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PubMed1965

WILD-TYPE VARIANTS OF EXOPENICILLINASE FROM STAPHYLOCOCCUS AUREUS.

1. Three variants of staphylococcal exopenicillinase (types A, B and C) can be distinguished on chemical, enzymological and immunological grounds. 2. Enzyme type A has a higher specific activity than that of type B, but has a similar combination affinity with anti-(exopenicillinase type A) serum. 3. Enzyme types A and C have a similar specific activity, but enzyme type C has a lower combination affinity for anti-(exopenicillinase type A) serum than has enzyme type A. 4. The sedimentation coefficients and amino acid analyses of the three enzyme types are similar. 5. All three enzyme types have small but significant differences in kinetics of action when hydrolysing benzylpenicillin, methicillin, cloxacillin and cephalosporin C. 6. Peptide maps, obtained from enzyme types A and C after digestion with trypsin, show that these two variants probably differ in the nature of only a very few amino acid residues. 7. Enzyme type B seems to be confined to staphylococci that are members of staphylococcal phage group II. Enzyme types A and C are produced by staphylococci that are members either of phage group I or III, but never group II. 8. The low specific enzyme activity and affinity of enzyme type B towards all penicillins tested suggest that this enzyme type has a lower ;efficiency' in hydrolysing penicillin and therefore in protecting bacteria from the action of penicillin. This could account for the low incidence among ;hospital staphylococci' of penicillin-resistant staphylococci that are members of phage group II.

Amino Acids↗

PubMed1965

Bacteriophage types and antibiotic sensitivity of staphylococci from bovine milk and human nares.

The number, phage types, and antibiotic sensitivity of coagulase-positive staphylococci from grade A raw milk samples produced on 40 farms in the Athens, Ga., milkshed were determined. Counts of mannitol-positive staphylococci in milk ranged from 100 to 3,580 per milliliter, with an arithmetic mean of 1,047. Examination of the nares of 48 dairymen on 34 of the farms also revealed that 13 (27%) were carriers of coagulase-positive staphylococci. Isolates from milk (412) and from nares (39) were tested against the Coles, Seto-Wilson, and International phage sets and 87, 68, and 56%, respectively, proved typable. Nine isolates were not typable. Each of the 33 phages used lysed one or more of the isolates. Staphylococcal phage types per milk sample ranged from 0 to 5, 0 to 7, and 0 to 8, with arithmetic means of 1.9, 2.3, and 2.3, respectively. Of the 13 narial carriers, 7 harbored staphylococci of one or more of the same phage types as those isolated from the milk at the respective farms. Randomly selected isolates were tested against high and low concentrations of 12 common antibiotics. All were either moderately sensitive or resistant to polymixin B. Over 30% were moderately sensitive or resistant to dihydrostreptomycin and penicillin individually. With but few exceptions, all isolates were sensitive to chlortetracycline, bacitracin, carbomycin, chloramphenicol, erythromycin, neomycin, novobiocin, oxytetracycline, and tetracycline individually.

Animals↗

PubMed2005

The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus.

This study concerns the cloning, characterization, and expression of the lysin (LysK) from staphylococcal phage K in Lactococcus lactis. Lactococcal lysates containing recombinant LysK were found to inhibit a range of different species of staphylococci isolated from bovine and human infection sources, including methicillin-resistant Staphylococcus aureus. LysK thus has potential as an antimicrobial for applications in the prevention and/or treatment of infections caused by staphylococci.

Animals↗

PubMed1979

Comparison of Staphylococcus aureus carriage and skin infection rates in hospital and office employees.

The incidence of Staphylococcus aureus in the nose, throat and superficial wound infections of 99 office staff, 129 psychiatry staff and 115 surgical staff was studied over a 4-week period with the purpose of assessing the potential risk to hospital personnel of staphylococcal infection. Incidence rates, both average and cumulative, were essentially similar in the three groups but certain differences in the ecology of the staphylococcal phage groups were observed. Surgical staff appeared to have a more labile pattern of carriage. As in other Scandinavian studies throat carriage rates were high. Staphylococcal carriage seems largely to depend on individual characteristics rather than environmental factors.

Adult↗

PubMed2007

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812.

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

A study on Staphylococcus aureus strains submitted to a reference laboratory.

