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.     

Bacteriophages on dairy foods

This review focuses on the impact of bacteriophages on the manufacture of dairy foods. Firstly, the impact of phages of lactic acid bacteria in the dairy industry, where they are considered enemies, is discussed. The sources of phage contamination in dairy plants are detailed, with special emphasis on the rise of phage infections related to the growing use of cheese whey as ingredient. Other topics include traditional and new methods of phage detection, quantification and monitoring, and strategies of phage control in dairy plants, either of physical, chemical or biological nature. Finally, the use of phages or purified phage enzymes as allies to control pathogenic bacteria in the food industry is reviewed.     

Presence of streptococcal pyrogenic exotoxin A and C genes in human isolates of group G streptococci

The bacteriophage-associated genes speA and speC encode streptococcal pyrogenic exotoxins of group A streptococci (GAS). Human isolates of group C and G streptococci (GCS and GGS) are commensals and the closest known genetic relatives of GAS; on occasion, GCS-GGS can cause infection that is clinically similar to GAS disease. Thirty-four human isolates of GCS-GGS were tested for speA and speC. Two GGS isolates harbored speA only, whereas a third GGS had both genes. All spe alleles found in GGS were identical to known spe alleles of GAS, except for one speA allele, which was unique. The presence of shared speA and speC alleles in GAS and GGS is highly suggestive of recent interspecies transfer. Acquisition of GAS-like virulence genes by GGS may lead to enhanced pathogenicity in this usually commensal-like organism.     

High diversity and novel species of Pseudomonas aeruginosa bacteriophages

The diversity of Pseudomonas aeruginosa bacteriophages was investigated using a collection of 68 phages isolated from Central Mexico. Most of the phages carried double-stranded DNA (dsDNA) genomes and were classified into 12 species. Comparison of the genomes of selected archetypal phages with extant sequences in GenBank resulted in the identification of six novel species. This finding increased the group diversity by ~30%. The great diversity of phage species could be related to the ubiquitous nature of P. aeruginosa.     

The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin

The viral population, including bacteriophages, is an important component of the human microbiota, yet is poorly understood. We aim to determine whether bacteriophages modulate the composition of the bacterial populations, thus potentially playing a role in health or disease. We investigated the diversity and host interactions of the bacteriophages of Propionibacterium acnes, a major human skin commensal implicated in acne pathogenesis. By sequencing 48 P. acnes phages isolated from acne patients and healthy individuals and by analyzing the P. acnes phage populations in healthy skin metagenomes, we revealed that P. acnes phage populations in the skin microbial community are often dominated by one strain. We also found phage strains shared among both related and unrelated individuals, suggesting that a pool of common phages exists in the human population and that transmission of phages may occur between individuals. To better understand the bacterium–phage interactions in the skin microbiota, we determined the outcomes of 74 genetically defined Propionibacterium strains challenged by 15 sequenced phages. Depending on the Propionibacterium lineage, phage infection can result in lysis, pseudolysogeny, or resistance. In type II P. acnes strains, we found that encoding matching clustered regularly interspaced short palindromic repeat spacers is insufficient to confer phage resistance. Overall, our findings suggest that the prey–predator relationship between bacteria and phages may have a role in modulating the composition of the microbiota. Our study also suggests that the microbiome structure of an individual may be an important factor in the design of phage-based therapy.     

Diversity of phage integrases in Enterobacteriaceae: development of markers for environmental analysis of temperate phages

Viruses are the most abundant biological entities in aquatic systems. Temperate bacteriophages have enormous influences on microbial diversity, genetic exchange and bacterial population dynamics. However, development of molecular tools for their detection in the environment has been problematic. The integrase gene is used here as a molecular marker to analyse the diversity of temperate bacteriophages in a population of freshwater bacteria. Interrogation of the GenBank database revealed 32 non-cryptic enteric phage integrase sequences, leading to the development of a suite of 11 degenerate primer sets specific to the extant sequences elucidated. Application of these primer sets to enterobacterial isolates recovered from a freshwater pond and the temperate phages induced from them revealed a number of diverse integrase genes, including novel integrase-like sequences not represented in the databases. This highlights the potential of utilizing the integrase gene family as a marker for phage diversity.     

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Endolysins are produced by bacteriophages to degrade the host cell wall and release new particles, but the implications of their diversity on phage-host adaptation and evolution is unknown. This study uses CRISPR-Cas9 genome editing to reveal novel insights into bacteriophage endolysin diversity and phage-bacteria interactions as well as into endolysin adaptation towards a new bacterial host.     

Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.     

Bacteriophage genomics

Comparative genomic studies of bacteriophages, especially the tailed phages, together with environmental studies, give a dramatic new picture of the size, genetic structure and dynamics of this population. Sequence comparisons reveal some of the detailed mechanisms by which these viruses evolve and influence the evolution of their bacterial and archaeal hosts. We see rampant horizontal exchange of sequences among genomes, mediated by both homologous and nonhomologous recombination. High frequency exchange among phages occupying similar ecological niches leads to a high degree of mosaic diversity in local populations. Horizontal exchange also takes place at lower frequency across the entire span of phage sequence space.     

Differentiation of lactic streptococcal phages into phage species by DNA-DNA homology

Twenty-five lactic streptococcal bacteriophages were differentiated by DNA homology into four species. Complete correlation was found between DNA homology groups and morphological characteristics of the phages except that two phage types, which differed in only the presence or absence of a collar, were one species by DNA homology. These findings were supported by serological data and differences in DNA molecular weights of the proposed species. The complete lack of homology between these phage species indicates that they are unlikely to have a recent common phage ancestor and that one morphological type of phage has not been derived by mutation from a phage of another morphological type.     

Differentiation of lactic streptococcal phages into phage species by DNA-DNA homology.

