Multi-species host range of staphylococcal phages isolated from wastewater.

The host range of bacteriophages defines their impact on bacterial communities and genome diversity. Here, we characterize 94 novel staphylococcal phages from wastewater and establish their host range on a diversified panel of 117 staphylococci from 29 species. Using this high-resolution phage-bacteria interaction matrix, we unveil a multi-species host range as a dominant trait of the isolated staphylococcal phages. Phage genome sequencing shows this pattern to prevail irrespective of taxonomy. Network analysis between phage-infected bacteria reveals that hosts from multiple species, ecosystems, and drug-resistance phenotypes share numerous phages. Lastly, we show that phages throughout this network can package foreign genetic material enclosing an antibiotic resistance marker at various frequencies. Our findings indicate a weak host specialism of the tested phages, and therefore their potential to promote horizontal gene transfer in this environment.

Staphylococcus epidermidis Phages Transduce Antimicrobial Resistance Plasmids and Mobilize Chromosomal Islands.

Staphylococcus epidermidis is a leading opportunistic pathogen causing nosocomial infections that is notable for its ability to form a biofilm and for its high rates of antibiotic resistance. It serves as a reservoir of multiple antimicrobial resistance genes that spread among the staphylococcal population by horizontal gene transfer such as transduction. While phage-mediated transduction is well studied in Staphylococcus aureus, S. epidermidis transducing phages have not been described in detail yet. Here, we report the characteristics of four phages, 27, 48, 456, and 459, previously used for S. epidermidis phage typing, and the newly isolated phage E72, from a clinical S. epidermidis strain. The phages, classified in the family Siphoviridae and genus Phietavirus, exhibited an S. epidermidis-specific host range, and together they infected 49% of the 35 strains tested. A whole-genome comparison revealed evolutionary relatedness to transducing S. aureus phietaviruses. In accordance with this, all the tested phages were capable of transduction with high frequencies up to 10-4 among S. epidermidis strains from different clonal complexes. Plasmids with sizes from 4 to 19 kb encoding resistance to streptomycin, tetracycline, and chloramphenicol were transferred. We provide here the first evidence of a phage-inducible chromosomal island transfer in S. epidermidis Similarly to S. aureus pathogenicity islands, the transfer was accompanied by phage capsid remodeling; however, the interfering protein encoded by the island was distinct. Our findings underline the role of S. epidermidis temperate phages in the evolution of S. epidermidis strains by horizontal gene transfer, which can also be utilized for S. epidermidis genetic studies.IMPORTANCE Multidrug-resistant strains of S. epidermidis emerge in both nosocomial and livestock environments as the most important pathogens among coagulase-negative staphylococcal species. The study of transduction by phages is essential to understanding how virulence and antimicrobial resistance genes spread in originally commensal bacterial populations. In this work, we provide a detailed description of transducing S. epidermidis phages. The high transduction frequencies of antimicrobial resistance plasmids and the first evidence of chromosomal island transfer emphasize the decisive role of S. epidermidis phages in attaining a higher pathogenic potential of host strains. To date, such importance has been attributed only to S. aureus phages, not to those of coagulase-negative staphylococci. This study also proved that the described transducing bacteriophages represent valuable genetic modification tools in S. epidermidis strains where other methods for gene transfer fail.

Examination of Staphylococcus aureus Prophages Circulating in Egypt.

Staphylococcus aureus infections are of growing concern given the increased incidence of antibiotic resistant strains. Egypt, like several other countries, has seen alarming increases in methicillin-resistant S. aureus (MRSA) infections. This species can rapidly acquire genes associated with resistance, as well as virulence factors, through mobile genetic elements, including phages. Recently, we sequenced 56 S. aureus genomes from Alexandria Main University Hospital in Alexandria, Egypt, complementing 17 S. aureus genomes publicly available from other sites in Egypt. In the current study, we found that the majority (73.6%) of these strains contain intact prophages, including Biseptimaviruses, Phietaviruses, and Triaviruses. Further investigation of these prophages revealed evidence of horizontal exchange of the integrase for two of the prophages. These Egyptian S. aureus prophages are predicted to encode numerous virulence factors, including genes associated with immune evasion and toxins, including the Panton-Valentine leukocidin (PVL)-associated genes lukF-PV/lukS-PV. Thus, prophages are likely to be a major contributor to the virulence of S. aureus strains in circulation in Egypt.

