Search

Abstract Background The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs). Results We transferred RD2 from GAS strain MGAS6180 (serotype M28) to serotype M1 and M4 GAS strains by filter mating. The copy number of the RD2 element was rapidly and significantly increased following treatment of strain MGAS6180 with mitomycin C, a DNA damaging agent. Using a PCR-based method, we also identified RD2-like regions in multiple group C and G strains of Streptococcus dysgalactiae subsp.equisimilis cultured from invasive human infections. Conclusions Taken together, the data indicate that the RD2 element has disseminated by lateral gene transfer to genetically diverse strains of human-pathogenic streptococci.

Abstract Background Bacteria employ multiple mechanisms to control gene expression and react to their constantly changing environment. Bacterial growth in rich laboratory medium is a dynamic process in which bacteria utilize nutrients from simple to complex and change physical properties of the medium, as pH, during the process. To determine which genes are differentially expressed throughout growth from mid log to stationary phase, we performed global transcript analysis. Results The S. agalactiae transcriptome is dynamic in response to growth conditions. Several genes and regulons involved in virulence factor production and utilization of alternate carbon sources were differentially expressed throughout growth. Conclusion These data provide new information about the magnitude of plasticity of the S. agalactiae transcriptome and its adaptive response to changing environmental conditions. The resulting information will greatly assist investigators studying S. agalactiae physiology and pathogenesis.

Objectives: Genetic characterization of the antibiotic resistance determinants and associated mobile genetic elements (MGEs) among Streptococcus pyogenes [Group A streptococci (GAS)] clinical isolates of an M77 serotype collected in Poland between 2003 and 2017., Methods: The genomes of 136 M77 GAS isolates were sequenced using Illumina, and selected with long-read approach (Oxford Nanopore). Whole genome sequences were analyzed to determine the presence of macrolide resistance determinants, and their genetic context., Results: The strains used in the study were collected in the two multicenter surveys from in- and outpatients. Sequencing data analysis revealed that all strains carried the tet(O) gene (100%, N=136). They were classified as a single sequence type ST63. For erythromycin resistance, the unique determinant was erm(TR) detected in 76.5% (N=104) isolates. A single appearance of tet(M) and erm(B) on Tn3872 was noticed. The mefA, mefE, and msr(D) genes were detected in neither of the genomes. This correlated with the detected strain phenotypes - 11 exhibited cMLSB, 93 - iMLSB, and no M phenotype.The erm(TR) gene was predominantly (N=74) found within a novel hybrid Integrative Conjugative Element composed of the ICESp1108-like sequence and ICESp2906 variant which was then named ICESp1109. However, in strains isolated before 2008, erm(TR) was located within ICESp2905 (N=27). The erm(TR) gene was detected within stand-alone ICESp1108-like sequences in 3 strains., Conclusions: Based on phylogenetic analysis results the clonal dissemination of the macrolide-resistant S. pyogenes M77/ST63 strain with hybrid ICESp1109 was observed between 2008 and 2017. ICESp1109 is the novel hybrid ICE in Gram-positive bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Streptococcus anginosus is considered an emerging opportunistic pathogen causing life-threatening infections, including abscesses and empyema. Noticeably, clinical data revealed that S. anginosus also constitutes an important component of polymicrobial infections. Here, we showed for the first time that S. anginosus inactivates the antibacterial potential of neutrophil extracellular traps (NETs). The process is determined by a cell wall-anchored nuclease referred to as SanA, which high expression dominates in clinical strains isolated from severe infections. Nuclease activity protects S. anginosus against the antibacterial activity of NETs, supporting at the same time the survival of coexisting highly pathogenic species of Enterobacteriales. Obtained data suggest that SanA nuclease should be recognized as a critical S. anginosus virulence factor determining severe monospecies purulent infections but also shielding other pathogens promoting the development of polymicrobial infections., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Streptococcus pyogenes (group A Streptococcus, GAS) is a major human pathogen and causes every year over 600 millions upper respiratory tract onfections worldwide. Untreated or repeated infections may lead to post-infectional sequelae such as rheumatic heart disease, a major cause of GAS-mediated mortality. There is no comprehensive, longitudinal analysis of the M type distribution of upper respiratory tract strains isolated in Poland. Single reports describe rather their antibiotic resistance patterns or focus on the invasive isolates. Our goal was to analyse the clonal structure of the upper respiratory tract GAS isolated over multiple years in Poland. Our analysis revealed a clonal structure similar to the ones observed in high-income countries, with M1, M12, M89, M28, and M77 serotypes constituting over 80% of GAS strains. The M77 serotype is a major carrier of erythromycin resistance and is more often correlated with upper respiratory tract infections than other serotypes., (© 2024. The Author(s).)

