Bacteriophage ISP eliminates Staphylococcus aureus in planktonic phase, but not in the various stages of the biofilm cycle.

Metal-implant associated bacterial infections are a major clinical problem due to antibiotic treatment failure. As an alternative, we determined the effects of bacteriophage ISP on clinical isolates of Staphylococcus aureus in various stages of its life cycle in relation to biofilm formation and maturation. ISP effectively eliminated all planktonic phase bacteria, whereas its efficacy was reduced against bacteria attached to the metal implant and bacteria embedded within biofilms. The biofilm architecture hampered the bactericidal effects of ISP, as mechanical disruption of biofilms improved the efficacy of ISP against the bacteria. Phages penetrated the biofilm and interacted with the bacteria throughout the biofilm. However, most of the biofilm-embedded bacteria were phage-tolerant. In agreement, bacteria dispersed from mature biofilms of all clinical isolates, except for LUH15394, tolerated the lytic activity of ISP. Lastly, persisters within mature biofilms tolerated ISP and proliferated in its presence. Based on these findings, we conclude that ISP eliminates planktonic phase Staphylococcus aureus while its efficacy is limited against bacteria attached to the metal implant, embedded within (persister-enriched) biofilms, and dispersed from biofilms.

Isolation and characterization of two Staphylococcus aureus lytic bacteriophages "Huma" and "Simurgh".

Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.

Biological and genomic characterization of 4 novel bacteriophages isolated from sewage or the environment using non-aureus Staphylococci strains.

Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44 %) and PT94 (28/54, 52 %) had broader lysis than PT1-4 (3/54, 6 %) and PT1-9 (10/54, 19 %), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60℃. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5 h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of "phage tail fiber." In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.

Bacteriophages avoid autoimmunity from cognate immune systems as an intrinsic part of their life cycles.

Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned off during prophage induction, balancing immunity and autoimmunity. Our results show that the fine regulation of these processes is essential for the correct development of prophages' life cycle.

Phage activity against Staphylococcus aureus is impaired in plasma and synovial fluid.

S. aureus is a pathogen that frequently causes severe morbidity and phage therapy is being discussed as an alternative to antibiotics for the treatment of S. aureus infections. In this in vitro and animal study, we demonstrated that the activity of anti-staphylococcal phages is severely impaired in 0.5% plasma or synovial fluid. Despite phage replication in these matrices, lysis of the bacteria was slower than phage propagation, and no reduction of the bacterial population was observed. The inhibition of the phages associated with a reduction in phage adsorption, quantified to 99% at 10% plasma. S. aureus is known to bind multiple coagulation factors, resulting in the formation of aggregates and blood clots that might protect the bacterium from the phages. Here, we show that purified fibrinogen at a sub-physiological concentration of 0.4 mg/ml is sufficient to impair phage activity. In contrast, dissolution of the clots by tissue plasminogen activator (tPA) partially restored phage activity. Consistent with these in vitro findings, phage treatment did not reduce bacterial burdens in a neutropenic mouse S. aureus thigh infection model. In summary, phage treatment of S. aureus infections inside the body may be fundamentally challenging, and more investigation is needed prior to proceeding to in-human trials.

The host phylogeny determines viral infectivity and replication across Staphylococcus host species.

Virus host shifts, where a virus transmits to and infects a novel host species, are a major source of emerging infectious disease. Genetic similarity between eukaryotic host species has been shown to be an important determinant of the outcome of virus host shifts, but it is unclear if this is the case for prokaryotes where anti-virus defences can be transmitted by horizontal gene transfer and evolve rapidly. Here, we measure the susceptibility of 64 strains of Staphylococcaceae bacteria (48 strains of Staphylococcus aureus and 16 non-S. aureus species spanning 2 genera) to the bacteriophage ISP, which is currently under investigation for use in phage therapy. Using three methods-plaque assays, optical density (OD) assays, and quantitative (q)PCR-we find that the host phylogeny explains a large proportion of the variation in susceptibility to ISP across the host panel. These patterns were consistent in models of only S. aureus strains and models with a single representative from each Staphylococcaceae species, suggesting that these phylogenetic effects are conserved both within and among host species. We find positive correlations between susceptibility assessed using OD and qPCR and variable correlations between plaque assays and either OD or qPCR, suggesting that plaque assays alone may be inadequate to assess host range. Furthermore, we demonstrate that the phylogenetic relationships between bacterial hosts can generally be used to predict the susceptibility of bacterial strains to phage infection when the susceptibility of closely related hosts is known, although this approach produced large prediction errors in multiple strains where phylogeny was uninformative. Together, our results demonstrate the ability of bacterial host evolutionary relatedness to explain differences in susceptibility to phage infection, with implications for the development of ISP both as a phage therapy treatment and as an experimental system for the study of virus host shifts.