BACKGROUND & OBJECTIVES: Staphylococcus aureus is regarded as one of the most devastating human pathogens. Recently there have been reports of increasing incidence of S. aureus strains resistant to methicillin (MRSA). A surveillance study was undertaken to record the occurrence of MRSA and to study the prevalence of various phage groups in India. METHODS: A total of 7574 strains of S. aureus received during 1992-98 at the National Staphylococcal Phage Typing Centre, New Delhi were tested for methicillin resistance and susceptibility to phages of the International basic set. The occurrence of various phage groups between MRSA and MSSA (methicillin sensitive S. aureus) was compared. Results were analyzed according to the geographical origin and source of isolation of the strains. RESULTS: The dominant phage group from different parts of the country was phage group III. Prevalence of phage group III among the MRSA and MSSA isolates was 62.32 and 33.95 per cent respectively. The highest isolation of phage group III strains was from nasal carriers (45.94%), phage group II strains from skin (8.74%), phage group I strains from blood (19.44%) and nontypable strains from the environment (80.68%). An increase in the occurrence of MRSA has been noticed from 9.83 per cent in 1992 to 45.44 per cent in 1998. INTERPRETATION & CONCLUSION: S. aureus strains of phage group III are prevalent in India. The increase in occurrence of MRSA indicates an alarming spread of these organisms. A constant monitoring is important to take appropriate and timely measures to control their spread.

Bacteriophage Typing↗

PubMed1978

The phage adsorption test: a simple method for differentiation between staphylococci and micrococci.

A simple phage adsorption test is recommended for differentiation between staphylococci and micrococci. All 132 strains of Staphylococcus tested inactivated phage U16 significantly, whereas of 123 strains of Micrococcus tested, only three strains of Micrococcus candicans were able to adsorb this staphylococcal phage. There are special problems in the taxonomy of M. candicans.

Adsorption↗

PubMedDate not supplied

[New type of DNA structural organization in the composition of bacteriophage Sb-1 particles].

Spectrophotometric melting and chemical modification procedures were used for comparative study of parameters of DNA structure in particles of phages Sb-1 and FI-5 possessing spherical symmetry of the heads. Like in other phages of this morphological group, some part of DNA structure in FI-5 phage particles is disarrayed and has changed reactive capacity of basic aminogroups. Another type of DNA structural organization in situ was found with staphylococcal phage Sb-1. According to the results of melting process and pattern of interaction with 4.5% formaldehyde, DNA in particles of this phage throughout had base stacking. Basic aminogroups of DNA in situ are most likely completely involved in intramolecular complementary interactions and are not subjected to oximethylation under reaction conditions.

Amino Acids↗

PubMed2004

Novel cassette-based shuttle vector system for gram-positive bacteria.

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

Artificial Gene Fusion↗

PubMed1978

Serological characterization of phages of coagulase-negative staphylococci.

Selected 22 phages of coagulase-negative staphylococci were tested against group-specific antisera for Staphylococcus aureus phages using a neutralization test. Fourteen of the tested phages were classified into the serological group B and one into the group F. All other phages were resistant to the antisera against S. aureus phages but cross-reacted with the antisera produced against themselves. Therefore they could be joined together into a new serological group of the staphylococcal phages.

Bacteriophage Typing↗

PubMed2005

Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme.

Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. The current treatment strategy is limited to intrapartum antibiotic prophylaxis in pregnant women to prevent early-onset neonatal diseases, but considering the potential for antibiotic resistance, the risk of losing control over the disease is high. To approach this problem, we have developed a bacteriophage (phage) lytic enzyme to remove colonizing GBS. Bacteriophage muralytic enzymes, termed lysins, are highly evolved molecules designed to degrade the cell wall of host bacteria to release phage particles from the bacterial cytoplasm. Several different lysins have been developed to specifically kill bacterial pathogens both on mucosal surfaces and in blood and represent a novel approach to control infection. A lysin cloned from a phage infecting GBS was found to contain two putative catalytic domains and one putative binding domain, which is similar to the domain organization of some staphylococcal phage lysins. The lysin (named PlyGBS) was recombinantly expressed in Escherichia coli, and purified PlyGBS efficiently killed all tested GBS serotypes in vitro. In a mouse model, a single dose of PlyGBS significantly reduced bacterial colonization in both the vagina and oropharynx. As an alternative strategy for intrapartum antibiotic prophylaxis, this approach may be used to reduce vaginal GBS colonization in pregnant women before delivery or to decontaminate newborns, thus reducing the incidence of GBS-associated neonatal meningitis and sepsis.