For many years, Streptococcus anginosus has been considered a commensal colonizing the oral cavity, as well as the gastrointestinal and genitourinary tracts. However, recent epidemiological and clinical data designate this bacterium as an emerging opportunistic pathogen. Despite the reported pathogenicity of S. anginosus, the molecular mechanism underpinning its virulence is poorly described. Therefore, our goal was to develop and optimize efficient and simple infection models that can be applied to examine the virulence of S. anginosus and to study host-pathogen interactions. Using 23 S. anginosus isolates collected from different infections, including severe and superficial infections, as well as an attenuated strain devoid of CppA, we demonstrate for the first time that Dictyostelium discoideum is a suitable model for initial, fast, and large-scale screening of virulence. Furthermore, we found that another nonvertebrate animal model, Galleria mellonella, can be used to study the pathogenesis of S. anginosus infection, with an emphasis on the interactions between the pathogen and host innate immunity. Examining the profile of immune defense genes, including antimicrobial peptides, opsonins, regulators of nodulation, and inhibitors of proteases, by quantitative PCR (qPCR) we identified different immune response profiles depending on the S. anginosus strain. Using these models, we show that S. anginosus is resistant to the bactericidal activity of phagocytes, a phenomenon confirmed using human neutrophils. Notably, since we found that the data from these models corresponded to the clinical severity of infection, we propose their further application to studies of the virulence of S. anginosus., Competing Interests: The authors declare no conflict of interest.

Three distinct streptococcal species: Streptococcus anginosus, Streptococcus intermedius , and Streptococcus constellatus , belonging to the Streptococcus anginosus group (SAG), also known as Streptococcus milleri group, have been attracting clinicians and microbiologists, not only as oral commensals but also as opportunistic pathogens. For years they have been simply classified as so called viridans streptococci, and distinct species were not associated with particular clinical manifestations. Therefore, description of SAG members are clearly underrepresented in the literature, compared to other medically relevant streptococci. However, the increasing number of reports of life-threatening infections caused by SAG indicates their emerging pathogenicity. The improved clinical data generated with the application of modern molecular diagnostic techniques allow for precise identification of individual species belonging to SAG. This review summarizes clinical reports on SAG infections and systematizes data on the occurrence of individual species at the site of infection. We also discuss the issue of proper microbiological diagnostics, which is crucial for further clinical treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pilarczyk-Zurek, Sitkiewicz and Koziel.)

Streptococcus agalactiae (Group B Streptococcus, GBS) is a common commensal bacterium in adults but remains a leading source of invasive infections in newborns, pregnant women, and the elderly, and more recently, causes an increased incidence of invasive disease in nonpregnant adults. Reduced penicillin susceptibility and emerging resistance to non-β-lactams pose challenges for the development and implementation of novel, nonantimicrobial strategies to reduce the burden of GBS infections. Antimicrobial photodynamic inactivation (aPDI) via the production of singlet oxygen or other reactive oxygen species leads to the successful eradication of pathogenic bacteria, affecting numerous cellular targets of microbial pathogens and indicating a low risk of resistance development. Nevertheless, we have previously reported possible aPDI tolerance development upon repeated sublethal aPDI applications; thus, the current work was aimed at investigating whether aPDI tolerance could be observed for GBS and what mechanisms could cause it. To address this problem, 10 cycles of sublethal aPDI treatments employing rose bengal as a photosensitizer, were applied to the S. agalactiae ATCC 27956 reference strain and two clinical isolates (2306/02 and 2974/07, serotypes III and V, respectively). We demonstrated aPDI tolerance development and stability after 5 cycles of subculturing with no aPDI exposure. Though the treatment resulted in a stable phenotype, no increases in mutation rate or accumulated genetic alterations were observed (employing a RIF-, CIP-, STR-resistant mutant selection assay and cyl sequencing, respectively). qRT-PCR analysis demonstrated that 10 sublethal aPDI exposures led to increased expression of all tested major oxidative stress response elements; changes in sodA, ahpC, npx, cylE, tpx and recA expression indicate possible mechanisms of developed tolerance. Increased expression upon sublethal aPDI treatment was reported for all but two genes, namely, ahpC and cylE. aPDI targeting cylE was further supported by colony morphology changes induced with 10 cycles of aPDI (increased SCV population, increased hemolysis, increased numbers of dark- and unpigmented colonies). In oxidant killing assays, aPDI-tolerant strains demonstrated no increased tolerance to hypochlorite, superoxide (paraquat), singlet oxygen (new methylene blue) or oxidative stress induced by aPDI employing a structurally different photosensitizer, i.e., zinc phthalocyanine, indicating a lack of cross resistance. The results indicate that S. agalactiae may develop stable aPDI tolerance but not resistance when subjected to multiple sublethal phototreatments, and this risk should be considered significant when defining efficient anti-S. agalactiae aPDI protocols., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Streptococcus pyogenes encodes multiple virulence factors and their presence is often related to the severity of the disease. We designed the system of four low-volume multiplex PCR reactions to detect genes encoding 20 virulence factors: spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, speK, speM, speC, speI, speA, speH, speG, speJ, smeZ, and ssa. Classification of strains based on the virulence factors absence or presence correlates with PFGE MLST and emm typing results. The typing/detection system is fast and cost-effective, can be used to detect GAS virulence factors and as a rapid tool to effectively differentiate between strains.