Screening for Highly Transduced Genes in Staphylococcus aureus Revealed Both Lateral and Specialized Transduction.

Bacteriophage-mediated transduction of bacterial DNA is a major route of horizontal gene transfer in the human pathogen, Staphylococcus aureus. Transduction involves the packaging of bacterial DNA by viruses and enables the transmission of virulence and resistance genes between cells. To learn more about transduction in S. aureus, we searched a transposon mutant library for genes and mutations that enhanced transfer mediated by the temperate phage, ϕ11. Using a novel screening strategy, we performed multiple rounds of transduction of transposon mutant pools selecting for an antibiotic resistance marker within the transposon element. When determining the locations of transferred mutations, we found that the screen had selected for just 1 or 2 transposon mutant(s) within each pool of 96 mutants. Subsequent analysis showed that the position of the transposon, rather than the inactivation of bacterial genes, was responsible for the phenotype. Interestingly, from multiple rounds, we identified a pattern of transduction that encompassed mobile genetic elements as well as chromosomal regions both upstream and downstream of the phage integration site. The latter was confirmed by DNA sequencing of purified phage lysates. Importantly, transduction frequencies were lower for phage lysates obtained by phage infection rather than induction. Our results confirmed previous reports of lateral transduction of bacterial DNA downstream of the integrated phage but also indicated a novel form of specialized transduction of DNA upstream of the phage. These findings illustrated the complexity of transduction processes and increased our understanding of the mechanisms by which phages transfer bacterial DNA. IMPORTANCE Horizontal transfer of DNA between bacterial cells contributes to the spread of virulence and antibiotic resistance genes in human pathogens. For Staphylococcus aureus, bacterial viruses play a major role in facilitating the horizontal transfer. These viruses, termed bacteriophages, can transfer bacterial DNA between cells by a process known as transduction, which despite its importance is only poorly characterized. Here, we employed a transposon mutant library to investigate transduction in S. aureus. We showed that the genomic location of bacterial DNA relative to where bacteriophages integrated into that bacterial genome affected how frequently that DNA was transduced. Based on serial transduction of transposon mutant pools and direct sequencing of bacterial DNA in bacteriophage particles, we demonstrated both lateral and specialized transduction. The use of mutant libraries to investigate the genomic patterns of bacterial DNA transferred between cells could help us understand how horizontal transfer influences virulence and resistance development.

Staphylococcal Phages Adapt to New Hosts by Extensive Attachment Site Variability.

Bacterial pathogens commonly carry prophages that express virulence factors, and human strains of Staphylococcus aureus carry Sa3int phages, which promote immune evasion. Recently, however, these phages have been found in livestock-associated, methicillin-resistant S. aureus (LA-MRSA). This is surprising, as LA-MRSA strains contain a mutated primary bacterial integration site, which likely explains why the rare integration events that do occur mostly happen at alternative locations. Using deep sequencing, we show that after initial integration at secondary sites, Sa3int phages adapt through nucleotide changes in their attachment sequences to increase homology with alternative bacterial attachment sites. Importantly, this homology significantly enhances integrations in new rounds of infections. We propose that promiscuity of the phage-encoded tyrosine recombinase is responsible for establishment of Sa3int phages in LA-MRSA. Our results demonstrate that phages can adopt extensive population heterogeneity, leading to establishment in strains lacking bona fide integration sites. Ultimately, their presence may increase virulence and zoonotic potential of pathogens with major implications for human health. IMPORTANCE A growing number of humans are being infected by antibiotic resistant Staphylococcus aureus originating from livestock. The preference of S. aureus for humans or animals is in part determined by factors encoded by viruses (phages) that reside in the bacterial genome. Here, we reveal a process by which phages adapt to and become integrated in new strains of S. aureus lacking the preferred phage integration site. We propose that this is due to the relaxed specificity of a phage-encoded enzyme called recombinase. As this recombinase is used by many other phages, our results might have implications for a broader range of phages. Importantly, the adaptation described here enables S. aureus to jump between host organisms and increases its zoonotic threat.

Dynamics of Antibacterial Drone Establishment in Staphylococcus aureus: Unexpected Effects of Antibiotic Resistance Genes.

