The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages

Two newly discovered immune modulators, chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3' end of beta-hemolysin (hlb)-converting bacteriophages (betaC-phis). Since these betaC-phis also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3' end of betaC-phi represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a betaC-phi with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5'-to-3' orientation and on the 3' end of a betaC-phi. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different betaI-phis. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of beta-hemolysin-converting bacteriophages.     

Anti-opsonic properties of staphylokinase

Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.     

Large variabilities in host strain susceptibility and phage host range govern interactions between lytic marine phages and their Flavobacterium hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.     

Bacteria, phages and septicemia

The use of phages is an attractive option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage ecology in bacterial infections is obscure. Here we surveyed the phage ecology in septicemia, the most severe type of bacterial infection. We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease. Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis-causing bacterial clones play a role in clonal selection during bacterial invasion.     

Differentiation of methicillin-resistant strains of Staphylococcus aureus by prophage specificity]

By inducing with mitomycin C the following phages were isolated from all the tested 32 methicillin resistant strains of S. aureus: the serogroup B phage was isolated from 2 strains, the serogroup B and F phages were isolated from 5 strains and the serogroup F phage was isolated from 25 strains. The phages were divided into 5 groups by the antiphage immunity. In group 1 of the phages 4 additional phages were specified. By the specificity of the prophages in the cultures all the strains were divided into 5 groups. Group 1 of the cultures was divided into 5 subgroups (A, B, C, D and E).     

Localization of prophages of serological group B and F on restriction fragments defined in the restriction map of Staphylococcus aureus NCTC 8325

Abstract Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages φ53, φ85) as well as by some of serological group F (phages φ77, φ84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the Sma I restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages φ53 and φ85 integrate into Sma I fragment B. On the other hand, phages φ77 and φ84 integrate into Smal fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us φM integrates in fragment A, whereas the integration site for phage φJ lies in fragment E. Phage φM was estimated to be genetically related to phages of serological group A and phage φJ to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.     

Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their Flavobacterium Hosts

Phages are a main mortality factor for marine bacterioplankton and are thought to regulate bacterial community composition through host-specific infection and lysis. In the present study we demonstrate for a marine phage-host assemblage that interactions are complex and that specificity and efficiency of infection and lysis are highly variable among phages infectious to strains of the same bacterial species. Twenty-three Bacteroidetes strains and 46 phages from Swedish and Danish coastal waters were analyzed. Based on genotypic and phenotypic analyses, 21 of the isolates could be considered strains of Cellulophaga baltica (Flavobacteriaceae). Nevertheless, all bacterial strains showed unique phage susceptibility patterns and differed by up to 6 orders of magnitude in sensitivity to the same titer of phage. The isolated phages showed pronounced variations in genome size (8 to >242 kb) and host range (infecting 1 to 20 bacterial strains). Our data indicate that marine bacterioplankton are susceptible to multiple co-occurring phages and that sensitivity towards phage infection is strain specific and exists as a continuum between highly sensitive and resistant, implying an extremely complex web of phage-host interactions. Hence, effects of phages on bacterioplankton community composition and dynamics may go undetected in studies where strain identity is not resolvable, i.e., in studies based on the phylogenetic resolution provided by 16S rRNA gene or internal transcribed spacer sequences.     

Genomic relatedness of Staphylococcus aureus phages of the International Typing Set and detection of serogroup A, B, and F prophages in lysogenic strains

On the basis of HindIII-restriction digest analysis of genomic DNAs, the S. aureus bacteriophages of the International Typing Set were divided into five clusters designated as A, F, Ba, Bb, and Bc. The clusters A and F include all the phages of serogroups A and F and correspond to species 3A and 77 proposed by Ackermann and DuBow (1987). On the other hand, the phages of serogroup B were divided into three clusters designated as Ba, Bb, and Bc that differ significantly each from the other in their restriction patterns. The clusters Ba and Bb may represent two separate species, while the cluster Bc may include more than one phage species. For each of the phage serogroups A, B, and F, common HindIII-restriction fragments of phage 3A (1700 bp), of 53 (4060 bp), and of 77 (8300 bp) were used for the preparation of probes specific to the phages of serogroups A, B, and F. These probes were very effective, making it possible to detect up to three different prophages in a given lysogenic strain at the same time. Restriction enzyme maps of phages 3A, 53, and 77, each representing a different serogroup, were constructed. The restriction maps of phage 3A and that of phage 77 are linear, whereas that of phage 53 is circular and exhibits a circular permutation. DNAs of the phages of serogroups A and F have cohesive ends. On each restriction map, the sites corresponding to specific probes are indicated. The size of intact genomic DNA of all phages estimated by PFGE varies within the range of 41.5-46.2 kb.     

Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.     

The highly virulent 2006 Norwegian EHEC O103:H25 outbreak strain is related to the 2011 German O104:H4 outbreak strain

In 2006, a severe foodborne EHEC outbreak occured in Norway. Seventeen cases were recorded and the HUS frequency was 60%. The causative strain, Esherichia coli O103:H25, is considered to be particularly virulent. Sequencing of the outbreak strain revealed resemblance to the 2011 German outbreak strain E. coli O104:H4, both in genome and Shiga toxin 2-encoding (Stx2) phage sequence. The nucleotide identity between the Stx2 phages from the Norwegian and German outbreak strains was 90%. During the 2006 outbreak, stx(2)-positive O103:H25 E. coli was isolated from two patients. All the other outbreak associated isolates, including all food isolates, were stx-negative, and carried a different phage replacing the Stx2 phage. This phage was of similar size to the Stx2 phage, but had a distinctive early phage region and no stx gene. The sequence of the early region of this phage was not retrieved from the bacterial host genome, and the origin of the phage is unknown. The contaminated food most likely contained a mixture of E. coli O103:H25 cells with either one of the phages.     

Staphylococcal and other Bacterial Species Associated with Intramammary Infections in Danish Dairy Herds

Four thousand six hundred forty– five quarter milk samples from 1179 cows from 20 commercial dairy herds were examined in order to determine the prevalence of bacterial species. A total of 859 isolates from 839 (18.1%) culture positive samples could be assigned to 34 different species and subspecies. Diagnostics of staphylococcal species was based on conventional procedures able to differentiate between all 36 species and subspecies presently acknowledged. Staphylococcus aureus was found in 10.2% of the samples and was the most common species isolated. Streptococcus dysgalactiae (1.6%) and Streptococcus uberis (1.4%) were the second and third most common species isolated. Seventeen different coagulase negative staphylococcal species (CNS) were found in 4.1% of the samples. The most frequently isolated CNS were S. epidermidis (1.3%), S. chromogenes (1.0%) and S. simulans (0.7%). Isolates of S. aureus were phage typed, and isolates of S. epidermidis were investigated by phage typing, antibiogram typing, and biotyping. A total of 378 (79.9%) isolates of S. aureus could be typed by phages, assigning them to 18 different phage types. However, 6 phage types accounted for 92.1% of the typable isolates. One to 2 phage types predominated within each herd. Eleven (18%) isolates of S. epidermidis could be typed by phages, assigning the isolates to 3 different types. Biotyping of S. epidermidis produced a total of 8 different types, the most common accounting for 29.5% of the isolates. A total of 6 different antibiogram types were observed among all isolates of S. epidermidis. Resistance towards penicillin (36.1%), tetracycline (9.8%) and streptomycin (9.8%), were recorded in the isolates of S. epidermidis. However, 35 (57.4%) of the isolates were susceptible to all 12 antibiotics tested.     

Cangene gold medal award lecture - Genomic analysis and modification of Burkholderia cepacia complex bacteriophages

The Burkholderia cepacia complex (Bcc) is a group of 17 Gram-negative predominantly environmental bacterial species that cause potentially fatal opportunistic infections in cystic fibrosis (CF) patients. Although its prevalence in these individuals is lower than that of Staphylococcus aureus and Pseudomonas aeruginosa , the Bcc remains a serious problem in the CF community because of the pathogenicity, transmissibility, and inherent antibiotic resistance of these organisms. An alternative treatment for Bcc infections that is currently being developed is phage therapy, the clinical use of viruses that infect bacteria. To assess the suitability of individual phage isolates for therapeutic use, the complete genome sequences of a panel of Bcc-specific phages were determined and analyzed. These sequences encode a broad range of proteins with a gradient of relatedness to phage and bacterial gene products from Burkholderia and other genera. The majority of these phages were found not to encode virulence factors, and despite their predominantly temperate nature, a proof-of-principle experiment has shown that they may be modified to a lytic form. Both the genomic characterization and subsequent engineering of Bcc-specific phages are fundamental to the development of an effective phage therapy strategy for these bacteria.     

