Inter- and intraspecies variations of the 16S-23S rDNA intergenic spacer region of various streptococcal species

The 16S-23S rDNA intergenic spacer regions (ISR) of different streptococcal species and subspecies were amplified with primers derived from the highly conserved flanking regions of the 16S rRNA and 23S rRNA genes. The single sized amplicons showed a uniform pattern for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. uberis, S. parauberis, S. pyogenes and S. equi subsp. equi, respectively. The amplicons of S. equi subsp. zooepidemicus, S. porcinus and S. suis appeared with 3, 5 and 3 different sizes, respectively. ISR of selected strains of each species or subspecies investigated were sequenced and multiple aligned. This allowed a separation of ISR into regions, with 7 regions for S. agalactiae, S. dysgalactiae subsp. dysgalactiae (serogroup C), S. dysgalactiae subsp. equisimilis (serogroup G), S. dysgalactiae subsp. dysgalactiae (serogroup L), S. canis, S. phocae, S. pyogenes and S. suis, 8 regions for S. uberis and S. parauberis and mostly 9 regions for S. equi subsp. equi, S. equi subsp. zooepidemicus and S. porcinus. Region 4, encoding the transfer RNA for alanine (tRNA(Ala)), was present and identical for all isolates investigated. The size and sequence of ISR appears to be a unique marker for streptococci of various species and subspecies and could be used for bacterial identification. In addition the size and sequence variations of ISR of S. equi subsp. zooepidemicus, S. porcinus and S. suis allows a molecular typing of isolates of these species possibly useful in epidemiological aspects.     

Prevalence of a Streptococcal Inhibitor of a Complement-Mediated Cell lysis-like Gene (sicG) in Streptococcus Dysgalactiae subsp. Equisimilis

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.     

High-level fluorescence labeling of gram-positive pathogens

Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb) promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.     

Production and properties of bacteriocin-like inhibitory substances from the swine pathogen Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a major pathogen found in the upper respiratory tract of swine. In this study, isolates of this bacterial species were tested for the production of bacteriocin-like inhibitory substances (BLIS). Of the 38 strains tested, four inhibited the growth of other S. suis isolates according to a deferred-antagonism plate assay. Interestingly, three of the strains were originally isolated from healthy carrier pigs and were considered nonvirulent. Three isolates (94-623, 90-1330, and AAH4) that produced BLIS in liquid broth were selected for further characterization. None of the inhibitory activities was related to the production of either organic acids or hydrogen peroxide. The BLIS produced by these strains were heat stable and proteinase K, pronase, and elastase sensitive but were trypsin and chymotrypsin resistant. They were stable at pH 2 and 12 and had molecular masses in the range of 14 to 30 kDa. Maximum production was observed during the mid-log phase. Following a curing procedure with novobiocin, only 90-1330 lost the ability to produce BLIS, suggesting that the BLIS might be plasmid encoded. Analysis of the inhibitory spectra revealed that the BLIS-producing strains also inhibited the growth of Actinobacillus minor, Actinobacillus porcinus, Enterococcus durans, Micrococcus luteus, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus equi subsp. zooepidemicus, and S. dysgalactiae subsp. equisimilis. This study reports for the first time the ability of the swine pathogen S. suis serotype 2 to produce BLIS with the characteristics of classic bacteriocins. Further studies are required to investigate the possibility of using bacteriocin-producing strains to prevent swine infections caused by virulent strains of S. suis serotype 2.     

Cores, Microbial Organelles Possibly Specific to Group D Streptococci

A long, thin, approximately cylindrical core spans the interior of cells of 24-hr cultures of all group D streptococci that were examined, five strains of Streptococcus faecalis, single strains of S. faecalis subsp. zymogenes and S. durans, and three strains of Streptococcus spp. In one strain of S. faecalis, serial section electron microscopy showed that most cells possess a core. The core is 0.10 to 0.16 mum thick and consists of a matrix and an axial array of ribosomelike particles. It resembles one of two types of cores present in a stable protoplast form of one of the S. faecalis strains. Cores were not present in single strains of S. pyogenes (beta-hemolytic group A), S. agalactiae (group B), S. dysgalactiae (group C), S. equisimilis (group C), and S. mitis (viridans group) that were examined; nor were cores observed in single strains of Staphylococcus aureus, Escherichia coli, and Bacillus megaterium. Cores may be useful, therefore, in identification of group D streptococci. For preservation and rapid recognition of cores, a glutaraldehyde-osmium tetroxide sequence of fixation appears superior to the osmium tetroxide method often employed in processing bacteria for electron microscopy.     