Characterization of two newly isolated Staphylococcus aureus bacteriophages from Japan belonging to the genus Silviavirus.

Two Staphylococcus aureus bacteriophages, KSAP7 and KSAP11, were isolated from sewage and characterized. Based on morphology and DNA sequences, they were assigned to the genus Silviavirus, subfamily Twortvirinae, family Herelleviridae, whose members are hypothesized to be suitable for bacteriophage therapy. The KSAP7 and KSAP11 genomes were 137,950 and 138,307 bp in size, respectively. Although their DNA sequences were almost identical, evidence of site-specific DNA rearrangements was found in two regions. Changes in the number of PIEPEK amino acid sequence repeats encoded by orf10 and the insertion/deletion of a 541-bp sequence that includes a possible tail-related gene were identified.

Bioprospecting Staphylococcus Phages with Therapeutic and Bio-Control Potential.

Emergence of antibiotic-resistant bacteria is a serious threat to the public health. This is also true for Staphylococcus aureus and other staphylococci. Staphylococcus phages Stab20, Stab21, Stab22, and Stab23, were isolated in Albania. Based on genomic and phylogenetic analysis, they were classified to genus Kayvirus of the subfamily Twortvirinae. In this work, we describe the in-depth characterization of the phages that electron microscopy confirmed to be myoviruses. These phages showed tolerance to pH range of 5.4 to 9.4, to maximum UV radiation energy of 25 µJ/cm2, to temperatures up to 45 °C, and to ethanol concentrations up to 25%, and complete resistance to chloroform. The adsorption rate constants of the phages ranged between 1.0 × 10-9 mL/min and 4.7 × 10-9 mL/min, and the burst size was from 42 to 130 plaque-forming units. The phages Stab20, 21, 22, and 23, originally isolated using Staphylococcusxylosus as a host, demonstrated varied host ranges among different Staphylococcus strains suggesting that they could be included in cocktail formulations for therapeutic or bio-control purpose. Phage particle proteomes, consisting on average of ca 60-70 gene products, revealed, in addition to straight-forward structural proteins, also the presence of enzymes such DNA polymerase, helicases, recombinases, exonucleases, and RNA ligase polymer. They are likely to be injected into the bacteria along with the genomic DNA to take over the host metabolism as soon as possible after infection.

Isolation of a Novel Lytic Bacteriophage against a Nosocomial Methicillin-Resistant Staphylococcus aureus Belonging to ST45.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide range of infections from mild to life-threatening conditions. Its enhanced antibiotic resistance often leads to therapeutic failures and therefore alternative eradication methods must be considered. Potential candidates to control MRSA infections are bacteriophages and their lytic enzymes, lysins. In this study, we isolated a bacteriophage against a nosocomial MRSA strain belonging to the ST45 epidemiologic group. The phage belonging to Caudovirales, Siphoviridae, showed a narrow host range and stable lytic activity without the emergence of resistant MRSA clones. Phylogenetic analysis showed that the newly isolated Staphylococcus phage R4 belongs to the Triavirus genus in Siphoviridae family. Genetic analysis of the 45 kb sequence of R4 revealed 69 ORFs. No remnants of mobile genetic elements and traces of truncated genes were observed. We have localized the lysin (N-acetylmuramoyl-L-alanine amidase) gene of the new phage that was amplified, cloned, expressed, and purified. Its activity was verified by zymogram analysis. Our findings could potentially be used to develop specific anti-MRSA bacteriophage- and phage lysin-based therapeutic strategies against major clonal lineages and serotypes.

Temperate Phages of Staphylococcus aureus.

