyogenes isolates with efflux-mediated erythromycin resistance were used. The isolates were typed using several genotypic approaches. Gene detection was performed by PCR using specific primer pairs. The mef(A) elements of the test strains were induced with mitomycin C and phage DNA was extracted. Induction was monitored by PCR using primers targeting mef(A). RESULTS: Six tetracycline-susceptible isolates had PCR evidence of all of the eight open reading frames (ORFs) of the Tn1207.1 element; their mef(A) element was consistent with the Tn1207.3 element in four isolates and with the 58.8 kb chimeric element in two. Five tetracycline-resistant isolates had no PCR evidence of orf1 and orf2 and showed variable patterns as to orf3, orf7, and orf8. Three ORFs placed along the conserved region downstream of Tn1207.1 in the 58.8 kb mef(A) chimeric element were detected in the six tetracycline-susceptible, but not in the five tetracycline-resistant isolates. Induction assays with mitomycin C demonstrated that the mef(A) elements of all strains tested were present in culture supernatants in a DNAse-resistant form, such as a phage capsid. CONCLUSIONS: All recognized mef(A) elements of S. pyogenes appear to be prophage-associated. Whereas the two elements detected in tetracycline-susceptible isolates (Tn1207.3 and the 58.8 kb one) were apparently inserted into the same prophage, the tet(O)-mef(A) element was inserted into a different prophage. Phage transfer is likely to play a critical role in the dissemination of erythromycin resistance in S. pyogenes populations

Analysis of a Streptococcus pyogenes puerperal sepsis cluster using whole-genome sequencing

Between June and November 2010, a concerning rise in the number of cases of puerperal sepsis, a postpartum pelvic bacterial infection contracted by women after childbirth, was observed in the New South Wales, Australia, hospital system. Group A streptococcus (GAS; Streptococcus pyogenes) isolates PS001 to PS011 were recovered from nine patients. Pulsed-field gel electrophoresis and emm sequence typing revealed that GAS of emm1.40, emm75.0, emm77.0, emm89.0, and emm89.9 were each recovered from a single patient, ruling out a single source of infection. However, emm28.8 GAS were recovered from four different patients. To investigate the relatedness of these emm28 isolates, whole-genome sequencing was undertaken and the genome sequences were compared to the genome sequence of the emm28.4 reference strain, MGAS6180. A total of 186 single nucleotide polymorphisms were identified, for which the phylogenetic reconstruction indicated an outbreak of a polyclonal nature. While two isolates collected from different hospitals were not closely related, isolates from two puerperal sepsis patients from the same hospital were indistinguishable, suggesting patient-to-patient transmission or infection from a common source. The results of this study indicate that traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates. Whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease

Contribution of Exogenous Genetic Elements to the Group A Streptococcus Metagenome

Variation in gene content among strains of a bacterial species contributes to biomedically relevant differences in phenotypes such as virulence and antimicrobial resistance. Group A Streptococcus (GAS) causes a diverse array of human infections and sequelae, and exhibits a complex pathogenic behavior. To enhance our understanding of genotype-phenotype relationships in this important pathogen, we determined the complete genome sequences of four GAS strains expressing M protein serotypes (M2, M4, and 2 M12) that commonly cause noninvasive and invasive infections. These sequences were compared with eight previously determined GAS genomes and regions of variably present gene content were assessed. Consistent with the previously determined genomes, each of the new genomes is ∼1.9 Mb in size, with ∼10% of the gene content of each encoded on variably present exogenous genetic elements. Like the other GAS genomes, these four genomes are polylysogenic and prophage encode the majority of the variably present gene content of each. In contrast to most of the previously determined genomes, multiple exogenous integrated conjugative elements (ICEs) with characteristics of conjugative transposons and plasmids are present in these new genomes. Cumulatively, 242 new GAS metagenome genes were identified that were not present in the previously sequenced genomes. Importantly, ICEs accounted for 41% of the new GAS metagenome gene content identified in these four genomes. Two large ICEs, designated 2096-RD.2 (63 kb) and 10750-RD.2 (49 kb), have multiple genes encoding resistance to antimicrobial agents, including tetracycline and erythromycin, respectively. Also resident on these ICEs are three genes encoding inferred extracellular proteins of unknown function, including a predicted cell surface protein that is only present in the genome of the serotype M12 strain cultured from a patient with acute poststreptococcal glomerulonephritis. The data provide new information about the GAS metagenome and will assist studies of pathogenesis, antimicrobial resistance, and population genomics

Targeted Curing of All Lysogenic Bacteriophage from Streptococcus pyogenes Using a Novel Counter-selection Technique

