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PubMed1975

Experiences and observations with the typing of Staphylococcus aureus phage 94.

During a 28-month period, 5,078 cultures from a variety of anatomical sites were received for staphylococcal phage typing. Of these, 503 (10%) were not suitable for the procedure requested. Of the 4,575 viable cultures, 1,030 (23%) of the microorganisms were nontypable at both the routine test dilution (RTD) and at 100 times RTD. Of the 3,545 typable organisms, 3,061 (86%) were lysed at RTD, whereas 484 (14%) were typed only at 100 times RTD. Observations pertaining to the typing efficacy of staphylococcal phage 94 indicate that 651 (18%) of the typable microorganisms were lysed only by phage 94 at RTD or at 100 times RTD. Without the addition of this new phage to the international basic set, the number of nontypable strains (1,030 or 23%) would have been 1,681 (39%). Data regarding the geographic distribution of Staphylococcus aureus phage 94 point to the occurrence of the host strain in 13 (68%) of 19 states and 18 (62%) of the 29 hospitals submitting specimens to this laboratory. The assumed origin and speculated mode of dissemination of this microorganism are discussed.

Aerospace Medicine↗
PubMed2005

Synonymous codon usage bias in 16 Staphylococcus aureus phages: implication in phage therapy.

To reveal the factors influencing architecture of protein-coding genes in staphylococcal phages, relative synonymous codon usage variation has been investigated in 920 protein-coding genes of 16 staphylococcal phages. As expected for AT rich genomes, there are predominantly A and T ending codons in all 16 phages. Both Nc plot and correspondence analysis on relative synonymous codon usage indicates that mutation bias influences codon usage variation in the 16 phages. Correspondence analysis also suggests that translational selection and gene length also influence the codon usage variation in the phages to some extent and codon usage in staphylococcal phages is phage-specific but not S. aureus-specific. Further analysis indicates that among 16 staphylococcal phages, 44AHJD, P68 and K may be extremely virulent in nature as most of their genes have high translation efficiency. If this is true, then above three phages may be useful for curing staphylococcal infections.

Animals↗
PubMed2005

Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli.

Double-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin-endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the -1 reading frame at the 3' end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage lambda Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3.... Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.

Amino Acid Sequence↗
PubMed1980

DNA-mediated transformation in mutants of phage group 2 Staphylococcus aureus.

A large pool of antibiotic resistant and auxotrophic mutants was isolated from the Staphylococcus aureus phage group 2 strains UT0002-19 and UT0017 by (1) antibiotic gradient plates, (2) trimethoprim selection, and (3) nitrosoguanidine mutagenesis, which sometimes was coupled by enrichment with either penicillin or methicillin. Strain UT0002-19 has a chromosomal determinant for exfoliative toxin (ET), which causes "scalded skin syndrome" in man. A few mutants were isolated from the phage group 1 strain UT0080, which also produces ET. Two transformation regimens, called the broth and plate methods, were devised for the phage group 2 strains. They employed 80 alpha as helper phage, and recipient cells were incubated with transforming DNA in the presence of Ca2+. Strain UT0080 was transformed using phage 55 as helper. Maximum competence of the phage group 2 strains occurred during early logarithmic growth in trypticase soy broth, but cells grown overnight on heart infusion agar were also competent. Transformation frequencies of all markers ranged from 10(-6) to 10(-8). For phage 80 alpha, a multiplicity of infection of 4 was optimal in transforming a mutant of strain UT0002-19. Transformation of gly, lin, met, ole, rif, and ser markers in S. aureus is reported for the first time. Ery and ole markers in all three strains exhibited cross-resistance. Mapping studies, similar to those performed by DNA-mediated transformation in the phage group 3 strain 8325, can now be commenced for phage group 2 strains of S. aureus in order to elucidate the molecular genetics of this medically important bacterium.

Anti-Bacterial Agents↗
PubMed2006

Extensive phage dynamics in Staphylococcus aureus contributes to adaptation to the human host during infection.