Most Staphylococcus aureus isolates carry multiple bacteriophages in their genome, which provide the pathogen with traits important for niche adaptation. Such temperate S. aureus phages often encode a variety of accessory factors that influence virulence, immune evasion and host preference of the bacterial lysogen. Moreover, transducing phages are primary vehicles for horizontal gene transfer. Wall teichoic acid (WTA) acts as a common phage receptor for staphylococcal phages and structural variations of WTA govern phage-host specificity thereby shaping gene transfer across clonal lineages and even species. Thus, bacteriophages are central for the success of S. aureus as a human pathogen.

Staphylococci phages display vast genomic diversity and evolutionary relationships.

BACKGROUND: Bacteriophages are the most abundant and diverse entities in the biosphere, and this diversity is driven by constant predator-prey evolutionary dynamics and horizontal gene transfer. Phage genome sequences are under-sampled and therefore present an untapped and uncharacterized source of genetic diversity, typically characterized by highly mosaic genomes and no universal genes. To better understand the diversity and relationships among phages infecting human pathogens, we have analysed the complete genome sequences of 205 phages of Staphylococcus sp. RESULTS: These are predicted to encode 20,579 proteins, which can be sorted into 2139 phamilies (phams) of related sequences; 745 of these are orphams and possess only a single gene. Based on shared gene content, these phages were grouped into four clusters (A, B, C and D), 27 subclusters (A1-A2, B1-B17, C1-C6 and D1-D2) and one singleton. However, the genomes have mosaic architectures and individual genes with common ancestors are positioned in distinct genomic contexts in different clusters. The staphylococcal Cluster B siphoviridae are predicted to be temperate, and the integration cassettes are often closely-linked to genes implicated in bacterial virulence determinants. There are four unusual endolysin organization strategies found in Staphylococcus phage genomes, with endolysins predicted to be encoded as single genes, two genes spliced, two genes adjacent and as a single gene with inter-lytic-domain secondary translational start site. Comparison of the endolysins reveals multi-domain modularity, with conservation of the SH3 cell wall binding domain. CONCLUSIONS: This study provides a high-resolution view of staphylococcal viral genetic diversity, and insights into their gene flux patterns within and across different phage groups (cluster and subclusters) providing insights into their evolution.

Genomic analyses of two novel biofilm-degrading methicillin-resistant Staphylococcus aureus phages.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) biofilm producers represent an important etiological agent of many chronic human infections. Antibiotics and host immune responses are largely ineffective against bacteria within biofilms. Alternative actions and novel antimicrobials should be considered. In this context, the use of phages to destroy MRSA biofilms presents an innovative alternative mechanism. RESULTS: Twenty-five MRSA biofilm producers were used as substrates to isolate MRSA-specific phages. Despite the difficulties in obtaining an isolate of this phage, two phages (UPMK_1 and UPMK_2) were isolated. Both phages varied in their ability to produce halos around their plaques, host infectivity, one-step growth curves, and electron microscopy features. Furthermore, both phages demonstrated antagonistic infectivity on planktonic cultures. This was validated in an in vitro static biofilm assay (in microtiter-plates), followed by the visualization of the biofilm architecture in situ via confocal laser scanning microscopy before and after phage infection, and further supported by phages genome analysis. The UPMK_1 genome comprised 152,788 bp coding for 155 putative open reading frames (ORFs), and its genome characteristics were between the Myoviridae and Siphoviridae family, though the morphological features confined it more to the Siphoviridae family. The UPMK_2 has 40,955 bp with 62 putative ORFs; morphologically, it presented the features of the Podoviridae though its genome did not show similarity with any of the S. aureus in the Podoviridae family. Both phages possess lytic enzymes that were associated with a high ability to degrade biofilms as shown in the microtiter plate and CLSM analyses. CONCLUSIONS: The present work addressed the possibility of using phages as potential biocontrol agents for biofilm-producing MRSA.

Analysis of phage resistance in Staphylococcus aureus SA003 reveals different binding mechanisms for the closely related Twort-like phages ɸSA012 and ɸSA039.