Animals↗

PubMed1963

INHIBITORY ACTION OF PHAGE K ON STAPHYLOCOCCAL DEHYDROGENASES. II. ITS POSSIBLE RELATIONSHIP TO SENSITIZATION AND CELL LYSIS.

Ralston, D. J. (University of California, Berkeley) and M. D. Perry. Inhibitory action of phage K on staphylococcal dehydrogenases. II. Its possible relationship to sensitization and cell lysis. J. Bacteriol. 86:740-748. 1963.-By measuring the reduction of the dye triphenyl tetrazolium chloride to the insoluble red formazan, an analysis was made of the inhibition by phage K of the dehydrogenase capacity of populations of Staphylococcus aureus K(1), to determine to what extent this might be associated with the ability of phage K to sensitize the cells-a reaction characterized by the conversion of the cell wall to susceptibility to lysis by the staphylococcal enzyme, virolysin. Increasing multiplicities of phage progressively increased the fractions of sensitized cells and also caused increasing inhibition of the dehydrogenase activity of the populations. The dehydrogenase activities of the sensitized fractions were compared with those of the nonsensitized fractions. Over a wide range of phage-bacterium ratios, the dehydrogenase activities of the sensitized fractions were found to be lower than those of the nonsensitized fractions. Microscopically, this was reflected by the appearance of large numbers of cells with a reduced amount of visible formazan granules. When lysin was added to the phage-treated cells, lysis occurred mainly from cell fractions which possessed little or no tetrazolium-reducing capacity. The data indicated that sensitization by phage was accompanied by a marked decrease in cellular dehydrogenase activity but was not associated with a complete inhibition of these enzymes. A comparison was made between the dehydrogenases of phage-sensitized cells and cells found to be "spontaneously" sensitive to virolysin, i.e., lysed by the enzyme without any prior exposure to phage. Like phage-sensitized cells, the spontaneously sensitized staphylococci possessed low dehydrogenase activity and lacked the capacity to support phage synthesis. In tests of a given cell preparation, the dehydrogenase levels of the phage-sensitized fractions were found to be close to, or even lower than, the level of the spontaneously sensitized fraction, suggesting that in S. aureus K(1) the sensitized state is associated in some manner with a reduction of the dehydrogenase activity to a critical level. There is as yet no evidence for any direct causal relationship between sensitization and dehydrogenase inhibition.

Bacteriophages↗

PubMedDate not supplied

Characterization of thirteen Staphylococcus epidermidis and S. saprophyticus bacteriophages.

Thirteen bacteriophages of coagulase-negative staphylococci belonging to the Siphoviridae family (morphotype B1) were compared by seroneutralization kinetics, protein profiles, G + C content, DNA size and DNA/DNA hybridization. A previous classification into three morphological groups was confirmed. The Staphylococcus epidermidis phage group, although morphologically homogeneous, was heterogeneous by DNA/DNA hybridization (27 to 100% homology) and seroneutralization kinetics. Two new phage "species", STA1139 (STA for Staphylococcus) and STA1154A, corresponding to two morphological types of S. saprophyticus phages, were identified. Species STA1154A was particularly interesting because the size of its capsid, only 46 nm in diameter, and of its DNA, evaluated as 13 kbp, were smaller than those of other staphylococcal phages.

Base Composition↗

PubMed1978

Serological studies of a host range mutant of a lactic streptococcal bacteriophage.

A host range mutant was isolated from a bacteriophage that attacked Streptococcus cremoris 114. The mutant was able to adsorb and grow on S. cremoris 266, where the parent phage could not. The mutant phage was unable to adsorb to the original bacterial host, S. cremoris 114. The change in host range was accompanied by an alteration in the neutralization antigen as shown by a change in neutralization rate by an anti-phage serum. Serum-blocking experiments confirmed the difference in neutralization antigen between parent and mutant phages. The two phages nevertheless had similar complement fixation antigens, confirming that one was a mutant derived from the other. A distinction between complement fixation and neutralization antigens, similar to that found for the coliphages and staphylococcal phages, has therefore been demonstrated for two lactic streptococcal phages.

Adsorption↗

PubMed1999

The complete genome sequence of the Streptomyces temperate phage straight phiC31: evolutionary relationships to other viruses.