Twenty-five lactic streptococcal bacteriophages were differentiated by DNA homology into four species. Complete correlation was found between DNA homology groups and morphological characteristics of the phages except that two phage types, which differed in only the presence or absence of a collar, were one species by DNA homology. These findings were supported by serological data and differences in DNA molecular weights of the proposed species. The complete lack of homology between these phage species indicates that they are unlikely to have a recent common phage ancestor and that one morphological type of phage has not been derived by mutation from a phage of another morphological type.     

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.     

Transfer of the Salmonella type III effector sopE between unrelated phage families.

Salmonella spp. are pathogenic enterobacteria that employ type III secretion systems to translocate effector proteins and modulate responses of host cells. The repertoire of translocated effector proteins is thought to define host specificity and epidemic virulence, and varies even between closely related Salmonella strains. Therefore, horizontal transfer of effector protein genes between Salmonella strains plays a key role in shaping the Salmonella-host interaction. Several effector protein genes are located in temperate phages. The P2-like phage SopE Phi encodes SopE and the lambda-like GIFSY phages encode several effector proteins of the YopM/IpaH-family. Lysogenic conversion with these phages is responsible for much of the diversity of the effector protein repertoires observed among Salmonella spp. However, free exchange of effector proteins by lysogenic conversion can be restricted by superinfection immunity. To identify genetic mechanisms that may further enhance horizontal transfer of effector genes, we have analyzed sopE loci from Salmonella spp. that do not harbor P2-like sequences of SopE Phi. In two novel sopE loci that were identified, the 723 nt sopE gene is located in a conserved 1.2 kb cassette present also in SopE Phi. Most strikingly, in Salmonella enterica subspecies I serovars Gallinarum, Enteritidis, Hadar and Dublin, the sopE-cassette is located in a cryptic lambda-like prophage with similarity to the GIFSY phages. This provides the first evidence for transfer of virulence genes between different phage families. We show that such a mechanism can circumvent restrictions to phage-mediated gene transfer and thereby enhances reassortment of the effector protein repertoires in Salmonella spp.     

Complete genomic nucleotide sequence of the temperate bacteriophage Aa Phi 23 of Actinobacillus actinomycetemcomitans

The entire double-stranded DNA genome of the Actinobacillus actinomycetemcomitans bacteriophage Aa Phi 23 was sequenced. Linear DNA contained in the phage particles is circularly permuted and terminally redundant. Therefore, the physical map of the phage genome is circular. Its size is 43,033 bp with an overall molar G+C content of 42.5 mol%. Sixty-six potential open reading frames (ORFs) were identified, including an ORF resulting from a translational frameshift. A putative function could be assigned to 23 of them. Twenty-three other ORFs share homologies only with hypothetical proteins present in several bacteria or bacteriophages, and 20 ORFs seem to be specific for phage Aa Phi 23. The organization of the phage genome and several genetic functions share extensive similarities to that of the lambdoid phages. However, Aa Phi 23 encodes a DNA adenine methylase, and the DNA packaging strategy is more closely related to the P22 system. The attachment sites of Aa Phi 23 (attP) and several A. actinomycetemcomitans hosts (attB) are 49 bp long.     

Synonymous codon usage in forty staphylococcal phages identifies the factors controlling codon usage variation and the phages suitable for phage therapy

The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2 may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be employed in curing staphylococcal infections.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of phiKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphology, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of phiKZ-like phages of the family Myoviridae designated in this manner according to the best studied phage phiKZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests the similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to the specific DNA package intrinsic to phages of phiKZ group. In the meantime, phages Lul11 and OBP do not exhibit resemblance among themselves or with any of earlier described phiKZ-like phages in respect to other traits; particularly, they have no detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa phiKZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the phiKZ-supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup phiKZ composition. The results of the study are discussed in terms of the evolution of these phages.     

A new group of cosmopolitan bacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus

A clonal population of pathogenic Vibrio parahaemolyticus O3 : K6 serovar has spread in coastal waters, causing outbreaks worldwide since 1996. Bacteriophage infection is one of the main factors affecting bacterial strain concentration in the ocean. We studied the occurrence and properties of phages infecting this V. parahaemolyticus pandemic strain in coastal waters. Analysing 143 samples, phages were found in 13. All isolates clustered in a closely related group of podophages with at least 90% nucleotide sequence identity in three essential genes, despite distant geographical origins. These bacteriophages were able to multiply on the V. parahaemolyticus pandemic strain, but the impact on host concentration and subsequent growth was negligible. Infected bacteria continued producing the phage but were not lysogenized. The phage genome of prototype strain VP93 is 43 931 nucleotides and contains 337 bp direct terminal repeats at both ends. VP93 is the first non‐Pseudomonas phage related to the ΦKMV‐like subgroup of the T7 supergroup. The lack of a major effect on host growth suggests that these phages exert little control on the propagation of the pandemic strain in the environment. This form of phage growth can be modelled if phage‐sensitive and ‐resistant cells that convert to each other with a high frequency are present in clonal cultures of pandemic V. parahaemolyticus.     

Comparison of new giant bacteriophages OBP and Lu11 of soil pseudomonads with bacteriophages of the ϕKZ-supergroup of Pseudomonas aeruginosa

Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens, respectively, demonstrated their similarity in morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of ?KZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage ?KZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an “inner body” was detected, which points to specific DNA package intrinsic to phages of ?KZ group. In the meantime, phages Lu11 and OBP do not exhibit resemblance among themselves or with any of earlier described ?KZ-like phages in respect to detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa ?KZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination (“gene shuffling”) between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the ?KZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup ?KZ composition. The results of the study are discussed in terms of the evolution of these phages.