We have previously generated strains of Staphylococcus aureus SA003 resistant to its specific phage ɸSA012 through a long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed eight genes that acquired mutations: six point mutations (five missense mutations and one nonsense mutation) and two deletions. Complementation of the phage-resistant strains by the wild-type alleles showed that five genes were linked to phage adsorption of ɸSA012, and two mutated host genes were linked to the inhibition of post-adsorption. Unlike ɸSA012, infection by ɸSA039, a close relative of ɸSA012, onto early coevolved phage-resistant SA003 (SA003R2) was impaired drastically. Here, we identified that ɸSA012 and ɸSA039 adsorb to the cell surface S. aureus SA003 through a different mechanism. ɸSA012 requires the backbone of wall teichoic acids (WTA), while ɸSA039 requires both backbone and the ß-GlcNAc residue. In silico analysis of the ɸSA039 genome revealed that several proteins in the tail and baseplate region were different from ɸSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ɸSA012 and ɸSA039.

Electrophoretic techniques for purification, separation and detection of Kayvirus with subsequent control by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and microbiological methods.

The bacteriophage K1/420 is a member of genus Kayvirus that was extensively studied as an alternative treatment to combat bacterial infections caused by antibiotic-resistant Staphylococcus aureus strains. Despite the promise of phage therapy, the development of clinical applications of phages is facing regulatory and technical hurdles before it can receive acceptance in the Western World. Suitable simple and accurate diagnostic techniques to control the quality of the phage, which would satisfy the requirements of regulatory authorities are still being discussed. Here, we present the conditions for the simultaneous separation and detection of phage K1/420 and S. aureus by CZE and by CIEF were found, and the phage isoelectric point was determined to be 3.6. After removing the cell debris, the phage was successfully purified from the crude phage lysate and pre-concentrated by preparative isoelectric focusing. Its zone was localized by the positions of colored pI markers in the cellulose bed. The phage from the harvested zone had a decreased ability to infect its host. However, it was suitable for its separation, detection and identification by capillary electrophoretic methods, MALDI-TOF MS and electron microscopy.

Prevalence and Genomic Structure of Bacteriophage phi3 in Human-Derived Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates from 2000 to 2015.

Whereas the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) in animal husbandry and its transmission to humans are well documented, less is known about factors driving the epidemic spread of this zoonotic lineage within the human population. One factor could be the bacteriophage phi3, which is rarely detected in S. aureus isolates from animals but commonly found among isolates from humans, including those of the human-adapted methicillin-susceptible S. aureus (MSSA) CC398 clade. The proportion of phi3-carrying MRSA spa-CC011 isolates, which constitute presumptively LA-MRSA within the multilocus sequence type (MLST) clonal complex 398, was systematically assessed for a period of 16 years to investigate the role of phi3 in the adaptation process of LA-MRSA to the human host. For this purpose, 632 MRSA spa-CC011 isolates from patients of a university hospital located in a pig farming-dense area in Germany were analyzed. Livestock-associated acquisition of MRSA spa-CC011 was previously reported as having increased from 1.8% in 2000 to 29.4% in 2014 in MRSA-positive patients admitted to this hospital. However, in this study, the proportion of phi3-carrying isolates rose only from 1.1% (2000 to 2006) to 3.9% (2007 to 2015). Characterization of the phi3 genomes revealed 12 different phage types ranging in size from 40,712 kb up to 44,003 kb, with four hitherto unknown integration sites (genes or intergenic regions) and several modified bacterial attachment (attB) sites. In contrast to the MSSA CC398 clade, phi3 acquisition seems to be no major driver for the readaptation of MRSA spa-CC011 to the human host.

Rapid Identification of Intact Staphylococcal Bacteriophages Using Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry.

Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.

Complete genome sequence analysis of a novel Staphylococcus phage StAP1 and proposal of a new species in the genus Silviavirus.

Bacteriophage StAP1 was isolated from a soil sample infecting Staphylococcus aureus and S. xylosus. Its genome was found to be 135,502 base pairs (bp) long with 30.00 mol% G+C content and 192 open reading frames. While no tRNA encoding genes were identified, 7 mobile elements were found to interrupt five StAP1 open reading frames. Comparative genomic and proteomic analysis consistently supports the establishment of a new species in the genus Silviavirus.

