High virulence gene diversity in Streptococcus pyogenes isolated in Central Italy.

Globally, Streptococcus pyogenes poses a continuous burden on human health, causing both self-limiting and life-threatening diseases. Therefore, studying the profile of virulence genes and their combinations is essential to monitor the epidemiology and pathogenic potential of this important species. Thus, the aim of this study was to analyze related genetic features of clinical strains collected in Italy in 2012 in order to obtain a valid picture of their virulence profile that could be compared to similar studies made in other countries approximately in the same period. We conducted emm typing and fibronectin-collagen-T antigen (FCT) region typing in 122 Streptococcus pyogenes strains. Furthermore, several additional virulence genes were screened by polymerase chain reaction. We found correlations between emm types and FCT region profiles. emm1 strains were mainly associated with FCT2 and FCT6, while emm89 and emm12 strains were associated with FCT4. FCT5 was mainly represented in emm4, emm6, and emm75 strains. Significantly, we defined subtypes for each FCT type based on the differences in single and double loci compared to the reference scheme used for the classification of the FCT region. In addition, new FCT-region variants with differences in multiple loci were also recorded. Cluster analysis based on virulence gene profiling showed a non-random distribution within each emm type. This study added new data to existing studies conducted worldwide and revealed new variability scores in circulating Streptococcus pyogenes strains and new assortments in well-established virulence gene signatures.

Low biological cost of carbapenemase-encoding plasmids following transfer from Klebsiella pneumoniae to Escherichia coli.

OBJECTIVES: The objective of this study was to determine the biological cost, stability and sequence of two carbapenemase-encoding plasmids containing blaKPC-2 (pG12-KPC-2) and blaVIM-1 (pG06-VIM-1) isolated from Klebsiella pneumoniae when newly acquired by uropathogenic Escherichia coli clinical isolates of different genetic backgrounds. METHODS: The two plasmids were transferred into plasmid-free E. coli clinical isolates by transformation. The fitness effect of newly acquired plasmids on the host cell was assessed in head-to-head competitions with the corresponding isogenic strain. Plasmid stability was estimated by propagating monocultures for ∼312 generations. Plasmid nucleotide sequences were determined using next-generation sequencing technology. Assembly, gap closure, annotation and comparative analyses were performed. RESULTS: Both plasmids were stably maintained in three of four E. coli backgrounds and resulted in low to moderate reductions in host fitness ranging from 1.1% to 3.6%. A difference in fitness cost was observed for pG12-KPC-2 between two different genetic backgrounds, while no difference was detected for pG06-VIM-1 between three different genetic backgrounds. In addition, a difference was observed between pG12-KPC-2 and pG06-VIM-1 in the same genetic background. In general, the magnitude of biological cost of plasmid carriage was both host and plasmid dependent. The sequences of the two plasmids showed high backbone similarity to previously circulating plasmids in K. pneumoniae. CONCLUSIONS: The low to modest fitness cost of newly acquired and stably maintained carbapenemase-encoding plasmids in E. coli indicates a potential for establishment and further dissemination into other Enterobacteriaceae species. We also show that the fitness cost is both plasmid and host specific.

Transcriptome Remodeling Contributes to Epidemic Disease Caused by the Human Pathogen Streptococcus pyogenes.