A GapC chimera retains the properties of the Streptococcus uberis wild-type GapC protein

The GapC products of Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis share considerable homology at the DNA and amino acid levels. The high similarity at the protein level suggests that one GapC protein might be used as a single antigen to protect dairy cows against infections with the contagious S. agalactiae and the environmental S. dysgalactiae and S. uberis strains. Despite their similarities, immunization with the S. dysgalactiae GapC did not protect dairy cows from a challenge with S. uberis, suggesting the presence of regions in GapC that are involved in species-specific protection. To produce a single antigen that can be used to protect against all streptococcal mastitis infections, we constructed a GapC chimeric protein using the S. uberis GapC product as the backbone followed by non-conserved peptide regions from the S. agalactiae and S. dysgalactiae GapC proteins. We report that the chimeric GapC protein retains the enzymatic activity of the S. uberis GapC protein. In addition, we fused the chimera to the OmpF and LipoF transport sequences of Escherichia coli and the GapC chimeras were present in membrane fractions of E. coli. These extracts could be the basis of an antigen preparation for use in mastitis vaccines.     

Inter-species genetic movement may blur the epidemiology of streptococcal diseases in endemic regions

Streptococcus dysgalactiae subsp. equisimilis (human group G streptococcus, GGS) is generally regarded as a commensal organism but can cause a spectrum of human diseases very similar to that caused by S. pyogenes (group A streptococcus, GAS). Lateral acquisition of genes between these two phylogenetically closely related species is well documented. However, the extent and mechanisms of lateral acquisitions is not known. We report here genomic subtraction between a pathogenic GGS isolate and a community GGS isolate and analyses of the gene sequences unique to the pathovar. Our results show that cross-species genetic transfers are common between GGS and two closely related human pathogens, GAS and the group B streptococcus. We also demonstrate that mobile genetic elements, such as phages and transposons, play an important role in the ongoing inter-species transfers of genetic traits between extant organisms in the community. Furthermore, lateral gene transfers between GAS and GGS may occur more frequently in geographical regions of high GAS endemicity. These observations may have important implications in understanding the epidemiology of streptococcal diseases in such regions.     

Superantigen-like gene(s) in human pathogenic Streptococcus dysgalactiae,subsp equisimilis: genomic localisation of the gene encoding streptococcal pyrogenic exotoxin G (speG(dys))

Streptococcus pyogenes (GAS) causes about 90% of streptococcal human infections while group C (GCS) and G (GGS) streptococci can be pathogenic for different mammalians. Especially the human pathogenic GCS and GGS, Streptococcus dysgalactiae, subsp. equisimilis, account for 5-8% of the human streptococcal diseases like wound infections, otitis media, purulent pharyngitis and also streptococcal toxic shock syndrome. A defined superantigen so far was not identified in GCS and GGS strains. In the present investigation we screened DNA of GCS and GGS human isolates for the presence of genes for streptococcal pyrogenic exotoxins (spe) by hybridisation with probes that stand for the GAS genes speA, speC, speZ (smeZ), speH, speG, speI, speJ and ssa. In many GCS and GGS strains we found positive reactions with the probes speG, speJ and ssa, but not with the probes for the remaining genes under investigation. PCR amplification with subsequent sequence analysis of the PCR fragments revealed only the presence of the gene speG in GCS and GGS strains, while no DNA fragments specific for speJ and ssa could be amplified. Additionally, the upstream and downstream regions flanking speG in GGS strain 39072 were sequenced. Remarkable differences were found in the neighbourhood of speG between GAS and GGS sequences. Downstream of speG we identified in strain GGS 39072 two new open reading frames encoding proteins with no similarity to protein sequences accessible in the databases so far. In the compared GAS strains SF370 and MGAS8232, this segment, apart from some small fragments, had been deleted. Our analysis suggests that a gene transfer from GGS to GAS has preceded following deletion of the two genes orf1 and orf2 in GAS.     

Region specific and worldwide distribution of collagen-binding M proteins with PARF motifs among human pathogenic streptococcal isolates

Some of the variety of Streptococcus pyogenes and Streptococcus dysgalactiae ssp. equisimilis (SDSE) M proteins act as collagen-binding adhesins that facilitate acute infection. Moreover, their potential to trigger collagen autoimmunity has been implicated in the pathogenesis of acute rheumatic fever and attributed to a collagen-binding motif called PARF (peptide associated with rheumatic fever). For the first time we determine the rate of clinical isolates with collagen-binding M proteins that use a PARF motif (A/T/E)XYLXX(L/F)N in a defined geographic region, Vellore in South India. In this region both, incidence of streptococcal infections and prevalence of acute rheumatic fever are high. M proteins with PARF motif conferred collagen-binding activity to 3.9% of 153 S. pyogenes and 10.6% of 255 SDSE clinical isolates from Vellore. The PARF motif occurred in three S. pyogenes and 22 SDSE M protein types. In one of the S. pyogenes and five of the SDSE M proteins that contained the motif, collagen-binding was impaired, due to influences of other parts of the M protein molecule. The accumulated data on the collagen binding activity of certain M protein types allowed a reanalysis of published worldwide emm-typing data with the aim to estimate the rates of isolates that bind collagen via PARF. The results indicate that M proteins, which bind collagen via a PARF motif, are epidemiologically relevant in human infections, not only in Vellore. It is imperative to include the most relevant collagen-binding M types in vaccines. But when designing M protein based vaccines it should be considered that collagen binding motifs within the vaccine antigen remain potential risk factors.     

