Evaluation of lytic activity of staphylococcal bacteriophage Sb‐1 against freshly isolated clinical pathogens

In recent decades the increase in antibiotic‐resistant bacterial strains has become a serious threat to the treatment of infectious diseases. Drug resistance of Staphylococcus aureus has become a major problem in hospitals of many countries, including developed ones. Today the interest in alternative remedies to antibiotics, including bacteriophage treatment, is gaining new ground. Here, we describe the staphylococcal bacteriophage Sb‐1 – a key component of therapeutic phage preparation that was successfully used against staphylococcal infections during many years in the Former Soviet Union. This phage still reveals a high spectrum of lytic activity in vitro against freshly isolated, genetically different clinical samples (including methicillin‐resistant S. aureus) obtained from the local hospitals, as well as the clinics from different geographical areas. The sequence analyses of phage genome showed absence of bacterial virulence genes. A case report describes a promising clinical response after phage application in patient with cystic fibrosis and indicates the efficacy of usage of Sb‐1 phage against various staphylococcal infections.     

Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin

The lysin LysGH15, which is derived from the staphylococcal phage GH15, demonstrates a wide lytic spectrum and strong lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we find that the lytic activity of the full-length LysGH15 and its CHAP domain is dependent on calcium ions. To elucidate the molecular mechanism, the structures of three individual domains of LysGH15 were determined. Unexpectedly, the crystal structure of the LysGH15 CHAP domain reveals an “EF-hand-like” calcium-binding site near the Cys-His-Glu-Asn quartet active site groove. To date, the calcium-binding site in the LysGH15 CHAP domain is unique among homologous proteins, and it represents the first reported calcium-binding site in the CHAP family. More importantly, the calcium ion plays an important role as a switch that modulates the CHAP domain between the active and inactive states. Structure-guided mutagenesis of the amidase-2 domain reveals that both the zinc ion and E282 are required in catalysis and enable us to propose a catalytic mechanism. Nuclear magnetic resonance (NMR) spectroscopy and titration-guided mutagenesis identify residues (e.g., N404, Y406, G407, and T408) in the SH3b domain that are involved in the interactions with the substrate. To the best of our knowledge, our results constitute the first structural information on the biochemical features of a staphylococcal phage lysin and represent a pivotal step forward in understanding this type of lysin.     

Potential of the Polyvalent Anti-Staphylococcus Bacteriophage K for Control of Antibiotic-Resistant Staphylococci from Hospitals

The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth.     

Effective Removal of Staphylococcal Biofilms by the Endolysin LysH5

Staphylococcal biofilms are a major concern in both clinical and food settings because they are an important source of contamination. The efficacy of established cleaning procedures is often hindered due to the ability of some antimicrobial compounds to induce biofilm formation, and to the presence of persister cells, a small bacterial subpopulation that exhibits multidrug tolerance. Phage lytic enzymes have demonstrated antimicrobial activity against planktonic and sessile bacteria. However, their ability to lyse and/or select persister cells remains largely unexplored so far. In this work, the lytic activity of the endolysin LysH5 against Staphylococcus aureus and Staphylococcus epidermidis biofilms was confirmed. LysH5 reduced staphylococcal sessile cell counts by 1–3 log units, compared with the untreated control, and sub-inhibitory concentrations of this protein did not induce biofilm formation. LysH5-surviving cells were not resistant to the lytic activity of this protein, suggesting that no persister cells were selected. Moreover, to prove the lytic ability of LysH5 against this subpopulation, both S. aureus exponential cultures and persister cells obtained after treatment with rifampicin and ciprofloxacin were subsequently treated with LysH5. The results demonstrated that besides the notable activity of endolysin LysH5 against staphylococcal biofilms, persister cells were also inhibited, which raises new opportunities as an adjuvant for some antibiotics.     

Romulus and Remus,Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus,Display Suitable Properties for Phage Therapy Applications

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.     

Staphylococcal Phage 2638A endolysin is lytic for Staphylococcus aureus and harbors an inter-lytic-domain secondary translational start site

Staphylococcus aureus is a notorious pathogen highly successful at developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic for S. aureus when exposed externally, making it a new candidate antimicrobial. It shares a common protein organization with more than 40 other reported staphylococcal peptidoglycan hydrolases. There is an N-terminal M23 peptidase domain, a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase), and a C-terminal SH3b cell wall-binding domain. It is the first phage endolysin reported with a secondary translational start site in the inter-lytic-domain region between the peptidase and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. Although it is common for one domain to demonstrate a dominant activity over the other, the 2638A endolysin is the first in this class of proteins to have a high-activity amidase domain (dominant over the N-terminal peptidase domain). The high activity amidase domain is an important finding in the quest for high-activity staphylolytic domains targeting novel peptidoglycan bonds.     