UNLABELLED: For over a century, a fundamental objective in infection biology research has been to understand the molecular processes contributing to the origin and perpetuation of epidemics. Divergent hypotheses have emerged concerning the extent to which environmental events or pathogen evolution dominates in these processes. Remarkably few studies bear on this important issue. Based on population pathogenomic analysis of 1,200 Streptococcus pyogenes type emm89 infection isolates, we report that a series of horizontal gene transfer events produced a new pathogenic genotype with increased ability to cause infection, leading to an epidemic wave of disease on at least two continents. In the aggregate, these and other genetic changes substantially remodeled the transcriptomes of the evolved progeny, causing extensive differential expression of virulence genes and altered pathogen-host interaction, including enhanced immune evasion. Our findings delineate the precise molecular genetic changes that occurred and enhance our understanding of the evolutionary processes that contribute to the emergence and persistence of epidemically successful pathogen clones. The data have significant implications for understanding bacterial epidemics and for translational research efforts to blunt their detrimental effects. IMPORTANCE: The confluence of studies of molecular events underlying pathogen strain emergence, evolutionary genetic processes mediating altered virulence, and epidemics is in its infancy. Although understanding these events is necessary to develop new or improved strategies to protect health, surprisingly few studies have addressed this issue, in particular, at the comprehensive population genomic level. Herein we establish that substantial remodeling of the transcriptome of the human-specific pathogen Streptococcus pyogenes by horizontal gene flow and other evolutionary genetic changes is a central factor in precipitating and perpetuating epidemic disease. The data unambiguously show that the key outcome of these molecular events is evolution of a new, more virulent pathogenic genotype. Our findings provide new understanding of epidemic disease.

Correlation between genetic features of the mef(A)-msr(D) locus and erythromycin resistance in Streptococcus pyogenes.

We investigated the correlation between the genetic variation within mef(A)-msr(D) determinants of efflux-mediated erythromycin resistance in Streptococcus pyogenes and the level of erythromycin resistance. Twenty-eight mef(A)-positive strains were selected according to erythromycin MIC (4-32 µg/mL), and their mef(A)-msr(D) regions were sequenced. Strains were classified according to the bacteriophage carrying mef(A)-msr(D). A new Φm46.1 genetic variant was found in 8 strains out of 28 and named VP_00501.1. Degree of allelic variation was higher in mef(A) than in msr(D). Hotspots for recombination were mapped within the locus that could have shaped the apparent mosaic structure of the region. There was a general correlation between mef(A)-msr(D) sequence and erythromycin resistance level. However, lysogenic conversion of susceptible strains by mef(A)-msr(D)-carrying Φm46.1 indicated that key determinants may not all reside within the mef(A)-msr(D) locus and that horizontal gene transfer could contribute to changes in the level of antibiotic resistance in S. pyogenes.

A study on erm(B)-mediated MLS resistance in Streptococcus pyogenes clinical isolates.

The constitutive or inducible macrolide-lincosamide-streptogramin (MLS) phenotype of 30 erm(B)-positive Streptococcus pyogenes isolates was determined by different methods and under various growth conditions and correlated to the sequence of the 5'-untranslated regions of erm(B). The MLS phenotype of one-third of the isolates could not be classified. In liquid medium, some of these isolates responded to induction only during the logarithmic phase of growth, while others expressed clindamycin resistance even under noninducing conditions. By increasing the growth rate, we observed a shift from a constitutive towards an inducible pattern of resistance. All data were confirmed by analysis of the 23S rRNA methylation level. The erm(B)-5'-untranslated region was 99% similar in sequence. In erm(B)-positive S. pyogenes, the MLS phenotype is strongly influenced by culture conditions and control of its expression does not depend exclusively on the sequence of the erm(B)-5'-untranslated region.

Lysogenic transfer of mef(A) and tet(O) genes carried by Phim46.1 among group A streptococci.

We report the ex vivo lysogenic transfer of erythromycin and tetracycline resistance genes among group A streptococci (GAS). Of 42 susceptible strains, 69% acquired erythromycin/tetracycline resistance when infected with purified supernatants from strain m46 culture containing the phage Φm46.1. A significant emm-type-dependent barrier to lysogenic transfer was not observed. The emm12 strains were the only strains susceptible to the lytic action of the bacteriophage preparation.

Distribution of phage-associated virulence genes in pharyngeal group a streptococcal strains isolated in Italy.

The presence and assortment of 16 known virulence/resistance genetic determinants carried by prophages or prophage-like elements were tested in 212 clinical group A Streptococcus (GAS) strains and related to available data from SmaI macrorestriction/pulsed-field gel electrophoresis analysis and emm typing. A strong correlation existed among the three analyses. This finding supports the substantial contribution to the evolution and diversification of the GAS genome attributed to phages.