The antibacterial drone (ABD) system is based on repurposing the phage-inducible staphylococcal pathogenicity islands (SaPIs) for use as antibacterial agents that are indifferent to antibiotic resistance. The ABDs were constructed by inserting tetM for tetracycline resistance (Tcr) selection, replacing the SaPI virulence genes with bactericidal or bacteriostatic genes such as CRISPR/cas9/agrA, whose expression kills by double-strand cleavage of agrA, or CRISPR/dcas9/agrP2P3, whose expression blocks the target organism's virulence. ABD DNA is packaged in phage-like particles that attack their staphylococcal targets in vivo as well as in vitro. We determine ABD titers by transfer frequency, enumerate surviving cells as a function of multiplicity, and analyze the fate of ABD DNA with green fluorescent protein. An initial study revealed surprisingly that many more cells were killed by the ABD than were measured by transduction. Our study of this phenomenon has revealed several important features of the ABD system: (i) a significant number of entering ABD DNA molecules do not go on to establish stable transductants (i.e., are abortive); (ii) ABD cargo genes are expressed immediately following entry, even by the abortive ABDs; (iii) immediate plating on Tc-containing agar seriously underestimates particle numbers, partly owing to Tc inhibition of protein synthesis; (iv) replacement of tetM with cadA (conferring resistance to CdCl2) provides more accurate particle enumeration; (v) ABDs expressing CRISPR/cas9/agrA kill ∼99.99% of infected cells and provide the most accurate measurement of particle numbers as well as proof of principle for the system; and (vi) surprisingly, TetM interferes with stable establishment of ABD DNA independently of Tcr. IMPORTANCE For a particulate therapeutic agent, such as the ABD, accurate enumeration of particles is critical to enable evaluation of preparative procedures and calculation of therapeutic dosages. It is equally important that a selective marker used for these two purposes be biologically inert. We have long used tetM for these purposes but show here that tetM not only underestimates particle titers, by over 20-fold in some experiments, but also seriously impedes stable establishment of the therapeutic particle DNA. Given that tetM is a very convenient and widely used selective marker, publication of these findings is of considerable importance to the microbiological community as well as an interesting illustration of the unpredictable biological effects of genes taken out of their native context.

Characterization of the genetic switch from phage ɸ13 important for Staphylococcus aureus colonization in humans.

Temperate phages are bacterial viruses that after infection either reside integrated into a bacterial genome as prophages forming lysogens or multiply in a lytic lifecycle. The decision between lifestyles is determined by a switch involving a phage-encoded repressor, CI, and a promoter region from which lytic and lysogenic genes are divergently transcribed. Here, we investigate the switch of phage ɸ13 from the human pathogen Staphylococcus aureus. ɸ13 encodes several virulence factors and is prevalent in S. aureus strains colonizing humans. We show that the ɸ13 switch harbors a cI gene, a predicted mor (modulator of repression) gene, and three high-affinity operator sites binding CI. To quantify the decision between lytic and lysogenic lifestyle, we introduced reporter plasmids that carry the 1.3 kb switch region from ɸ13 with the lytic promoter fused to lacZ into S. aureus and Bacillus subtilis. Analysis of ß-galactosidase expression indicated that decision frequency is independent of host factors. The white "lysogenic" phenotype, which relies on the expression of cI, could be switched to a stable blue "lytic" phenotype by DNA damaging agents. We have characterized lifestyle decisions of phage ɸ13, and our approach may be applied to other temperate phages encoding virulence factors in S. aureus.

A Case of In Situ Phage Therapy against Staphylococcus aureus in a Bone Allograft Polymicrobial Biofilm Infection: Outcomes and Phage-Antibiotic Interactions.

Phage therapy (PT) shows promising potential in managing biofilm infections, which include refractory orthopedic infections. We report the case of a 13-year-old girl who developed chronic polymicrobial biofilm infection of a pelvic bone allograft after Ewing's sarcoma resection surgery. Chronic infection by Clostridium hathewayi, Proteus mirabilis and Finegoldia magna was worsened by methicillin-susceptible Staphylococcus aureus exhibiting an inducible Macrolides-Lincosamides-Streptogramin B resistance phenotype (iMLSB). After failure of conventional conservative treatment, combination of in situ anti-S. aureus PT with surgical debridement and intravenous antibiotic therapy led to marked clinical and microbiological improvement, yet failed to prevent a recurrence of infection on the midterm. This eventually led to surgical graft replacement. Multiple factors can explain this midterm failure, among which incomplete coverage of the polymicrobial infection by PT. Indeed, no phage therapy against C. hathewayi, P. mirabilis or F. magna could be administered. Phage-antibiotic interactions were investigated using OmniLog® technology. Our results suggest that phage-antibiotic interactions should not be considered "unconditionally synergistic", and should be assessed on a case-by-case basis. Specific pharmacodynamics of phages and antibiotics might explain these differences. More than two years after final graft replacement, the patient remains cured of her sarcoma and no further infections occurred.