The truncated phage lysin CHAP(k) eliminates Staphylococcus aureus in the nares of mice

The endolysin LysK derived from staphylococcal phage K has previously been shown to have two enzymatic domains, one of which is an N-acetylmuramoyl-L-alanine amidase and the other a cysteine/histidine-dependant amidohydrolase/peptidase designated CHAP(k). The latter, when cloned as a single-domain truncated enzyme, is conveniently overexpressed in a highly-soluble form. This enzyme was shown to be highly active in vitro against live cell suspensions of S. aureus. In the current study, the IVIS imaging system was used to demonstrate the effective elimination of a lux labeled S. aureus from the nares of BALB/c mice.     

Characterization of the staphylococcal bacteriophage lysin CHAP(K)

Aims: To develop an efficient purification strategy for the bacteriophage lysin CHAP_K. To evaluate its antibacterial spectrum_, enzymatic properties, optimal reaction conditions and lytic activity against live Staphlyococcus aureus. Methods and Results: Recombinant CHAP_K was purified to homogeneity by cation exchange chromatography, with yields of up to 10 mg from 1 l of Escherichia coli culture. The lytic spectrum of CHAP_K includes all staphylococcal species and also members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Carnobacterium. The enzyme was active from pH 6 to 11 with an optimum activity at pH 9, from 5 to 40°C, with an optimum activity at 15°C. When cell lysis by CHAP_K and lysostaphin was compared over a concentration range of 2·5–10 μg ml^?1 using live Staph. aureus for 5 min at 37°C, CHAP_K gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Conclusions: This study describes in detail the purification and characteristics of the novel phage‐derived enzyme CHAP_K demonstrating that it has excellent biochemical properties as an anti‐staphylococcal agent. Significance and Impact of the Study: Currently, there is a need for new antimicrobial agents due to the increasing worldwide prevalence of antibiotic resistance. Our findings demonstrate the potential for development of CHAP_K as an alternative therapeutic against pathogenic staphylococci including MRSA.     

Relative Rates of Lysis of Staphylococcal Cell Walls by Lytic Enzymes from Various Bacteriophage Types

Lytic enzymes were isolated from 14 strains of phage-infected Staphylococcus aureus. Cell walls were prepared from the same uninfected strains of bacteria. Comparison of the lytic rates was made for each enzyme, with each of the cell walls as substrate. Differences in the rate of substrate utilization of the various cell wall types exceeded 10-fold. Cell walls from strains 42E, 29, and 77 were the best substrates, whereas cell walls from strains 3C, 80, and 187 were the poorest substrates. The cell wall amino acid composition is discussed as related to lytic enzyme specificity. A possible explanation of phage typing of staphylococcal cells, based on enzyme activity and cell wall composition, is presented.     

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3–45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00934-6.     

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.     

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.     

Isolation and characterization of two anti-staphylococcal bacteriophages specific for pathogenic Staphylococcus aureus associated with bovine infections

AIMS: The aim of this study was to isolate and characterize bacteriophages against bovine Staphylococcus aureus associated with mastitis. METHODS AND RESULTS: We describe the isolation of two anti-staphylococcal phages namely DW2 and CS1 from farmyard slurry. Both phages were characterized by electron microscopy and restriction analysis and shown to belong to the Siphoviridae family. CS1 and DW2 were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. These phages were compared with the previously characterized Myoviridae phage K. Infusion of a cocktail of all three phages at 10(8) PFU ml(-1) into live cow teats resulted in no detectable increase in somatic cell counts in milks indicating that the phages did not irritate the animal. CONCLUSION: Two new anti-staphylococcal phages CS1 and DW2 were isolated and characterized and tested for immunogenicity in animal teats. SIGNIFICANCE AND IMPACT OF THE STUDY: The phages isolated in this study are active against pathogenic S. aureus and may be incorporated into teat-dips or teat-washes as a non-antibiotic prophylaxis against staphylococcal bovine mastitis.     

Two Phages,phiIPLA-RODI and phiIPLA-C1C,Lyse Mono- and Dual-Species Staphylococcal Biofilms

Phage therapy is a promising option for fighting against staphylococcal infections. Two lytic phages, vB_SauM_phiIPLA-RODI (phiIPLA-RODI) and vB_SepM_phiIPLA-C1C (phiIPLA-C1C), belonging to the Myoviridae family and exhibiting wide host ranges, were characterized in this study. The complete genome sequences comprised 142,348 bp and 140,961 bp and contained 213 and 203 open reading frames, respectively. The gene organization was typical of Spounavirinae members, with long direct terminal repeats (LTRs), genes grouped into modules not clearly separated from each other, and several group I introns. In addition, four genes encoding tRNAs were identified in phiIPLA-RODI. Comparative DNA sequence analysis showed high similarities with two phages, GH15 and 676Z, belonging to the Twort-like virus genus (nucleotide identities of >84%); for phiIPLA-C1C, a high similarity with phage phiIBB-SEP1 was observed (identity of 80%). Challenge assays of phages phiIPLA-RODI and phiIPLA-C1C against planktonic staphylococcal cells confirmed their lytic ability, as they were able to remove 5 log units in 8 h. Exposure of biofilms to phages phiIPLA-RODI and phiIPLA-C1C reduced the amount of adhered bacteria to about 2 log units in both monospecies and dual-species biofilms, but phiIPLA-RODI turned out to be as effective as the mixture of both phages. Moreover, the frequencies of bacteriophage-insensitive mutants (BIMs) of Staphylococcus aureus and S. epidermidis with resistance to phiIPLA-RODI and phiIPLA-C1C were low, at 4.05 × 10⁻⁷ ± 2.34 × 10⁻⁹ and 1.1 × 10⁻⁷ ± 2.08 × 10⁻⁹, respectively. Overall, a generally reduced fitness in the absence of phages was observed for BIMs, which showed a restored phage-sensitive phenotype in a few generations. These results confirm that lytic bacteriophages can be efficient biofilm-disrupting agents, supporting their potential as antimicrobials against staphylococcal infections.     

Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent against Bacillus cereus

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The catalytic domain of LysPBC1 when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus, even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus.     

Development of a broad-host-range phage cocktail for biocontrol

The aims of this study were to investigate the incidence of different resistance mechanisms to phage K in a bank of Irish Staph aureus hospital strains; and to develop a broad host-range phage cocktail with enhanced lytic activity against those strains which were previously phage resistant. A bank of 180 Staph aureus strains, which included all the sequence types currently in existence in Ireland, were tested for sensitivity to phage K. Twenty nine strains were identified, which did not permit plaque formation. The phage resistance systems in the 29 strain were investigated and it was found that restriction modification (r-m) was evident in 24, an adsorption inhibition mechanism was evident in three, while two were resistant by an unidentified mechanism. Seventeen modified derivatives of phage K were developed which could circumvent all the r-m systems. Nevertheless, six of the modified phage were considered superior in terms of their individual host ranges. These six were pooled as a cocktail with phage K, which then lysed 24 of the 29 resistant strains (97.2% of the entire staphylococcal bank). In conclusion, phage resistant systems affecting phage K are common in Staph. aureus but it is possible to significantly broaden the host-range of this phage for biocontrol applications.     

Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci

Developing chimeric lysins with a wide lytic spectrum would be important for treating some infections caused by multiple pathogenic bacteria. In the present work, a novel chimeric lysin (Ply187N-V12C) was constructed by fusing the catalytic domain (Ply187N) of the bacteriophage lysin Ply187 with the cell binding domain (146-314aa, V12C) of the lysin PlyV12. The results showed that the chimeric lysin Ply187N-V12C had not only lytic activity similar to Ply187N against staphylococcal strains but also extended its lytic activity to streptococci and enterococci, such as Streptococcus dysgalactiae, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium and Enterococcus faecalis, which Ply187N could not lyse. Our work demonstrated that generating novel chimeric lysins with an extended lytic spectrum was feasible through fusing a catalytic domain with a cell-binding domain from lysins with lytic spectra across multiple genera.     

Restriction and Modification in Group N Streptococci: Effect of Heat on Development of Modified Lytic Bacteriophage

The appearance of lytic bacteriophage against newly introduced starter strains used during commercial cheese manufacture occurs rapidly, and their origin is not well understood. In this study, members of the group N streptococci were examined for the presence of bacteriophage restriction and modification systems. Two streptococcal phages from Streptococcus cremoris TR and Streptococcus lactis C2 (phage designations tr and c2) showed restricted lytic development on S. cremoris 799 and KH, respectively. Efficiency of plaquing was 1.9 × 10⁻⁷ for tr plaqued on 799 and 2.1 × 10⁻⁷ for c2 plaqued on KH. After passage through the restrictive hosts, these phages demonstrated high lytic ability for formerly restrictive hosts. Stress of the restrictive host strains at temperatures of 40 to 50°C resulted in a significant increase in the efficiency of plaquing of restricted bacteriophages. Elevated temperatures are encountered during commercial cheese manufacture. The results suggested that the temporary loss of host restriction activity with the resulting modification of nonspecific bacteriophage may contribute directly to the appearance of lytic phage against new starter strains.     

LysGH15 reduces the inflammation caused by lethal methicillin-resistant Staphylococcus aureus infection in mice

The endolysin LysGH15, derived from staphylococcal phage GH15, has a wide lytic spectrum and strong lytic activity against Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), in vitro and in vivo. Here, the ability of lethal MRSA to induce mRNA levels of interleukin-6 (IL-6), interleukin-4 (IL-4), and interferon-γ (IFN-γ) in spleen tissues of mice was studied. A large number of bacteria were detected in spleens. The bacteria caused elevated expression levels of these three cytokines. Administration of LysGH15 significantly reduced the number of bacteria and the levels of IL-6, IL-4, and IFN-γ mRNA in spleen cells compared with those in untreated mice at 24 h (P < 0.05). LysGH15 can eliminate a large number of bacteria and effectively alleviate inflammation induced by infection with lethal MRSA.     

Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds

Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.