The completed genome sequence of the temperate Streptomyces phage straight phiC31 is reported. straight phiC31 contains genes that are related by sequence similarities to several other dsDNA phages infecting many diverse bacterial hosts, including Escherichia, Arthrobacter, Mycobacterium, Rhodobacter, Staphylococcus, Bacillus, Streptococcus, Lactobacillus and Lactococcus. These observations provide further evidence that dsDNA phages from diverse bacterial hosts are related and have had access to a common genetic pool. Analysis of the late genes was particularly informative. The sequences of the head assembly proteins (portal, head protease and major capsid) were conserved between straight phiC31, coliphage HK97, staphylococcal phage straight phiPVL, two Rhodobacter capsulatus prophages and two Mycobacterium tuberculosis prophages. These phages and prophages (where non-defective) from evolutionarily diverse hosts are, therefore, likely to share a common head assembly mechanism i.e. that of HK97. The organisation of the tail genes in straight phiC31 is highly reminiscent of tail regions from other phage genomes. The unusual organisation of the putative lysis genes in straight phiC31 is discussed, and speculations are made as to the roles of some inessential early gene products. Similarities between certain phage gene products and eukaryotic dsDNA virus proteins were noted, in particular, the primase/helicases and the terminases (large subunits). Furthermore, the complete sequence clarifies the overall transcription map of the phage during lytic growth and the positions of elements involved in the maintenance of lysogeny.

Amino Acid Sequence↗

PubMedDate not supplied

Phage-typing results of Staphylococcus aureus isolated from poultry in Japan and Europe.

Phage typing was performed on 795 strains of Staphylococcus aureus isolated from poultry, a turkey, pigeons, and birds of prey in Japan and 4 countries in Europe, using the avian phage set of typing phages plus 6 others. Six hundred and seventy-three (89.1%) of 755 strains from poultry were typable by one or more of the phages, 617 strains at routine test dilution (RTD) and 56 strains at 100 RTD. Of these typable strains, 494 (73.4%) belonged to group I, 90 (13.4%) to group II, 50 (7.4%) to group III, 29 (4.3%) to group IV, and 10 (1.5%) to mixed types. There was no significant relation between staphylococcal phage types and the origin of the strains, such as types of disease. Type I strains were commonly found in staphylococci isolated from poultry farms regardless of districts or countries, and thus they were considered to be distributed widely among poultry in Japan and Europe. On the other hand, type III or IV strains were obtained from very limited farms or areas. Four (66.7%) of 6 strains of S. aureus isolated from birds of prey were typed by the additional phage CH39 at RTD. None of 33 pigeon and 1 turkey strain was sensitive to any of the avian phages used.

Animals↗

PubMed1973

Characterization of Staphylococcus aureus in a pediatric burn unit.

A one-year study on an endemic strain of Staphylococcus aureus phage type 84/85 in a children's burn unit is described. The endemic strain rapidly colonized the burns and nares of acute patients after admission but was not isolated from a patient on admission. Nonendemic strains of S. aureus found on some new patients were mostly non-phage typable and did not prevail in burns. The endemic strain was rarely isolated from the nares and skin of reconstructive patients or from the nares of hospital personnel. The endemic strain did colonize the oral cavity, normal skin, and intestinal tract of some acute patients. Endemic and nonendemic strains of S. aureus from the burned children were compared in their biochemical activities and antibiotic sensitivities to two groups of S. aureus from one other local and one Danish burns unit. The latter groups of strains represented different combinations of staphylococcal phage group III strains. Each of the four groups of strains differed in production of hemolysins, Tween 80 hydrolysis, egg yolk reaction, and proteolysis of casein and gelatin. All of the strains were uniformly sensitive to gentamicin, oxacillin, and cephalothin. Only 4 of 162 strains tested were methicillin resistant. The endemic S. aureus strains of phage type 84/85 were uniformly resistant to eight other antibiotics including lincomycin and clindamycin. The endemic strain was not the known cause of a clinically documented infection in a group of 82 acute patients studied. The possible role of S. aureus strains of phage group III in burn grafting problems is discussed.

Adolescent↗

PubMed2005

The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli.

From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes (holWMY and lytWMY) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and pI were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli, lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N-acetylmuramoyl-L-alanine amidases encoded by the staphylococcal phages phi11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for D-alanyl-glycyl endopeptidase), amidase (L-muramoyl-L-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys-His-Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and pI of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, phi11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (phiWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY-lysWMY genes in the phage genome.

Amidohydrolases↗