Bacteriophages serve as a driving force in microbial evolution, adaptation to new environments and the pathogenesis of human bacterial infections. In Staphylococcus aureus phages encoding immune evasion molecules (SAK, SCIN, CHIPS), which integrate specifically into the beta-haemolysin (Hlb) gene, are widely distributed. When comparing S. aureus strain collections from infectious and colonizing situations we could detect a translocation of sak-encoding phages to atypical genomic integration sites in the bacterium only in the disease-related isolates. Additionally, significantly more Hlb producing strains were detected in the infectious strain collection. Extensive phage dynamics (intragenomic translocation, duplication, transfer between hosts, recombination events) during infection was shown by analysing cocolonizing and consecutive isolates of patients. This activity leads to the splitting of the strain population into various subfractions exhibiting different virulence potentials (Hlb-production and/or production of immune evasion molecules). Thus, phage-inducing conditions and strong selection for survival of the bacterial host after phage movement are typical for the infectious situation. Further in vitro characterization of phages revealed that: (i) SAK is encoded not only on serogroup F phages showing a conserved tropism for hlb but also on serogroup B phages which always integrate in a distinct intergenic region, (ii) the level of sak transcription correlates to phage inducibility but is independent of the phage localization in the chromosome, and (iii) phages can be stabilized extra-chromosomally during their life cycle.

Attachment Sites, Microbiological↗
PubMed1980

[Periodicity in the rise and fall of the numbers of the principal Staphylococcus aureus phage groups].

The results of the phage typing of 5, 168 Staph. aureus strains isolated in a surgical hospital between 1959 and 1977 are analyzed for each year of this period. The wave of increase in the number of staphylococci belonging to phage group II which began, as discovered in this study, in 1965 and still showed no tendency towards decrease in 1977, as well as the periodicity of rises and falls in the number of staphylococci belonging to phage groups I and III are discussed and compared with the data contained in the literature. The authors come to the conclusion that Staph. aureus is subject to wave-like rises and falls in the number of strains belonging to the main phage groups of the species, and among them the strains belonging to phage groups I and III seem to be inversely related in respect of rises and falls in number, such changes occurring periodically at an interval of 10-12 years, while in the strains belonging to phage group II changes in number occur at a slower rate. The constant account of the percentage of phage groups I, II and III is recommended to ensure rational antibiotic therapy.

Anti-Bacterial Agents↗
PubMed1987

Polypeptide synthesis during lytic induction of phage 11 of Staphylococcus aureus.

Staphylococcus aureus phage 11 was induced to replicate by treatment of lysogens with mitomycin C. Lysis was complete in 80 min at 37 degrees C in complete synthetic medium. Labelling with L-[3H]lysine showed a stimulation of protein synthesis during phage replication, followed after 30 min by shut-down of host protein synthesis while virion protein synthesis continued. The synthesis of approximately 16 virus-specific proteins was detected during replication, with seven of these recovered in purified virions after lysis. The remainder appeared to be synthesized earlier in phage replication and thus may represent precursors of virion proteins, or regulatory proteins, or enzymes associated with phage replication. Protein synthesis during replication of three suppressible morphological (head, tail) mutants of phage 11 did not differ significantly from that seen in the wild-type lysogen, indicating that the mutation in each case affected a protein whose synthesis was not detectable, or which was synthesized but did not fulfil its role in virion maturation or assembly. However, in a suppressible 'early' mutant which did not lyse when treated with mitomycin C, the synthesis of the 'late' (virion) proteins (with one exception) did not occur. 'Early' proteins were apparently made normally in this mutant, and there was no shut-down of host protein synthesis. This mutant phage presumably encodes a defective 'early' protein involved in the regulation of replication, or a key precursor polypeptide of 'late' protein synthesis. The morphological mutants provided a means to analyse and tentatively to allocate six of the virion proteins to head or tail/baseplate structures.

Mitomycin↗
PubMed2004

Lactobacillus plantarum bacteriophage LP65: a new member of the SPO1-like genus of the family Myoviridae.