Background: Cholera is one of the most deadly diarrheal diseases that require new treatments. We investigated the neutralization of cholera toxin by five plant extracts obtained from the Rosaceae family that have been traditionally used in Poland to treat diarrhea (of unknown origin)., Methods: Hot water extracts were prepared from the dried plant materials and lyophilized before phytochemical analysis and assessment of antimicrobial activity using microdilution assays. The ability of the plant extracts to neutralize cholera toxin was analyzed by measurement of cAMP levels in cell cultures, enzyme-linked immunosorbent assay and electrophoresis, as well as flow cytometry and fluorescence microscopy studies of fluorescent-labeled cholera toxins with cultured human fibroblasts., Results: The antimicrobial assays displayed modest bacteriostatic potentials. We found that the plant extracts modulate the effects of cholera toxin on intracellular cAMP levels. Three plant extracts (Agrimonia eupatoria L., Rubus fruticosus L., Fragaria vesca L.) suppressed the binding of subunit B of cholera toxin to the cell surface and immobilized ganglioside GM 1 while two others (Rubus idaeus L., Rosa.canina L.) interfered with the toxin internalization process., Conclusions: The traditional application of the Rosaceae plant infusions for diarrhea appears relevant to cholera, slowing the growth of pathogenic bacteria and either inhibiting the binding of cholera toxin to receptors or blocking toxin internalization. The analyzed plant extracts are potential complements to standard antibiotic treatment and Oral Rehydration Therapy for the treatment of cholera.

For the last 40 years, "Sanger sequencing" allowed to unveil crucial secrets of life. However, this method of sequencing has been time-consuming, laborious and remains expensive even today. Human Genome Project was a huge impulse to improve sequencing technologies, and unprecedented financial and human effort prompted the development of cheaper high-throughput technologies and strategies called next-generation sequencing (NGS) or whole genome sequencing (WGS). This review will discuss applications of high-throughput methods to study bacteria in a much broader context than simply their genomes. The major goal of next-generation sequencing for a microbiologist is not really resolving another circular genomic sequence. NGS started its infancy from basic structural and functional genomics, to mature into the molecular taxonomy, phylogenetic and advanced comparative genomics. Today, the use of NGS expended capabilities of diagnostic microbiology and epidemiology. The use of RNA sequencing techniques allows studying in detail the complex regulatory processes in the bacterial cells. Finally, NGS is a key technique to study the organization of the bacterial life-from complex communities to single cells. The major challenge in understanding genomic and transcriptomic data lies today in combining it with other sources of global data such as proteome and metabolome, which hopefully will lead to the reconstruction of regulatory networks within bacterial cells that allow communicating with the environment (signalome and interactome) and virtual cell reconstruction.

Streptococcus anginosus group (SAG) is Gram-positive bacteria responsible for a number of purulent human infections such as brain and liver abscesses, which have been on the rise for last few decades. Although some virulence factors of SAG are described, they are mostly undefined and there are almost no methods for genetic manipulations of clinical SAG. Therefore, we presented various approaches to produce engineered strains of this poorly known group of streptococci. We developed a procedure of transformation characterized by transformation efficiency at the level of 10 4 per 1 μg DNA for certain strains. Moreover, mutagenesis for many SAG strain is possible based on the process of natural transformation. However, the usefulness of methods and their effectiveness are strain dependent.

Group A Streptococcus (GAS) is a Gram-positive human pathogen that causes a variety of diseases ranging from pharyngitis to life-threatening streptococcal toxic shock syndrome. Recently, several global gene expression analyses have yielded extensive new information regarding the regulation of genes encoding known and putative virulence factors in GAS. A microarray analysis found that transcription of the GAS gene M5005_Spy_1343 was significantly increased in response to interaction with human polymorphonuclear leukocytes. M5005_Spy_1343 is predicted to encode a member of the LysR family of transcriptional regulators and is located upstream of a putative operon containing six genes. Five of these genes have sequence similarity to genes involved in short-chain fatty acid metabolism, whereas the sixth gene (luxS) is found in many bacterial species and is involved in quorum sensing. Unexpectedly, inactivation of the M5005_Spy_1343 gene resulted in hypervirulence in an intraperitoneal mouse model of infection. Increased virulence was not due to changes in luxS gene expression. We postulate that short-chain fatty acid metabolism is involved in GAS pathogenesis.

We compared association of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Enterococcus faecalis with nine vascular implants after co-culture. Vascular implants were composed of various materials such as warp knitted polyester (with or without gelatin and silver ions), expanded polytetrafluoroethylene and biological materials - surface treated porcine pericardial patch and Omniflow II. The lowest overall number of associated bacteria was detected for polytetrafluoroethylene implants and porcine pericardial patch. The highest overall number of associated bacteria was detected for Omniflow II implant. The major source of variation, i.e. primary factor influencing colonization, is the implant type (56.22%), bacterial species is responsible for only 1.81%, and interaction of those two factors - 13.09% of variation.

Streptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus form a group of related streptococcal species, namely the Streptococcus Anginosus Group (SAG). The group, previously called "milleri" had been rarely described until 1980/1990 as source of infections. Nowadays SAG bacteria are often described as pathogens causing predominantly purulent infections. The number of infections is highly underestimated, as SAG strains are often classified in the microbiology laboratory as less virulent "viridans streptococci" Epidemiological situation regarding SAG infections in Poland has been unrecognized, therefore we performed a retrospective analysis of strains isolated between 1996 and 2012. Strains suspected of belonging to SAG were re-identified using an automated biochemical approach (Vitek2) and MALDI-TOF MS. We performed first analysis of antibiotic resistance among SAG strains isolated in Poland using automated methods (Vitek2), disk diffusion tests and E-Tests. We also performed PCR detection of resistance determinants in antibiotic resistant strains. Clonal structure of analyzed strains was evaluated with PFGE and MLVF methods. All three species are difficult to distinguish using automated diagnostic methods and the same is true for automated MIC evaluation. Our analysis revealed SAG strains are rarely isolated in Poland, predominantly from purulent infections. All isolates are very diverse on the genomic level as estimated by PFGE and MLVF analyses. All analyzed strains are sensitive to penicillin, a substantial group of strains is resistant to macrolides and the majority of strains are resistant to tetracycline.

We developed a new method of typing for anginosus group streptococci (SAG). It is the first SAG-dedicated, PCR-based method, which allows to determine the relationship between strains. The method is based on the detection of tandem repeats among 9 genomic loci and is classified as multilocus variable number tandem repeats fingerprint (MLVF) type of analysis. Using the described method, it is possible to detect over half million MLVF patterns, which correlate with pulsed-field gel electrophoresis profiles. The other advantage of the method is relatively short time from "cell to data", low costs, and easy application for epidemiological and evolutionary studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A steady increase in macrolide resistance in Streptococcus pyogenes, group A streptococci (GAS) was reported in Serbia during 2004-2009 (9.9%). However, there are no data on the molecular epidemiology of pharyngeal macrolide resistance GAS (MRGAS) isolates. Therefore, the aims of this first nationwide study were to examine the prevalence of macrolide resistance in Serbian GAS and to determine their resistance phenotypes, genotypes and clonal relationships. Overall 3893 non-duplicate pharyngeal S. pyogenes isolates from outpatients with GAS infection were collected throughout country during 2008 and 2009. Among 486 macrolide resistant pharyngeal isolates collected, 103 were further characterized. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion test and PCR, respectively. Strain relatedness was determined by emm typing, multilocus sequence typing (MLST), multilocus variable tandem repeat analysis (MLVA), phage profiling (PP) and virulence factor profiling (VFP). Overall, macrolide resistance among GAS isolates in Serbia was 12.5%. M phenotype was the most common (71.8%), followed by iMLS (18.4%) and cMLS (9.7%). Three clonal complexes--emm75/mefA/ST49, emm12/mefA/ST36 and emm77/ermA/tetO/ST63 comprised over 90% of the tested strains. Although MLVA, PP and VFP distinguished 10, 20 and 12 different patterns, respectively, cluster analysis disclosed only small differences between strains which belonged to the same emm/ST type. Our data indicate dominance of three major internationally widely disseminated macrolide resistant clones and a high genetic homogeneity among the Serbian MRGAS population. Continued surveillance of macrolide resistance and clonal composition in MRGAS in Serbia in future is necessary to determine stability of MRGAS clones and to guide therapy strategies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Streptococcus anginosus (milleri) is a diverse group of gram positive bacteria. Molecular methods to establish relationship between strains are poorly developed. Therefore, main tool to study genetic variability is restriction fragment length polymorphism combined with pulsed field gel electrophoresis (RFLP-PFGE). In this communication, we present optimized protocol for S. anginosus PFGE analysis.

Poland's first Enterobacteriaceae isolate producing the New Delhi metallo-β-lactamase (NDM) was identified in August 2011. Escherichia coli sequence type ST410 NDM-1 was cultured from a critically ill patient who had been transferred directly from the Congo. The blaNDM-1 gene was carried by conjugative IncFII-type plasmid pMC-NDM (87,619 bp), which showed structural similarity to plasmid pGUE-NDM, which was identified earlier in France in an E. coli ST131 isolate of Indian origin.

Plants contain a broad spectrum of small molecules with potential antimicrobial properties. Here, we review the antimicrobial activities of plant extracts against enterotoxic bacteria encoding AB5 toxins, including Vibrio cholerae, Shigella dysenteriae and enterotoxic Escherichia coli strains. Several plant extracts have strong antimicrobial effects and the potential to boost Oral Rehydration Therapy, which is the first line of treatment for acute diarrhea.