Characterization and complete genome of the virulent Myoviridae phage JD007 active against a variety of Staphylococcus aureus isolates from different hospitals in Shanghai, China.

BACKGROUND: The implementation of phage therapy is re-emerging with the increase in widespread antibiotic-resistant bacteria. METHODS: Staphylococcus phage JD007 was characterized and its complete genome sequence analysed. RESULTS: Staphylococcus phage JD007 was classified as belonging to the Myoviridae family based on its morphology, as observed by transmission electron microscopy. Its lytic activity was stable between pH 5-11 and below 42 °C; moreover, an absorbance curve showed that nearly 90% of the viral particles had adsorbed to its host after a 20 min co-incubation. The complete genome size is 141,836 bp, making JD007 one of the largest Staphylococcus phages of Myoviridae. No identifiable resistance or virulence genes were found in the JD007 genome. JD007 was able to lyse 95% of S. aureus isolates, including the prevalent ST239-MRSA and ST59-MRSA strains isolated from different hospitals in Shanghai, China, and inhibition assays showed that JD007 could inhibit S. aureus growth at a multiplicity of infection of 0.1. CONCLUSIONS: The results suggested that Staphylococcus phage JD007 can potentially be used in phage therapy or for the detection of S. aureus.

Phage Conversion for ß-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-ß-lactamase enzymes that degrade ß-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-ß-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a ß-lactam antibiotic susceptibility test, the transductant was more highly resistant to ß-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of ß-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Genomic analysis of Staphylococcus phage Stau2 isolated from medical specimen.

Stau2 is a lytic myophage of Staphylococcus aureus isolated from medical specimen. Exhibiting a broad host range against S. aureus clinical isolates, Stau2 is potentially useful for topical phage therapy or as an additive in food preservation. In this study, Stau2 was firstly revealed to possess a circularly permuted linear genome of 133,798 bp, with low G + C content, containing 146 open reading frames, but encoding no tRNA. The genome is organized into several modules containing genes for packaging, structural proteins, replication/transcription and host-cell-lysis, with the structural proteins and DNA polymerase modules being organized similarly to that in Twort-like phages of Staphylococcus. With the encoded DNA replication genes, Stau2 can possibly use its own system for replication. In addition, analysis in silico found several introns in seven genes, including those involved in DNA metabolism, packaging, and structure, while one of them (helicase gene) is experimentally confirmed to undergo splicing. Furthermore, phylogenetic analysis suggested Stau2 to be most closely related to Staphylococcus phages SA11 and Remus, members of Twort-like phages. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 14 structural proteins of Stau2 and N-terminal sequencing identified three of them. Importantly, this phage does not encode any proteins which are known or suspected to be involved in toxicity, pathogenicity, or antibiotic resistance. Therefore, further investigations of feasible therapeutic application of Stau2 are needed.

Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97.

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus.

Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus.

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

Characterization and complete genome sequence analysis of Staphylococcus aureus bacteriophage JS01.

Staphylococcus aureus is a primary pathogen that causes bovine mastitis resulting in serious economic losses and herd management problems in dairy cows. A novel bacteriophage, JS01, specifically infecting bovine S. aureus, was isolated from milk of mastitis-affected cattle. TEM observation showed that it belonged to the family Siphovirus. The JS01 strain demonstrated a broad host range. The prediction result of PHACTS suggested that the JS01 strain was temperate phage. The JS01 genome is 43,458 bp long, with a GC content of 33.32% and no tRNAs. Annotation and functional analysis of the predicted ORFs revealed six functional groups: structure and morphology, DNA replication and regulation, packaging, lysogeny, lysis, and pathogenicity. Comparative analysis between JS01, S. aureus MSSA476, and S. aureus prophage PVL was also performed. The characterization and genomic analysis of JS01 provide a better understanding of S. aureus-targeting bacteriophages and useful information for the development of phage-based biocontrol agents against S. aureus.