Horizontal gene transfer and host specificity of beta-haemolytic streptococci: the role of a putative composite transposon containing scpB and lmb

Beta-haemolytic streptococci are important human and animal pathogens: their genetic traits that are associated with the ability to infect human hosts remain, however, unclear. The surface protein, Lmb, mediates the adherence of Streptococcus agalactiae to human laminin. For further analysis of the corresponding gene, the adjacent genomic regions were sequenced. Lmb is localized on a putative composite transposon of 16 kb and is flanked by two copies of a novel insertion sequence element (ISSag2). It harbours the genes scpB and lmb, which are 98% identical with the respective genes of Streptococcus pyogenes. Analysis of the distribution of these genes and ISSag2 among 131 streptococcal strains revealed that all of the human isolates, but only 20% (12 of 61) of the animal isolates, contained scpB and lmb or their homologues. To investigate if the putative transposon can be mobilized, an erythromycin resistance marker was incorporated into the lmb gene of S. agalactiae. Screening for mutant strains with a regained susceptibility for erythromycin identified strains with a deletion of scpB, lmb, and one copy of ISSag2. We hypothesize that a horizontal gene transfer caused the exchange of scpB and lmb and that the ability of S. pyogenes, S. agalactiae and group C and G streptococcal strains to colonize or infect human hosts is dependent on their presence.     

Characterization of acid phosphatase activities in the equine pathogen Streptococcus equi

Acid phosphatases hydrolyse phosphomonoesters at acidic pH in a variety of physiological contexts. The recently defined class C family of acid phosphatases includes the 32 kDa LppC lipoprotein of Streptococcus equisimilis. To define further the distribution of acid phosphatases in the genus Streptococcus we have examined the equine pathogens Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus. Whole cell assays indicated that these organisms possess two acid phosphatases with activity optima at pH 5.0 and pH 6.0-6.5 and that only the former of these was, like LppC, resistant to EDTA. Western blotting with a polyclonal anti-LppC antiserum revealed the presence of a cross-reactive 32 kDa protein in both organisms. The cross-reactive protein in S. equi was shown to be a surface accessible lipoprotein as its processing was inhibited by the antibiotic globomycin and it was released from whole cells by treatment with trypsin. The presence of DNA sequences homologous to the S. equisimilis lppC gene were confirmed by PCR. These data strongly suggest that Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus produce a lipoprotein acid phosphatase homologous to LppC of S. equisimilis.     

Role of α2-macroglobulin in phagocytosis of group A and C streptococci

Binding of alpha 2-macroglobulin (alpha 2M) to streptococci and its effects on phagocytosis were investigated. Two types of streptococcal binding sites for alpha 2M were observed: Streptococcus pyogenes from human infections interacted only with native alpha 2M whereas S. dysgalactiae from bovine and S. equi from equine infections bound only a complex of alpha 2M with trypsin (alpha 2M-T). Preincubation of S. pyogenes with native alpha 2M substantially enhanced their phagocytosis by human polymorphonuclear neutrophils (PMN) whereas preincubation with alpha 2M-T was without any effect. On the other hand, incubation of S. dysgalactiae and S. equi with alpha 2M-T markedly reduced their phagocytosis by PMN from the respective host species. Native alpha 2M did not affect the phagocytosis of these streptococci. Digestion of the streptococcal binding sites for alpha 2M and alpha 2M-T pronase abolished the enhancement of phagocytosis of S. pyogenes by native alpha 2M as well as the inhibition of phagocytosis of S. dysgalactiae and S. equi by alpha 2M-T. Thus, binding of alpha 2M or its complexes appeared to play a role in streptococcal pathogenicity.     

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively.     

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.     

Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis

The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 α-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest that human and animal isolates of S. dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.     