Streptococcus pyogenes (GAS) is a human pathogen that causes multiple infections worldwide. The pathogenic properties of GAS strains are often linked to the production of virulence factors such as toxins, proteases or DNases. Detection of virulence factors produced by GAS strains can be used to either determine pathogenic potential of the strain or as a rapid screening and typing method. We recently developed a method to detect simultaneously 20 GAS virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) in four low volume multiplex PCR reactions (Borek et al., 2011) and below we present a detailed protocol describing the method.

We recently developed a method that allows fast differentiation between Streptococcus pyogenes (GAS) strains. The method named phage profiling (PP) is based on a simple assumption that a regular PCR reaction with Taq polymerase and relatively short elongation time is not able to yield long DNA fragment, such as ~40-50 kb integrated prophage. Only fragments without any integrated DNA or short fragments inserted between integration sites can be efficiently amplified. We designed primers that anneal upstream and downstream prophage integration sites, so in simple PCR reaction we can test if any additional DNA is integrated into particular site. Profiling of integrated elements can be used as rapid, high resolution typing method, with the resolution as high as PFGE and is excellent predictor of PFGE type.

Streptococcus pyogenes (GAS) is a human pathogen that causes millions of infections worldwide. Comparison between GAS strains isolated in different laboratories all over the world is often difficult. Three usually used methods have either low resolution (emm typing), are expensive (MLST) or time- and labor-consuming (PFGE). Our laboratory recently developed new, inexpensive methods of GAS typing-VF (virulence factor profiling) and PP (phage profiling). To improve the typing scheme developed for GAS, we recently proposed a new typing method. Here we present detailed protocol for MLVF analysis.

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

We tested 21 polymorphic loci encoded by the genome of Streptococcus pyogenes (group A Streptococcus, GAS). Seven of them were chosen for the MLVA scheme. The primer pairs, designed for selected loci, detect from few to several alleles, and the method has a Simpson's Index of diversity of 0.957. To test the overall performance of the method, multiplex PCR reactions were carried out for over 700 GAS strains. Using the method we were able to detect differences between highly clonal strains that share the same emm, MLST and PFGE types. The most diverse strains were M4, M2, M3 and M28. We developed a typing method that can be employed to differentiate between GAS strains. The method has high resolution and measures diversity of the GAS core chromosome, on the contrary to methods such as PFGE., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Advancements in high-throughput, high-volume data generating techniques increasingly present us with opportunities to probe new areas of biology. In this work we assessed the extent to which four closely related and genetically representative strains of group A Streptococcus causing epidemic disease have differentiated from one another. Comparative genome sequencing, expression microarray analysis, and proteomic studies were used in parallel to assess strain variation. The extent of phenotypic differentiation was unexpectedly large. We found significant associations between genetic polymorphisms and alterations in gene expression allowing us to estimate the frequency with which specific types of polymorphisms alter gene transcription. We identified polymorphisms in the gene (ropB) encoding the RopB regulator that associate with altered transcription of speB and production of the SpeB protein, a critical secreted protease virulence factor. Although these four epidemic strains are closely related, a key discovery is that accumulation of modest genetic changes has rapidly resulted in significant strain phenotypic differentiation, including the extracellular proteome that contains multiple virulence factors. These data provide enhanced understanding of genetic events resulting in strain variation in bacterial epidemics., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs)., Results: We transferred RD2 from GAS strain MGAS6180 (serotype M28) to serotype M1 and M4 GAS strains by filter mating. The copy number of the RD2 element was rapidly and significantly increased following treatment of strain MGAS6180 with mitomycin C, a DNA damaging agent. Using a PCR-based method, we also identified RD2-like regions in multiple group C and G strains of Streptococcus dysgalactiae subsp.equisimilis cultured from invasive human infections., Conclusions: Taken together, the data indicate that the RD2 element has disseminated by lateral gene transfer to genetically diverse strains of human-pathogenic streptococci.

Staphylococcus aureus is an opportunistic pathogen responsible for various infections in humans and animals. It causes localized and systemic infections, such as abscesses, impetigo, cellulitis, sepsis, endocarditis, bone infections, and meningitis. S. aureus virulence factors responsible for the initial contact with host cells (MSCRAMMs-microbial surface components recognizing adhesive matrix molecules) include three Sdr proteins. The presence of particular sdr genes is correlated with putative tissue specificity. The transcriptional organization of the sdr region remains unclear. We tested expression of the sdrC, sdrD, or sdrE genes in various in vitro conditions, as well as after contact with human blood. In this work, we present data suggesting a separation of the sdr region into three transcriptional units, based on their differential reactions to the environment. Differential reaction of the sdrD transcript to environmental conditions and blood suggests dissimilar functions of the sdr genes. SdrE has been previously proposed to play role in bone infections, whilst our results can indicate that sdrD plays a role in the interactions between the pathogen and human immune system, serum or specifically reacts to nutrients/other factors present in human blood.

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.

