Host range, morphological and genomic characterisation of bacteriophages with activity against clinical Streptococcus agalactiae isolates.
ABSTRACT: Streptococcus agalactiae or Group B Streptococcus (GBS) is a leading cause of sepsis in neonates. As a preventative measure prophylactic antibiotic administration is common in pregnant women colonised with GBS, but antibiotic-resistance and adverse effects on neonatal microbiomes may result. Use of bacteriophages (phages) is one option for targeted therapy. To this end, four phages (LF1 -LF4) were isolated from wastewater. They displayed lytic activity in vitro against S. agalactiae isolates collected from pregnant women and neonates, with 190/246 isolates (77.2%) and 10/10 (100%) isolates susceptible to at least one phage, respectively. Phage genomes ranged from 32,205-44,768 bp and all phages were members of the Siphoviridae family. High nucleotide identity (99.9%) was observed between LF1 and LF4, which were closely related to a putative prophage of S. agalactiae. The genome organisation of LF2 differed, and it showed similarity to a different S. agalactiae prophage, while LF3 was more closely related to a Streptococcus pyogenes phage. Lysogenic gene presence (integrase, repressor and regulatory modules), was suggestive of temperate phages. In a therapeutic context, temperate phages are not ideal candidates, however, the broad host range activity of these phages observed on clinical isolates in vitro is promising for future therapeutic approaches including bioengineered phage or lysin applications.
Project description:Streptococcus agalactiae, or Group B Streptococcus (GBS), is a major pathogen causing neonatal sepsis and meningitis, bovine mastitis, and fish meningoencephalitis. CC23, including its namesake ST23, is not only the predominant GBS strain derived from human and cattle, but also can infect a variety of homeothermic and poikilothermic species. However, it has never been characterized in fish. This study aimed to determine the pathogenicity of ST23 GBS to fish and explore the mechanisms causing the difference in the pathogenicity of ST23 GBS based on the genome analysis. Infection of tilapia with 10 human-derived ST23 GBS isolates caused tissue damage and the distribution of pathogens within tissues. The mortality rate of infection was ranged from 76 to 100%, and it was shown that the mortality rate caused by only three human isolates had statistically significant difference compared with fish-derived ST7 strain (P < 0.05), whereas the mortality caused by other seven human isolates did not show significant difference compared with fish-derived ST7 strain. The genome comparison and prophage analysis showed that the major genome difference between virulent and non-virulent ST23 GBS was attributed to the different prophage sequences. The prophage in the P1 region contained about 43% GC and encoded 28-39 proteins, which can mediate the acquisition of YafQ/DinJ structure for GBS by phage recombination. YafQ/DinJ belongs to one of the bacterial toxin-antitoxin (TA) systems and allows cells to cope with stress. The ST23 GBS strains carrying this prophage were not pathogenic to tilapia, but the strains without the prophage or carrying the pophage that had gene mutation or deletion, especially the deletion of YafQ/DinJ structure, were highly pathogenic to tilapia. In conclusion, human ST23 GBS is highly pathogenic to fish, which may be related to the phage recombination.
Project description:<i>Streptococcus agalactiae</i> (group B Streptococcus, GBS) represents a leading cause of invasive bacterial infections in newborns and is also responsible for diseases in older and immunocompromised adults. Prophages represent an important factor contributing to the genome plasticity and evolution of new strains. In the present study, prophage content was analyzed in human GBS isolates. Thirty-seven prophages were identified in genomes of 20 representative sequenced strains. On the basis of the sequence comparison, we divided the prophages into eight groups named A-H. This division also corresponded to the clustering of phage integrase, even though several different integration sites were observed in some relative prophages. Next, PCR method was used for detection of the prophages in 123 GBS strains from adult hospitalized patients and from pregnancy screening. At least one prophage was present in 105 isolates (85%). The highest prevalence was observed for prophage group A (71%) and satellite prophage group B (62%). Other groups were detected infrequently (1-6%). Prophage distribution did not differ between clinical and screening strains, but it was unevenly distributed in MLST (multi locus sequence typing) sequence types. High content of full-length and satellite prophages detected in present study implies that prophages could be beneficial for the host bacterium and could contribute to evolution of more adapted strains.
Project description:Streptococcus agalactiae (group B streptococcus [GBS]) is a leading cause of invasive diseases in neonates and severe infections in elderly individuals. GBS serine-rich repeat glycoprotein 1 (Srr1) acts as a critical virulence factor by facilitating GBS invasion into the central nervous system through interaction with the fibrinogen A? chain. This study revealed that srr1 is highly conserved, with 86.7% of GBS clinical isolates expressing the protein. Vaccination of mice with different Srr1 truncated peptides revealed that only Srr1 truncates containing the latch domain protected against GBS meningitis. Furthermore, the latch peptide alone was immunogenic and elicited protective antibodies, which efficiently enhanced antibody-mediated opsonophagocytic killing of GBS by HL60 cells and provided heterogeneous protection against 4 different GBS serogroups. Taken together, these findings indicated that the latch domain of Srr1 may constitute an effective peptide vaccine candidate for GBS.