We thank the members of the Laboratory of Microbial Pathogenesis and Immunology, especially Annette Nelkenbaum and Ben Winer for their technical assistance. We also thank Estee Colleen Cervantes and Sutapa Banerjee from Hunter College for their technical contribution to this project. We are grateful to Joseph Ferretti for S. pyogenes strain SF370.Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.Yeshttp://www.plosone.org/static/editorial#pee

SmaI Typeability and Tetracycline Susceptibility and Resistance in Streptococcus pyogenes Isolates with Efflux-Mediated Erythromycin Resistance▿

These findings contribute to conclusively clarifying why, among S. pyogenes isolates with the M phenotype, the tetracycline- resistant ones are, as a rule, SmaI typeable, while the tetracycline-susceptible ones are not. Previous studies have shown that, while the two related genetic elements (Tn1207.3 and 10394.4) typical of tetracycline-susceptible isolates are inserted into the same prophage, the tet(O)-mef(A) element typical of tetracycline-resistant isolates is inserted into a different prophage (6). The present results show that the R-M cassette encoding, in Tn1207.3-10394.4, the methyltransferase responsible for DNA resistance to cleavage by SmaI is absent in the tet(O)-mef(A) element where, together with an adjacent ORF, it is replaced by three new ORFs flanked, both upstream and downstream, by homologous regions

Two distinct genetic elements are responsible for erm(TR)-mediated erythromycin resistance in tetracycline-susceptible and tetracycline-resistant strains of Streptococcus pyogenes.

In Streptococcus pyogenes, inducible erythromycin (ERY) resistance is due to posttranscriptional methylation of an adenine residue in 23S rRNA that can be encoded either by the erm(B) gene or by the more recently described erm(TR) gene. Two erm(TR)-carrying genetic elements, showing extensive DNA identities, have thus far been sequenced: ICE10750-RD.2 (∼49 kb) and Tn1806 (∼54 kb), from tetracycline (TET)-susceptible strains of S. pyogenes and Streptococcus pneumoniae, respectively. However, TET resistance, commonly mediated by the tet(O) gene, is widespread in erm(TR)-positive S. pyogenes. In this study, 23 S. pyogenes clinical strains with erm(TR)-mediated ERY resistance-3 TET susceptible and 20 TET resistant-were investigated. Two erm(TR)-carrying elements sharing only a short, high-identity erm(TR)-containing core sequence were comprehensively characterized: ICESp1108 (45,456 bp) from the TET-susceptible strain C1 and ICESp2905 (65,575 bp) from the TET-resistant strain iB21. While ICESp1108 exhibited extensive identities to ICE10750-RD.2 and Tn1806, ICESp2905 showed a previously unreported genetic organization resulting from the insertion of separate erm(TR)- and tet(O)-containing fragments in a scaffold of clostridial origin. Transferability by conjugation of the erm(TR) elements from the same strains used in this study had been demonstrated in earlier investigations. Unlike ICE10750-RD.2 and Tn1806, which are integrated into an hsdM chromosomal gene, both ICESp1108 and ICESp2905 shared the chromosomal integration site at the 3' end of the conserved rum gene, which is an integration hot spot for several mobile streptococcal elements. By using PCR-mapping assays, erm(TR)-carrying elements closely resembling ICESp1108 and ICESp2905 were shown in the other TET-susceptible and TET-resistant test strains, respectively

Phim46.1, the main Streptococcus pyogenes element carrying mef(A) and tet(O) genes.

Phi-m46.1, the recognized representative of the most common variant of mobile, prophage-associated genetic elements carrying resistance genes mef(A) (which confers efflux-mediated erythromycin resistance) and tet(O) (which confers tetracycline resistance) in Streptococcus pyogenes, was fully characterized. Sequencing of the m46.1 genome (55,172 bp) demonstrated a modular organization typical of tailed bacteriophages. Electron microscopic analysis of mitomycin-induced m46.1 revealed phage particles with the distinctive icosahedral head and tail morphology of the Siphoviridae family. The chromosome integration site was within a 23S rRNA uracil methyltransferase gene. BLASTP analysis revealed that the proteins of m46.1 had high levels of amino acid sequence similarity to the amino acid sequences of proteins from other prophages, especially 10394.4 of S. pyogenes and Sa04 of S. agalactiae. Phage DNA was present in the host cell both as a prophage and as free circular DNA. The lysogeny module appears to have been split due to the insertion of a segment containing tet(O) (from integrated conjugative element 2096-RD.2) and mef(A) (from a Tn1207.1-like transposon) into the unintegrated phage DNA. The phage attachment sequence lies in the region between tet(O) and mef(A) in the unintegrated form. Thus, whereas in this form tet(O) is 5.5 kb upstream of mef(A), in the integrated form, tet(O), which lies close to the right end of the prophage, is 46.3 kb downstream of mef(A), which lies close to the left end of the prophage