Background: For more than 100 years, group A Streptococcus has been identified as a cause of severe and, in many cases, fatal infections of the female urogenital tract. Due to advances in hospital hygiene and the advent of antibiotics, this type of infection has been virtually eradicated. However, within the last three decades there has been an increase in severe intra- and post-partum infections attributed to GAS., Methodology: We hypothesized that GAS alters its transcriptome to survive in human amniotic fluid (AF) and cause disease. To identify genes that were up or down regulated in response to growth in AF, GAS was grown in human AF or standard laboratory media (THY) and samples for expression microarray analysis were collected during mid-logarithmic, late-logarithmic, and stationary growth phases. Microarray analysis was performed using a custom Affymetrix chip and normalized hybridization values derived from three biological replicates were collected at each growth point. Ratios of AF/THY above a 2-fold change and P-value <0.05 were considered significant., Principal Findings: The majority of changes in the GAS transcriptome involved down regulation of multiple adhesins and virulence factors and activation of the stress response. We observed significant changes in genes involved in the arginine deiminase pathway and in the nucleotide de novo synthesis pathway., Conclusions/significance: Our work provides new insight into how pathogenic bacteria respond to their environment to establish infection and cause disease.

Understanding the fine-structure molecular architecture of bacterial epidemics has been a long-sought goal of infectious disease research. We used short-read-length DNA sequencing coupled with mass spectroscopy analysis of SNPs to study the molecular pathogenomics of three successive epidemics of invasive infections involving 344 serotype M3 group A Streptococcus in Ontario, Canada. Sequencing the genome of 95 strains from the three epidemics, coupled with analysis of 280 biallelic SNPs in all 344 strains, revealed an unexpectedly complex population structure composed of a dynamic mixture of distinct clonally related complexes. We discovered that each epidemic is dominated by micro- and macrobursts of multiple emergent clones, some with distinct strain genotype-patient phenotype relationships. On average, strains were differentiated from one another by only 49 SNPs and 11 insertion-deletion events (indels) in the core genome. Ten percent of SNPs are strain specific; that is, each strain has a unique genome sequence. We identified nonrandom temporal-spatial patterns of strain distribution within and between the epidemic peaks. The extensive full-genome data permitted us to identify genes with significantly increased rates of nonsynonymous (amino acid-altering) nucleotide polymorphisms, thereby providing clues about selective forces operative in the host. Comparative expression microarray analysis revealed that closely related strains differentiated by seemingly modest genetic changes can have significantly divergent transcriptomes. We conclude that enhanced understanding of bacterial epidemics requires a deep-sequencing, geographically centric, comparative pathogenomics strategy.

Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis ("flesh-eating disease"). Working from this clinical observation, we find that wild-type mtsR function is required for group A Streptococcus to cause necrotizing fasciitis in mice and nonhuman primates. Expression microarray analysis revealed that mtsR inactivation results in overexpression of PrsA, a chaperonin involved in posttranslational maturation of SpeB, an extracellular cysteine protease. Isogenic mutant strains that overexpress prsA or lack speB had decreased secreted protease activity in vivo and recapitulated the necrotizing fasciitis-negative phenotype of the DeltamtsR mutant strain in mice and monkeys. mtsR inactivation results in increased PrsA expression, which in turn causes decreased SpeB secreted protease activity and reduced necrotizing fasciitis capacity. Thus, a naturally occurring single-nucleotide mutation dramatically alters virulence by dysregulating a multiple gene virulence axis. Our discovery has broad implications for the confluence of population genomics and molecular pathogenesis research.

Beta-hemolytic, pyogenic streptococci are classified according to type of major surface antigen into A (Streptococcus pyogenes), B (Streptococcus agalactiae), C (multiple species including Streptococcus dysagalactiae) and G (multiple species including Streptococcus canis) Lancefield groups. Group A Streptococcus causes each year hundreds of thousands deaths globally as a result of infections and post-infectional sequelae. An increasing number of severe, invasive infections is caused by selected, specialized pathogenic clones. Within the last 50 years, an increasing number of human infections caused by groups B, C and G Streptococcus (GBS, GCS, GGS) has been observed worldwide. GBS was first identified as animal pathogen but the spectrum of diseases caused by GBS quickly shifted to human infections. Groups C and G Streptococcus are still regarded mostly as animal pathogens, however, an increased number of severe infections caused by these groups is observed. The increasing number of human infections caused worldwide by GCS/GGS can be a sign of similar development from animal to human pathogen as observed in case of GBS and this group will gain much more clinical interest in the future.The situation in Poland regarding invasive infections caused by pyogenic streptococci is underestimated.