A regulatory cascade controls Staphylococcus aureus pathogenicity island activation.

Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage-encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA-immR-str'. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl-str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.

Characterization of staphylococcal endolysin LysSAP33 possessing untypical domain composition.

Endolysin, a peptidoglycan hydrolase derived from bacteriophage, has been suggested as an alternative antimicrobial agent. Many endolysins on staphylococcal phages have been identified and applied extensively against Staphylococcus spp. Among them, LysK-like endolysin, a well-studied staphylococcal endolysin, accounts for most of the identified endolysins. However, relatively little interest has been paid to LysKunlike endolysin and a few of them has been characterized. An endolysin LysSAP33 encoded on bacteriophage SAP33 shared low homology with LysK-like endolysin in sequence by 41% and domain composition (CHAP-unknown CBD). A green fluorescence assay using a fusion protein for LysSAP33_CBD indicated that the CBD domain (157-251 aa) was bound to the peptidoglycan of S. aureus. The deletion of LysSAP33_CBD at the C-terminal region resulted in a significant decrease in lytic activity and efficacy. Compared to LysK-like endolysin, LysSAP33 retained its lytic activity in a broader range of temperature, pH, and NaCl concentrations. In addition, it showed a higher activity against biofilms than LysK-like endolysin. This study could be a helpful tool to develop our understanding of staphylococcal endolysins not belonging to LysK-like endolysins and a potential biocontrol agent against biofilms.

Comparative Genomics of Three Novel Jumbo Bacteriophages Infecting Staphylococcus aureus.

The majority of previously described Staphylococcus aureus bacteriophages belong to three major groups, namely, P68-like podophages, Twort-like or K-like myophages, and a more diverse group of temperate siphophages. Here, we present the following three novel S. aureus "jumbo" phages: MarsHill, Madawaska, and Machias. These phages were isolated from swine production environments in the United States and represent a novel clade of S. aureus myophage. The average genome size for these phages is ∼269 kb with each genome encoding ∼263 predicted protein-coding genes. Phage genome organization and content are similar to those of known jumbo phages of Bacillus sp., including AR9 and vB_BpuM-BpSp. All three phages possess genes encoding complete virion and nonvirion RNA polymerases, multiple homing endonucleases, and a retron-like reverse transcriptase. Like AR9, all of these phages are presumed to have uracil-substituted DNA which interferes with DNA sequencing. These phages are also able to transduce host plasmids, which is significant as these phages were found circulating in swine production environments and can also infect human S. aureus isolates. IMPORTANCE This study describes the comparative genomics of the following three novel S. aureus jumbo phages: MarsHill, Madawaska, and Machias. These three S. aureus myophages represent an emerging class of S. aureus phage. These genomes contain abundant introns which show a pattern consistent with repeated acquisition rather than vertical inheritance, suggesting intron acquisition and loss are active processes in the evolution of these phages. These phages have presumably hypermodified DNA which inhibits sequencing by several different common platforms. Therefore, these phages also represent potential genomic diversity that has been missed due to the limitations of standard sequencing techniques. In particular, such hypermodified genomes may be missed by metagenomic studies due to their resistance to standard sequencing techniques. Phage MarsHill was found to be able to transduce host DNA at levels comparable to that found for other transducing S. aureus phages, making it a potential vector for horizontal gene transfer in the environment.

Synergistic action of phage phiIPLA-RODI and lytic protein CHAPSH3b: a combination strategy to target Staphylococcus aureus biofilms.

