The morphology and nucleotide composition of DNA of Citrobacter phages

Citrobacter phages 38/37, 31/37, 40/1 and 8/5, isolated from lysogenic cultures, were concentrated and purified by 2 cycles of differential centrifugation. Electron microscopy of the phages has shown that their particles have similar morphology and that they relate to the morphological group A1. The heads of the phages are hexagonal, 50 +/- 2 nm in diameter. The tail of the phage is straight, 112-152 nm in length, with a contracting sheath 11.5-12.5 nm wide. The tails of the phages 38/37 and 40/1 were found to be slightly longer in comparison with the phages 31/37 and 8/5. Chromatographic investigation of DNA preparations of the phages revealed the presence of 4 nitrous bases. Identification of the latter permitted us to relate them to common nitrous bases. DNA of the phages is double-stranded and belongs to a weakly expressed guanine-cytosine type. The content of guanine and cytosine in DNA of the phage 38/37 amounts to 56.68%, that of the phage 31/37 to 56.75, of the phage 40/1 to 57.36% and of the phage 8/5 to 55.58%. No substantial variations were observed in the DNA composition of the phages.     

Isolation and partial characterization of phages infectingCitrobacter intermedius C3

Several new bacteriophages infecting strain C3 ofCitrobacter intermedius have been isolated from fresh waters. The physicochemical properties, plaque morphology, growth characteristics, virion structure, and immunology of eight bacteriophage isolates are reported. The phages are classified into three groups according to their characteristics.     

The level of spontaneous phage production and sensitivity to melioidosis phages of museum cultures of Pseudomonas pseudomallei]

The phage-producing activity and sensitivity of museum cultures of a melioidosis agent to melioidosis phages have been studied. Most cultures show spontaneous phage-production, though its level differs in strains. Some of the melioidosis cultures demonstrate lack of phages. The fact that these strains have different sensitivity to chloroform lysate of cultures producing phages indicates their possible lysogenic state. Electron microscopy has shown the presence of at least tow morphological types of phages in P. pseudomallei C-141. These data confirm existence of polysogeny in the melioidosis agent.     

Ten new temperate bacteriophages of<Emphasis Type="Italic">Citrobacter youngae</Emphasis>

In a cross-test, we examined 55 strains of Citrobacter youngae against each other as potential producers of temperate bacteriophages and as potential sensitive indicators for them. Ten strains (18.2 %) showed the production of phages. Seven different strain-specific spectra of activity (from 1 to 11 strains each) were found. Phage production by 6 strains was inducible with mitomycin C, in 4 strains it was not inducible. The plaques of the phages were more or less turbid, without a lytic halo, tiny to small, 0.2-1.3 mm in diameter. Using a polyclonal, specific anti-lambda serum, all 10 phages were found to be clearly distinct from E. coli lambda phage, the phage 31/47 showing the highest neutralization titre of all. Interspecific tests with 15 strains of 8 species of Enterobacteriaceae revealed not a single case of activity of Citrobacter phages towards any of them. Five phage-immune clones lysogenized with 5 of the phages kept their remaining phage sensitivity spectra, though extended by sensitivity to 1-3 phages; 2 of these strains acquired also sensitivity to phage lambda. The phages belong to the morphotypes of Myoviridae (6 phages) and Siphoviridae (4 phages), with head diameters of 51-58 nm and tail length of 97-173 nm. Three strains produced corpuscular bacteriocins.     

Phage typing of the coagulase-positive staphylococci isolated from various species of animals

More than 200 coagulase-positive strains of animal origin have been studied by means of Staphylococcus aureus typing phages, belonging to two international sets and intended for typing staphylococci isolated from large cattle and humans, and experimental "chicken" phage A 1591. Among S. aureus strains the cultures isolated from swine, cows, chickens, and belonging to biotypes B1, C1, B2, respectively, have been mostly (in 78.5-90.0% of cases) determined by phage typing. The strains belonging to one biotype have proved to be sensitive predominantly to the same phages. In this connection further differentiation of staphylococci within individual biotypes by means of the phages used in these experiments seems to be impracticable. S. intermedius strains have been found to be completely resistant to the above phages, which confirms that S. intermedius is rightly considered to be an independent species of coagulase-positive staphylococci.     