Because passage of the bacterium to blood is a crucial step in the pathogenesis of many group B Streptococcus (GBS) invasive infections, we recently conducted a whole-genome transcriptome analysis during GBS incubation ex vivo with human blood. In the current work, we sought to analyze in detail the difference in GBS gene expression that occurred in one blood sample (donor A) relative to other blood samples. We incubated GBS strain NEM316 with fresh heparinized human blood obtained from healthy volunteers, and analyzed GBS genome expression and cytokine production. Principal component analysis identified extensive clustering of the transcriptome data among all samples at time 0. In striking contrast, the whole bacterial gene expression in the donor A blood sample was significantly different from the gene expression in all other blood samples studied, both after 30 and 90 min of incubation. More genes were up-regulated in donor A blood relative to the other samples, at 30 min and 90 min. Furthermore, there was significant variation in transcript levels between donor A blood and other blood samples. Notably, genes with the highest transcript levels in donor A blood were those involved in carbohydrate metabolism. We also discovered an unusual production of proinflammatory and immunomodulatory cytokines: MIF, tPAI-1 and IL-1beta were produced at higher levels in donor A blood relative to the other blood samples, whereas GM-CSF, TNF-alpha, IFN-gamma, IL-7 and IL-10 remained at lower levels in donor A blood. Potential reasons for our observations are that the immune response of donor A significantly influenced the bacterial transcriptome, or both GBS gene expression and immune response were influenced by the metabolic status of donor A.

Background: Streptococcus agalactiae (group B Streptococcus) is a bacterial pathogen that causes severe intrauterine infections leading to fetal morbidity and mortality. The pathogenesis of GBS infection in this environment is poorly understood, in part because we lack a detailed understanding of the adaptation of this pathogen to growth in amniotic fluid. To address this knowledge deficit, we characterized the transcriptome of GBS grown in human amniotic fluid (AF) and compared it with the transcriptome in rich laboratory medium., Methods: GBS was grown in Todd Hewitt-yeast extract medium and human AF. Bacteria were collected at mid-logarithmic, late-logarithmic and stationary growth phase. We performed global expression microarray analysis using a custom-made Affymetrix GeneChip. The normalized hybridization values derived from three biological replicates at each growth point were obtained. AF/THY transcript ratios representing greater than a 2-fold change and P-value exceeding 0.05 were considered to be statistically significant., Principal Findings: We have discovered that GBS significantly remodels its transcriptome in response to exposure to human amniotic fluid. GBS grew rapidly in human AF and did not exhibit a global stress response. The majority of changes in GBS transcripts in AF compared to THY medium were related to genes mediating metabolism of amino acids, carbohydrates, and nucleotides. The majority of the observed changes in transcripts affects genes involved in basic bacterial metabolism and is connected to AF composition and nutritional requirements of the bacterium. Importantly, the response to growth in human AF included significant changes in transcripts of multiple virulence genes such as adhesins, capsule, and hemolysin and IL-8 proteinase what might have consequences for the outcome of host-pathogen interactions., Conclusions/significance: Our work provides extensive new information about how the transcriptome of GBS responds to growth in AF, and thus new leads for pathogenesis research.

To enhance understanding of how Streptococcus agalactiae (group B streptococcus, GBS) adapts during invasive infection, we performed a whole-genome transcriptome analysis after incubation with whole human blood. Global changes occurred in the GBS transcriptome rapidly in response to blood contact following shift from growth in a rich laboratory medium. Most (83%) of the significantly altered transcripts were down-regulated after 30 minutes of incubation in blood, and all functional categories of genes were abundantly represented. We observed complex dynamic changes in the expression of transcriptional regulators and stress response genes that allow GBS to rapidly adapt to blood. The transcripts of relatively few proven virulence genes were up-regulated during the first 90 minutes. However, a key discovery was that genes encoding proteins involved in interaction with the host coagulation/fibrinolysis system and bacterial-host interactions were rapidly up-regulated. Extensive transcript changes also occurred for genes involved in carbohydrate metabolism, including multi-functional proteins and regulators putatively involved in pathogenesis. Finally, we discovered that an incubation temperature closer to that occurring in patients with severe infection and high fever (40 degrees C) induced additional differences in the GBS transcriptome relative to normal body temperature (37 degrees C). Taken together, the data provide extensive new information about transcriptional adaptation of GBS exposed to human blood, a crucial step during GBS pathogenesis in invasive diseases, and identify many new leads for molecular pathogenesis research.

Background: To act as a commensal bacterium and a pathogen in humans and animals, Streptococcus agalactiae (group B streptococcus, GBS) must be able to monitor and adapt to different environmental conditions. Temperature variation is a one of the most commonly encountered variables., Methodology/principal Findings: To understand the extent to which GBS modify gene expression in response to temperatures encountered in the various hosts, we conducted a whole genome transcriptome analysis of organisms grown at 30 degrees C and 40 degrees C. We identified extensive transcriptome remodeling at various stages of growth, especially in the stationary phase (significant transcript changes occurred for 25% of the genes). A large proportion of genes involved in metabolism was up-regulated at 30 degrees C in stationary phase. Conversely, genes up-regulated at 40 degrees C relative to 30 degrees C include those encoding virulence factors such as hemolysins and extracellular secreted proteins with LPXTG motifs. Over-expression of hemolysins was linked to larger zones of hemolysis and enhanced hemolytic activity at 40 degrees C. A key theme identified by our study was that genes involved in purine metabolism and iron acquisition were significantly up-regulated at 40 degrees C., Conclusion/significance: Growth of GBS in vitro at different temperatures resulted in extensive remodeling of the transcriptome, including genes encoding proven and putative virulence genes. The data provide extensive new leads for molecular pathogenesis research.

Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization show growth phase-dependent expression in human saliva. We sought to determine whether the putative polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and the levels increased significantly during growth in human saliva compared to the levels during growth in glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization proteins in the parental serotype M1 strain MGAS5005 and its DeltamalR isogenic mutant derivative. The transcript levels of all eight genes were significantly increased in the DeltamalR strain compared to the parental strain, especially during growth in human saliva. Following experimental infection, the DeltamalR strain persistently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers of DeltamalR strain CFU recovered were significantly lower than the numbers of the parental strain CFU recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.

Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart.

Phospholipases are ubiquitous and diverse enzymes that induce changes in membrane composition, activate the inflammatory cascade and alter cell signaling pathways. Recent evidence suggests that certain bacterial pathogens have acquired genes encoding secreted phospholipase A2 enzymes through lateral gene transfer events. The two best-studied members of this class of enzyme are ExoU and SlaA, which are produced by Pseudomonas aeruginosa and group A Streptococcus, respectively. These enzymes modulate the host inflammatory response, increase the severity of disease and otherwise alter host-pathogen interactions. We propose that a key function of ExoU and SlaA is to increase the fitness of the subclones expressing these enzymes, thereby increasing the population size of the PLA2-positive strains and enhancing the likelihood of encountering an at-risk host.

The molecular basis of pathogen clone emergence is relatively poorly understood. Acquisition of a bacteriophage encoding a previously unknown secreted phospholipase A(2) (designated SlaA) has been implicated in the rapid emergence in the mid-1980s of a new hypervirulent clone of serotype M3 group A Streptococcus. Although several lines of circumstantial evidence suggest that SlaA is a virulence factor, this issue has not been addressed experimentally. We found that an isogenic DeltaslaA mutant strain was significantly impaired in ability to adhere to and kill human epithelial cells compared with the wild-type parental strain. The mutant strain was less virulent for mice than the wild-type strain, and immunization with purified SlaA significantly protected mice from invasive disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We conclude that transductional acquisition of the ability of a GAS strain to produce SlaA enhanced the spread and virulence of the serotype M3 precursor strain. Hence, these studies identified a crucial molecular event underlying the evolution, rapid emergence, and widespread dissemination of unusually severe human infections caused by a distinct bacterial clone.

Analysis of multiple group A Streptococcus (GAS) genomes shows that genes encoding proteins involved in carbohydrate utilization comprise some 15% of the core GAS genome. Yet there is a limited understanding of how carbohydrate utilization contributes to GAS pathogenesis. Previous genome-wide GAS studies led us to a focused investigation of MalE, a putative maltodextrin-binding protein. Analysis of 28 strains of 22 distinct M protein serotypes showed that MalE is highly conserved among diverse GAS strains. malE transcript levels were significantly increased during growth in human saliva compared to growth in a chemically defined glucose-containing medium or a nutrient-rich medium. MalE was accessible to antibody binding, indicating that it is expressed on the GAS cell surface. Moreover, growth in human saliva appeared to increase MalE surface expression compared to growth in a nutrient-rich medium. Analysis of a delta malE isogenic mutant strain revealed decreased growth in human saliva compared to wild-type GAS. Radiolabeled carbohydrate binding assays showed that MalE was required for the binding of maltose but not glucose. The delta malE isogenic mutant strain colonized a lower percentage of GAS-challenged mice compared to wild-type and genetically complemented strains. Furthermore, decreased numbers of CFU were recovered from mice infected with the delta malE strain compared to those infected with wild-type GAS. These data demonstrate that maltodextrin acquisition is likely to be a key factor in the ability of GAS to successfully infect the oropharynx. Further investigation into carbohydrate transport and metabolism pathways may yield novel insights into GAS pathogenesis.

Group A Streptococcus (GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28&#95;Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.

High segregational stability of the streptococcal plasmid pSM19035 is achieved by the concerted action of systems involved in plasmid copy number control, multimer resolution, and postsegregational killing. In this study, we demonstrate the role of two genes, delta and omega, in plasmid stabilization by a partition mechanism. We show that these two genes can stabilize the native pSM19035 replicon as well as other theta- and sigma-type plasmids in Bacillus subtilis. In contrast to other known partition systems, in this case the two genes are transcribed separately; however, they are coregulated by the product of the parB-like gene omega. Analysis of mutants of the parA-like gene delta showed that the Walker A ATPase motif is necessary for plasmid stabilization. The ParB-like product of the omega gene binds to three regions containing repeated WATCACW heptamers, localized in the copS (regulation of plasmid copy number), delta, and omega promoter regions. We demonstrate that all three of these regions can cause partition-mediated incompatibility. Moreover, our data suggest that each of these could play the role of a centromere-like sequence. We conclude that delta and omega constitute a novel type of plasmid stabilization system.