Staphylococcus aureus is considered a priority pathogen due to its increasing acquisition of antibiotic resistance determinants. Additionally, this microbe has the ability to form recalcitrant biofilms on different biotic and inert surfaces. In this context, bacteriophages and their derived lytic proteins may be a forward-looking strategy to help combat staphylococcal biofilms. However, these antimicrobials exhibit individual limitations that may be overcome by combining them with other compounds. This work investigates the combination of a phage-derived lytic protein, CHAPSH3b, and the virulent bacteriophage phiIPLA-RODI. The obtained results show the synergy between both antimicrobials for the treatment of 24-h-old S. aureus biofilms, with greater reductions in viable cell counts observed when phage and lysin are applied together compared to the individual treatments. Time-kill curves and confocal microscopy revealed that the fast antibacterial action of CHAPSH3b reduces the population up to 7 hours after initial exposure, which is subsequently followed by phage predation, limiting regrowth of the bacterial population. Moreover, at least 90% of bacteriophage insensitive mutants are susceptible to the lytic protein. Therefore, CHAPSH3b might help curtail the development of phage resistance during treatment. The combination of the lysin and phiIPLA-RODI also showed promising results in an ex vivo pig skin model of wound infection. Overall, the results of this study demonstrate that the combination of phage-derived lytic proteins and bacteriophages can be a viable strategy to develop improved antibiofilm products.

Potential of bacteriophages as disinfectants to control of Staphylococcus aureus biofilms.

BACKGROUND: Staphylococcus aureus is the causative agent of chronic mastitis, and can form a biofilm that is difficult to completely remove once formed. Disinfectants are effective against S. aureus, but their activity is easily affected by environmental factors and they are corrosive to equipment and chemically toxic to livestock and humans. Therefore, we investigated the potential utility of a bacteriophage as a narrow-spectrum disinfectant against biofilms formed by S. aureus. In this study, we isolated and characterized bacteriophage vB_SauM_SDQ (abbreviated to SDQ) to determine its efficacy in removing S. aureus biofilms. RESULTS: SDQ belongs to the family Myoviridae and consists of a hexagonal head, long neck, and short tail. This phage can sterilize a 109 CFU/mL culture of S. aureus in 12 h and multiply itself 1000-fold in that time. Biofilms formed on polystyrene, milk, and mammary-gland tissue were significantly reduced after SDQ treatment. Fluorescence microscopy and scanning electron microscopy showed that SDQ destroyed the biofilm structure. Moreover, the titer of SDQ remained relatively high after the lysis of the bacteria and the removal of the biofilm, exerting a continuous bacteriostatic effect. SDQ also retained its full activity under conditions that mimic common environments, i.e., in the presence of nonionic detergents, tap water, or organic materials. A nonionic detergent (Triton X-100) enhanced the removal of biofilm by SDQ. CONCLUSIONS: Our results suggest that SDQ, a specific lytic S. aureus phage, can be used to control biofilm infections. SDQ maintains its full activity in the presence of nonionic detergents, tap water, metal chelators, and organic materials, and can be used in combination with detergents. We propose this phage as a narrow-spectrum disinfectant against S. aureus, to augment or supplement the use of broad-spectrum disinfectants in the prevention and control of the mastitis and dairy industry contamination caused by S. aureus.

Genes Influencing Phage Host Range in Staphylococcus aureus on a Species-Wide Scale.

Staphylococcus aureus is a human pathogen that causes serious diseases, ranging from skin infections to septic shock. Bacteriophages (phages) are both natural killers of S. aureus, offering therapeutic possibilities, and important vectors of horizontal gene transfer (HGT) in the species. Here, we used high-throughput approaches to understand the genetic basis of strain-to-strain variation in sensitivity to phages, which defines the host range. We screened 259 diverse S. aureus strains covering more than 40 sequence types for sensitivity to eight phages, which were representatives of the three phage classes that infect the species. The phages were variable in host range, each infecting between 73 and 257 strains. Using genome-wide association approaches, we identified putative loci that affect host range and validated their function using USA300 transposon knockouts. In addition to rediscovering known host range determinants, we found several previously unreported genes affecting bacterial growth during phage infection, including trpA, phoR, isdB, sodM, fmtC, and relA We used the data from our host range matrix to develop predictive models that achieved between 40% and 95% accuracy. This work illustrates the complexity of the genetic basis for phage susceptibility in S. aureus but also shows that with more data, we may be able to understand much of the variation. With a knowledge of host range determination, we can rationally design phage therapy cocktails that target the broadest host range of S. aureus strains and address basic questions regarding phage-host interactions, such as the impact of phage on S. aureus evolution.IMPORTANCEStaphylococcus aureus is a widespread, hospital- and community-acquired pathogen, many strains of which are antibiotic resistant. It causes diverse diseases, ranging from local to systemic infection, and affects both the skin and many internal organs, including the heart, lungs, bones, and brain. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent. One alternative therapy to antibiotics is phage therapy, in which viruses specific to infecting bacteria clear infection. In this work, we identified and validated S. aureus genes that influence phage host range-the number of strains a phage can infect and kill-by testing strains representative of the diversity of the S. aureus species for phage host range and associating the genome sequences of strains with host range. These findings together improved our understanding of how phage therapy works in the bacterium and improve prediction of phage therapy efficacy based on the predicted host range of the infecting strain.