Experimental phages for differentiating nontypable methicillin-resistant Staphylococcus aureus

The possibility of using phages, isolated from the lysogenic cultures of methicillin-resistant staphylococci and modified in methicillin-resistant cultures of phage 85, for the differentiation of nontypable staphylococcal strains has been studied. The variants of phage 85 cannot be used for the determination of differences between the strains of methicillin-resistant cultures; they are not suited for typing. For this purpose the collection of phages isolated from lysogenic methicillin-resistant cultures should be used.     

Results of typing staphylococci isolated from cows and their milk products using the basic set of phages and local phages]

The basic set of phages recommended for typing staphylococci from cattle and also of local phages were approbated. Staphylococci cultures (950 in all) isolated in various regions of the Soviet Union from milk, milk produce and from cows suffering from mastitis were studied. Percentage of cultures typed by the phages of the basic set proved to be 78.3. Thirty different phage patterns were revealed among staphylococcal cultures lysed by phages. Lytic activity was the greatest in phages of group IV of the basic set. It is suggested that local phage 34k can be used as an additional phage permitting to subdivide the prevailing phage types within group IV into a number of new ones.     

Phenomena in mixed cultures ofSalmonella paratyphi B; serological changes and adaptations of the bacteriophage; incomplete natural phages; transformation of phage types

Summary The phenomenon that natural phages are only released in mixed cultures and are not found in pure cultures of bacterial strains has been discussed. It was described how an infecting phage absorbs material from the natural phage in the bacterium and the other way round, that the natural phage absorbs material from the infecting phage. Hereby the bacteriophages can change serologically. Simultaneously the bacteriophage can be adapted. The phenomenon that natural phages are only released in mixed cultures is in some cases explained by assuming that the prophage is incompletely present in the bacterium and is completed by material from an infecting phage.     

Sensitivity of nosocomial microflora circulating in a transplantation clinic to medicinal bacteriophages

The sensitivity of 239 isolates obtained from patients with postoperative infectious complications to phagolysis was determined. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were found to have the highest sensitivity to phages. Variations in the sensitivity of the same cultures to phages from different producers and even from the same producer were established. The sensitivity of cultures to phages may serve as an additional criterion of the biological properties of strains and their marker.     

Persistense of mono- and associated cultures of conditionally pathogenic enterobacteria

The comparative study of adhesive, hemolytic, DNA-ase, lecithinase, antilysozymic, anticomplementary activities of mono- and associated cultures of 57 Enterobacter spp., 61 Citrobacter spp. and 55 Serratia spp. strains, isolated from patients with pyoinflammatory, intestinal and urological diseases is carried out. Different variations of cocultivated bacteria including Enterobacter and Citrobacter, Enterobacter and Serratia, Citrobacter and Serratia are used. It was shown, that cocultivated Enterobacter, Citrobacter and Serratia bacteria increased the persistent properties of mixt cultures.     

Comparative study of moderate and virulent phages of mesophilous lactic acid streptococci]

A method has been developed to identify phage containing mesophilous lactic acid streptococci. The method is based on the chloroform treatment of young cultures. The isolated moderate phages P4St and P6St are morphologically identical and serologically related to one another and to virulent phages P1Sv, P2Sv, P3Sv, P12Sv, P13Sv that can be isolated in industrial phagolysis.     

Sensitivity to virulent bacteriophages of Pseudomonas aeruginosa strains of different serogroups

The data on the sensitivity of P. aeruginosa clinical strains to pyocyaneum, a therapeutic and prophylactic bacteriophage preparation, and to individual groups of phages contained in this preparation are presented. Out of 549 P. aeruginosa strains, 16% have proved to be nonlysing cultures. The proportion of phage-sensitive strains prevailed in serogroups 01, 03, 06, 09, while phage-resistant strains prevailed in serogroups 04, 07, 011, as well as among O-nontyped cultures. The expediency of introducing P. aeruginosa strains of different serotypes into the collection of cultures used for the production of pyocyaneum has been shown.     