Gene repertoire evolution of Streptococcus pyogenes inferred from phylogenomic analysis with Streptococcus canis and Streptococcus dysgalactiae

Streptococcus pyogenes, is an important human pathogen classified within the pyogenic group of streptococci, exclusively adapted to the human host. Our goal was to employ a comparative evolutionary approach to better understand the genomic events concomitant with S. pyogenes human adaptation. As part of ascertaining these events, we sequenced the genome of one of the potential sister species, the agricultural pathogen S. canis, and combined it in a comparative genomics reconciliation analysis with two other closely related species, Streptococcus dysgalactiae and Streptococcus equi, to determine the genes that were gained and lost during S. pyogenes evolution. Genome wide phylogenetic analyses involving 15 Streptococcus species provided convincing support for a clade of S. equi, S. pyogenes, S. dysgalactiae, and S. canis and suggested that the most likely S. pyogenes sister species was S. dysgalactiae. The reconciliation analysis identified 113 genes that were gained on the lineage leading to S. pyogenes. Almost half (46%) of these gained genes were phage associated and 14 showed significant matches to experimentally verified bacteria virulence factors. Subsequent to the origin of S. pyogenes, over half of the phage associated genes were involved in 90 different LGT events, mostly involving different strains of S. pyogenes, but with a high proportion involving the horse specific pathogen S. equi subsp. equi, with the directionality almost exclusively (86%) in the S. pyogenes to S. equi direction. Streptococcus agalactiae appears to have played an important role in the evolution of S. pyogenes with a high proportion of LGTs originating from this species. Overall the analysis suggests that S. pyogenes adaptation to the human host was achieved in part by (i) the integration of new virulence factors (e.g. speB, and the sal locus) and (ii) the construction of new regulation networks (e.g. rgg, and to some extent speB).     

Streptococcus dysgalactiae-derived mitogen (SDM), a novel bacterial superantigen: characterization of its biological activity and predicted tertiary structure

A mitogenic substance, designated Streptococcus dysgalactiae-derived mitogen (SDM), was purified from S. dysgalactiae culture supernatant, and the gene encoding the mitogen was cloned. Both native and recombinant SDM expressed in Escherichia coli significantly activated human V beta 1+ and V beta 23+ T cells in association with major histocompatibility complex (MHC) class II molecules on accessory cells, indicating that SDM possesses superantigenic properties. The sdm gene consists of two segments encoding a signal peptide and a mature 25 kDa protein composed of 212 amino acids. Three of 34 S. dysgalactiae strains but none of 28 Streptococcus pyogenes strains examined carried sdm. Phylogenetic analysis indicated that SDM belongs to a family distinct from established bacterial superantigens. SDM showed around 30% homology with other superantigens at the amino acid sequence level. The tertiary structure of SDM was predicted by modelling onto streptococcal pyrogenic exotoxin C and streptococcal mitogenic exotoxin Z-2, both of which share highly homologous structure-determining regions. SDM showed overall structural similarity to both these superantigens. This is the first study to characterize fully a bacterial superantigen from S. dysgalactiae.     

Determination of 16S ribosomal RNA gene copy number in Streptococcus uberis, S. agalactiae, S. dysgalactiae and S. parauberis

Abstract Species-specific oligonucleotide probes and a universal oligonucleotide probe derived from sequences of 16S rRNA were hybridised to chromosomal DNA from Streptococcus agalactiae, S. dysgalactiae, S. parauberis and S. uberis following digestion with Eco RI. Due to the presence of a unique Eco RI site in each 16S rRNA gene, the number of hybridised fragments was indicative of the number of 16S rRNA genes. Southern hybridisation indicated six 16S rRNA genes in ten isolates of S. agalactiae , five genes in ten isolates of S. uberis , five genes in six isolates and six in another isolate of S. dysgalactiae , and six genes in four isolates of S. parauberis . For a fifth isolate of S. parauberis , six 16S rRNA genes were indicated by the universal probe but only five when hybridised to the species-specific probe, indicating sequence variation (microheterogeneity) within the probe target region.     

Occurrence and drug-resistance of beta-hemolytic streptococci

The aim of this study was the analysis of drug-resistance and frequency appearance of beta-hemolytic streptococci strains which were isolated in 2003-2005 in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz University of Nicolaus Copernicus in Toruń. Among investigeted beta-hemolytic streptococci the most frequency isolated species was S. agalactiae. All isolates examined in our study were susceptible to penicillin, the higest rate of resistance was found for tetracycline. The rates of resistence to macrolide-lincosamide-streptogramin B (phenotyp MLS(B)) were as follows: S. agalactiae (18.7%), S. pyogenes (10.1%), group G streptococci (10.6%) and group C streptococci (8.0%). In our study we presented also a special case patient from which in investigeted period S. agalactiae was isolated twenty eight times. For ten chromosomal DNA isolated from this patient three different PFGE profiles were obtained.