Isolation and Preliminary Characterization of Bacteriophages for Bdellovibrio bacteriovorus

Ten bacteriophages that attack and lyse saprophytic strains of Bdellovibrio bacteriovorus were isolated. Morphological, serological, and host-range studies revealed that there were four different bdellovibrio phages present among the isolates. One of the phages lysed a strain of B. bacteriovorus that requires the presence of a suitable bacterial host for growth. The phage attached to the bdellovibrio cells in the absence of the bacterial host cells; lysis occurred only in the presence of host cells. The 19 saprophytic bdellovibrio strains employed in the phage host-range studies were grouped on the basis of their susceptibility to phage lysis.     

Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin

Lytic bacteriophages (phages) have been investigated as treatments for bacterial infectious diseases. An induced phage, SAP-26, was isolated from a clinical isolate of Staphylococcus aureus. It belongs to the family Siphoviridae and its genome consists of double-stranded 41,207 bp DNA coding for 63 open reading frames. The phage SAP-26 showed a wide spectrum of lytic activity against both methicillin-resistant S. aureus and methicillin-susceptible S.aureus. Furthermore, combined treatment with a phage and antimicrobial agents showed a strong biofilm removal effect which induced structural changes in the biofilm matrix and a substantial decrease in the number of bacteria. Such a broad host range in S. aureus and biofilm removal activity of the phage SAP-26 suggests the possibility of its use as a therapeutic phage in combination with appropriate antimicrobial agent(s). Among the three antimicrobial agents combined with phage, the combination of rifampicin showed the best biofilm removal effect. To the authors' knowledge, this study showed for the first time that S. aureus biofilm could be efficiently eradicated with the mixture of phage and an antimicrobial agent, especially rifampicin.     

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.     

Characterization of Deoxyribonucleic Acid of Virulent Bacteriophage and its Infectivity to Host Bacteria, Pseudomonas solanacearum

Virulent bacteriophage PK-101 was isolated from soil infested with strain K-101 of Pseudomonas solanacearum and nucleic acid was prepared from the phages. Some chemical properties of phage nucleic acid and its infectivities to various strains of P. solanacearum were examined in the present study. By digestion with restriction endonucleases, phage nucleic acid was shown to be linear duplex DNA approximately 35 kb long. Restriction fragment length polymorphism was observed when electrophoresis patterns of enzyme-digested PK-101 DNA were compared with those of DNA prepared from different phage isolates. Transfection of host strains by PK-101 DNA was carried out, and it was infectious not only to host strain K-101, but also to other strains which were resistant to phage particles. Transfection efficiency was considerably enhanced by directly introducing phage DNA into bacterial cells by means of an electroporation. The electroporation technique was also effective to transform P. solanacearum with large-size plasmid DNA.     

VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi

We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested. The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V. cholerae and Ff phages of Escherichia coli. The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide. VGJ phi, like other filamentous phages, uses a type IV pilus to infect V. cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin. VGJ phi-infected V. cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages. Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state. Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts. Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V. cholerae El Tor.     

Bacteriophage translocation

The occurrence of phages in the human body, especially in the gastrointestinal tract, raises the question of their potential role in the physiology and pathology of this system. Especially important is the issue of whether phages can pass the intestinal wall and migrate to lymph, peripheral blood, and internal organs and, if so, the effects such a phenomenon could have (such passage by bacteria, known as bacterial translocation, has been shown to cause various disturbances in humans, from immune defects to sepsis). Available data from the literature support the assumption that phage translocation can take place and may have some immunomodulatory effects. In addition, phages of the gut may play a protective role by inhibiting local immune reactions to antigens derived from gut flora.     

Relationship between capsular types 5 and 8 and phage types in Staphylococcus aureus isolates from cow, goat and ewe milk

Abstract In order to check the relationship between capsular polysaccharide (CP) type 5 and 8 and certain phage patterns, previously described in human Staphylococcus aureus clinical isolates, we typed 100 CP types 5 and 8 S. aureus strains isolated from cow, goat and ewe milk, with the human set of phages. The proportion of typable strains was much less than that found with human strains. The association between CP types 5 and 8 and phage patterns reported for human isolates was only partially confirmed and an original correlation between susceptibility to group III phages and CP type 5 was found.