The virulent Lactobacillus plantarum myophage LP65 was isolated from industrial meat fermentation. Tail contraction led to reorganization of the tail sheath and the baseplate; a tail tube was extruded. In ultrathin section the phage adsorbed via its baseplate to the exterior of the cell, while the tail tube tunneled through the thick bacterial cell wall. Convoluted membrane structures were induced in the infected cell. Progeny phage was detected 100 min postinfection, and lysis occurred after extensive digestion of the cell wall. Sequence analysis revealed a genome of 131,573 bp of nonredundant DNA. Four major genome regions and a large tRNA gene cluster were observed. One module corresponded to DNA replication genes. Helicase/primase and two replication/recombination enzymes represented the only links to T4-like Myoviridae from gram-negative bacteria. Another module corresponded to the structural genes. Sequence relatedness identified links with Listeria phage A511, Staphylococcus phage K, and Bacillus phage SPO1. LP65 structural proteins were identified by two-dimensional proteome analysis and mass spectrometry. The putative tail sheath protein showed a shear-induced change in electrophoretic migration behavior. The genome organization of the structural module in LP65 resembled that of Siphoviridae from the lambda supergroup.

Genome, Viral↗
PubMed2005

A change in Belgian epidemic methicillin-resistant Staphylococcus aureus phage types in 2000: phenotypic and genotypic characterization of isolates from a general hospital.

During 2000, new methicillin-resistant Staphylococcus aureus (MRSA) epidemic phage types became preponderant in Belgium. In the present study, phenotypic and genotypic characteristics of 130 MRSA isolates from a general Belgian hospital were investigated. The MRSA nature of the isolates was confirmed by coagulase test, oxacillin screen plate test and detection of the mecA gene by polymerase chain reaction. Phage typing categorized the MRSA strains into two main groups: the [O]* types and the [J]* types. SmaI macrorestriction analysis by pulsed-field gel electrophoresis gave the same pulsotype in the majority of strains. All strains of the [O]* and [J]* groups, except one, belonged to this pulsotype. Aminoglycoside-modifying-enzyme genes could only be detected in a minority of strains. Although the epidemic phage types of the mid-1990s appear to have been supplanted by the [O]* and [J]* groups, the MRSA population examined showed a remarkably uniform profile corresponding to the previous major clone B.

Adult↗
PubMed2002

Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort.

We have recently described three group I introns inserted into a single gene, orf142, of the staphylococcal bacteriophage Twort and suggested the presence of at least two additional self-splicing introns in this phage genome. Here we report that two previously uncharacterized introns, 429 and 1087 nt in length, interrupt the Twort gene coding for the large subunit of ribonucleotide reductase (nrdE). Reverse transcription-polymerase chain reaction (RT-PCR) of RNA isolated from Staphylococcus aureus after phage infection indicates that the introns are removed from the primary transcript in vivo. Both nrdE introns show sequence similarity to the Twort orf142 introns I2 and I3, suggesting either a common origin of these introns or shuffling of intron structural elements. Intron 2 encodes a DNA endonuclease, I-TwoI, with similarity to homing endonucleases of the HNH family. Like I-HmuI and I-HmuII, intron-encoded HNH endonucleases in Bacillus subtilis phages SPO1 and SP82, I-TwoI nicks only one strand of its DNA recognition sequence. However, whereas I-HmuI and I-HmuII cleave the template strand in exon 2, I-TwoI cleaves the coding strand in exon 1. In each case, the 3' OH created on the cut strand is positioned to prime DNA synthesis towards the intron, suggesting that this reaction contributes to the mechanism of intron homing. Both nrdE introns are inserted in highly conserved regions of the ribonucleotide reductase gene, next to codons for functionally important residues.

Amino Acid Sequence↗
PubMedDate not supplied

[Increasing the effectiveness of the method of Staphylococcus phage-typing].

Eight nutrient media used for St. aureus phage-typing were assessed in parallel experiments. 22 test strains of St. aureus with previously known bacteriophage formulas and 44 strains isolated from different sources during food poisonings were subjected to typing. Bacteriophages were employed in the test dose (TD) and in 10 TD. Marked relationship was established between the degree of lytic reactions of bacteriophages used in the TD and the type of solid nutrient media. The best results of phage-typing as regards the strength of lysis were obtained on agar for Brucella isolation (erythritol agar) with all bacteriophages applied in the TD. The results appeared similar to those obtained with Hottinger's medium. In order to unify the method of Staphylococcus phage-typing, it is suggested that dry erythritol agar manufactured by the Dagestan Research Institute for Nutrient Media should be used as a solid medium. In addition, two procedures for St. aureus cultivation before phage-typing are offered, raising the percentage of cultures lysed by bacteriophages.

Bacteriophage Typing↗