Project description:Group B Streptococcus (GBS) is a gram-positive pathogen mainly affecting humans, cattle, and fishes. Mobile genetic elements play an important role in the evolution of GBS, its adaptation to host species and niches, and its pathogenicity. In particular, lysogenic prophages have been associated with a high virulence of certain strains and with their ability to cause invasive infections in humans. It is therefore important to be able to accurately detect and classify prophages in GBS genomes. Several bioinformatic tools for the identification of prophages in bacterial genomes are available on-line. However, genome searches for most of these programs are affected by the composition of their reference database. Lack of databases specific to GBS results in failure to recognize all prophages in the species. Additionally, performance of these programs is affected by genome fragmentation in the case of draft genomes, leading to underestimation of the number of phages. They also prove impractical when dealing with large genome datasets and they do not offer a quick way of classifying bacteriophages. We developed a GBS-specific method to screen genome assemblies for the presence of prophages and to classify them based on a reproducible typing scheme. This was achieved through an extensive search of a vast number of high-quality GBS sequences (n = 572) originating from different host species and countries in order to build a database of phage integrase types, on which the scheme is based. The proposed typing scheme comprises 12 integration sites and sixteen prophage integrase types, including multiple subtypes per integration site and integrase genes that were not site-specific. Two putative phage-inducible chromosomal islands (PICI) and their insertion sites were also identified during the course of these analyses. Phages were common and diverse in all major clonal complexes associated with human disease and detected in isolates from every animal species and continent included in the study. This database will facilitate further work on the prevalence and role of prophages in GBS evolution, and identifies the roles of PICIs in GBS and of prophage in hypervirulent ST283 as areas for further research.
Project description:BACKGROUND: Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is a frequent resident of the rectovaginal tract in humans, and a major cause of neonatal infection. In addition, S. agalactiae is a known fish pathogen, which compromises food safety and represents a zoonotic hazard. The complete genome sequence of the piscine S. agalactiae isolate GD201008-001 was compared with 14 other piscine, human and bovine strains to explore their virulence determinants, evolutionary relationships and the genetic basis of host tropism in S. agalactiae. RESULTS: The pan-genome of S. agalactiae is open and its size increases with the addition of newly sequenced genomes. The core genes shared by all isolates account for 50 ~ 70% of any single genome. The Chinese piscine isolates GD201008-001 and ZQ0910 are phylogenetically distinct from the Latin American piscine isolates SA20-06 and STIR-CD-17, but are closely related to the human strain A909, in the context of the clustered regularly interspaced short palindromic repeats (CRISPRs), prophage, virulence-associated genes and phylogenetic relationships. We identified a unique 10 kb gene locus in Chinese piscine strains. CONCLUSIONS: Isolates from cultured tilapia in China have a close genomic relationship with the human strain A909. Our findings provide insight into the pathogenesis and host-associated genome content of piscine S. agalactiae isolated in China.
Project description:CC17 <i>Streptococcus agalactiae</i> carrying group-A prophages is increasingly responsible for neonatal infections. To investigate the impact of the genetic features of a group-A prophage, we first conducted an in silico analysis of the genome of 12/111phiA, a group-A prophage carried by a strain responsible for a bloodstream infection in a parturient. This revealed a Restriction Modification system, suggesting a prophage maintenance strategy and five ORFs of interest for the host and encoding a type II toxin antitoxin system RelB/YafQ, an endonuclease, an S-adenosylmethionine synthetase MetK, and an StrP-like adhesin. Using the WT strain cured from 12/111phiA and constructing deleted mutants for the ORFs of interest, and their complemented mutants, we demonstrated an impact of prophage features on growth characteristics, cell morphology and biofilm formation. Our findings argue in favor of 12/111phiA domestication by the host and a role of prophage features in cell autoaggregation, glycocalyx and biofilm formation. We suggest that lysogeny may promote GBS adaptation to the acid environment of the vagina, consequently colonizing and infecting neonates.
Project description:Streptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in nonpregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to the loss of a synergistic effect. We therefore performed a multicenter study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centers in four countries, 1,128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. However, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon designated Tn3706. For the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing plasmid-mediated HLGR in GBS. Thus, in our clinical GBS isolates, HLGR is mediated both chromosomally and extrachromosomally.
Project description:Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.
Project description:Background:Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of meningitis, sepsis and pneumonia in neonates in the United States. GBS also causes invasive disease in older infants, pregnant women, children and young adults with underlying medical conditions, and older adults. Resistance to lincosamides in the absence of erythromycin resistance is rare in GBS, but has been previously reported in clinical isolates, both on its own or in combination with resistance to streptogramins A and pleuromutilins (L/LSA/LSAP phenotypes). Objectives:To retrospectively screen the Active Bacterial Core surveillance (ABCs) GBS isolate collection for these phenotypes in order to identify the causal genetic determinants and determine whether their frequency is increasing. Methods:Based on MIC data, 65 (0.31%) isolates susceptible to erythromycin (MIC ?0.25?mg/L) and non-susceptible to clindamycin (MIC ?0.5?mg/L) were identified among 21?186 GBS isolates. Genomic DNA was extracted and WGS was performed. The presence of 10 genes previously associated with LSA resistance was investigated by read mapping. Results:Forty-nine (75%) isolates carried the lsa (C) gene and expressed the LSAP phenotype, and 12 (18%) carried both the lnu (B) and lsa (E) genes and expressed the LSAP phenotype. The four remaining isolates were negative for all determinants investigated. Conclusions:While the overall observed frequency of these phenotypes among our GBS isolates was quite low (0.31%), this frequency has increased in recent years. To the best of our knowledge, this is the first time the LSAP phenotype has been reported among GBS isolates from the USA.
Project description:Streptococcus anginosus is an often overlooked and understudied emerging pathogen inhabiting many areas of the human body. Through our sequencing of S. anginosus strains isolated from the female bladder microbiota, we detected numerous prophage sequences. Bioinformatic analysis of these sequences identified 17 distinct groups of S. anginosus prophages. The majority of these phages exhibit no sequence homology to previously characterized temperate or virulent phage sequences, indicating an unexplored diversity of Streptococcus phages. By culturing these bacterial isolates, we confirmed that the prophages of five of these groups are capable of induction. One of these putative phages was imaged, the first such evidence of an S. anginosus virus-like particle; it exhibits morphological characteristics of siphoviruses.