Staphylococcal Phage in Combination with Staphylococcus Epidermidis as a Potential Treatment for Staphylococcus Aureus-Associated Atopic Dermatitis and Suppressor of Phage-Resistant Mutants.

Atopic dermatitis is accompanied by the abnormal overgrowth of Staphylococcus aureus, a common cause of skin infections and an opportunistic pathogen. Although administration of antibiotics is effective against S. aureus, the resulting reduction in healthy microbiota and the emergence of drug-resistant bacteria are of concern. We propose that phage therapy can be an effective strategy to treat atopic dermatitis without perturbing the microbiota structure. In this study, we examined whether the S. aureus phage SaGU1 could be a tool to counteract the atopic exacerbation induced by S. aureus using an atopic mouse model. Administration of SaGU1 to the back skin of mice reduced both S. aureus counts and the disease exacerbation caused by S. aureus. Furthermore, the S. aureus-mediated exacerbation of atopic dermatitis with respect to IgE plasma concentration and histopathological findings was ameliorated by the application of SaGU1. We also found that Staphylococcus epidermidis, a typical epidermal symbiont in healthy skin, significantly attenuated the emergence of SaGU1-resistant S. aureus under co-culture with S. aureus and S. epidermidis in liquid culture infection experiments. Our results suggest that phage therapy using SaGU1 could be a promising clinical treatment for atopic dermatitis.

The Protective Effect of Staphylococcus epidermidis Biofilm Matrix against Phage Predation.

Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms' high tolerance to antibiotics, virulent bacteriophages were previously tested as novel therapeutic agents. However, several staphylococcal bacteriophages were shown to be inefficient against biofilms. In this study, the previously characterized S. epidermidis-specific Sepunavirus phiIBB-SEP1 (SEP1), which has a broad spectrum and high activity against planktonic cells, was evaluated concerning its efficacy against S. epidermidis biofilms. The in vitro biofilm killing assays demonstrated a reduced activity of the phage. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct planktonic and biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse planktonic cells in different physiological states, suggesting that the inefficacy for biofilm control resulted from the biofilm 3D structure and the protective effect of the matrix. To assess the impact of the biofilm architecture on phage predation, SEP1 was tested in disrupted biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells after 6 h of infection. The interaction between SEP1 and the biofilm matrix was further assessed by the addition of matrix to phage particles. Results showed that the matrix did not inactivate phages nor affected phage adsorption. Moreover, confocal laser scanning microscopy data demonstrated that phage infected cells were less predominant in the biofilm regions where the matrix was more abundant. Our results provide compelling evidence indicating that the biofilm matrix can work as a barrier, allowing the bacteria to be hindered from phage infection.

Milk microbial composition of Brazilian dairy cows entering the dry period and genomic comparison between Staphylococcus aureus strains susceptible to the bacteriophage vB_SauM-UFV_DC4.

Brazil has the second-largest dairy cattle herd in the world, and bovine mastitis still can cause significant losses for dairy farmers. Despite this fact, little information is available about milk microbial composition of Brazilian dairy cows, as well as the potential use of bacteriophages in the control of S. aureus. Here, we investigated milk bacterial composition of 28 Holstein Fresian cows (109 teats), selected in the dry-off period, using 16S rRNA analysis. Furthermore, a representative S. aureus strain (UFV2030RH1) was obtained at drying-off for isolation of a bacteriophage (vB_SauM-UFV_DC4, UFV_DC4) and bacterial genomic comparison purposes. Our outcomes revealed that Staphylococcus was the third most prevalent genus and positively correlated with subclinical mastitis events. As a major finding, genomic analyses showed the presence of adhesive matrix molecules that recognize microbial surface components (MSCRAMM) in UFV2030RH1 and might indicate great biofilm formation capability. A minimum inhibitory concentration (MIC) assay showed that resistance to ampicillin was the highest among the antibiotic tested in S. aureus 3059 and UFV2030RH1, displaying values four and sixteen times greater than MIC resistance breakpoint, respectively. Together, our results suggest that Staphylococcus is highly prevalent in dairy cows at drying-off and the use of the phage UFV_DC4 as a biocontrol agent must be investigated in future studies.