Shiga Toxin 2-Encoding Bacteriophages in Human Fecal Samples from Healthy Individuals

Shiga toxin-converting bacteriophages (Stx phages) carry the stx gene and convert nonpathogenic bacterial strains into Shiga toxin-producing bacteria. Previous studies have shown that high densities of free and infectious Stx phages are found in environments polluted with feces and also in food samples. Taken together, these two findings suggest that Stx phages could be excreted through feces, but this has not been tested to date. In this study, we purified Stx phages from 100 fecal samples from 100 healthy individuals showing no enteric symptoms. The phages retrieved from each sample were then quantified by quantitative PCR (qPCR). In total, 62% of the samples carried Stx phages, with an average value of 2.6 × 10⁴ Stx phages/g. This result confirms the excretion of free Stx phages by healthy humans. Moreover, the Stx phages from feces were able to propagate in enrichment cultures of stx-negative Escherichia coli (strains C600 and O157:H7) and in Shigella sonnei, indicating that at least a fraction of the Stx phages present were infective. Plaque blot hybridization revealed lysis by Stx phages from feces. Our results confirm the presence of infectious free Stx phages in feces from healthy persons, possibly explaining the environmental prevalence observed in previous studies. It cannot be ruled out, therefore, that some positive stx results obtained during the molecular diagnosis of Shiga toxin-producing Escherichia coli (STEC)-related diseases using stool samples are due to the presence of Stx phages.     

Sewage coliphages studied by electron microscopy.

Sewage was enriched with 35 Escherichia coli strains, and sediments of enrichment cultures were studied in the electron microscope. They contained up to 10 varieties of morphologically different particles. T-even-type phages predominated in 14 samples. Thirteen phages were enriched, representing the families Myoviridae (seven), Styloviridae (two), Podoviridae (three), and Microviridae (one). Twelve of these corresponded to known enterobacterial phage species, namely, 121, K19, FC3-9, O1, 9266, T2, 16-19, kappa, beta 4, N4, T7, and phi X174. Cubic RNA phages and filamentous phages were not detected. Types 121 and 9266 have previously been observed only in Romania and South Africa. Identification by morphology is usually simple. Our investigative technique is qualitative and will not detect all phages present. Most enrichment strains are polyvalent, and electron microscopy is always required for phage identification. In a general way, electron microscopy seems to be the method of choice for investigation of phage geography and ecology.     

Isolation and partial characterization of normal and defective bacteriophages of gram-negative hydrogen bacteria

Widespread defective lysogeny was detected in Alcaligenes eutrophus by electron microscopic analysis of cultures. Mitomycin C treatment of the cultures resulted in the production of defective (inco-) particles. Polysheaths were produced both with and without induction.With the simultaneous isolation technique six phages were isolated for hydrogen-oxidizing strains of the new species Pseudomonas pseudoflava. The phages were able to replicate under autotrophic conditions and were found to have a very restricted host range. Electron microscopic analysis allowed classification into two structural groups. Group I contained phages with contractile tails; group II contained phages with flexible, noncontractile tails. All but one (gb) of the new phages were shown to be temperate by isolation of lysogens and induction with mitomycin C.Non-Standard Abbreviations OD optical density - SM-medium standard mineral medium - KS-medium Kaserer medium - NB nutrient broth - p.f.u. plaque forming unit - c.f.u. colony forming unit - e.o.p. efficiency of plating - Do-phage buffer Dopatka-phage buffer - PTA phosphotungstic acid     

Evaluation of the usefulness of three collections of bacteriophages for typing methicillin-resistant Staphylococcus aureus,isolated in Moscow hospitals

The typing of S. aureus methicillin-resistant strains, isolated in different hospitals of Moscow; was carried out with the use of three collections of phages: the International Set of Phages; the set of phages of the International Center of S. aureus phage typing in London (L); and the experimental collection of phages of the Gamaleya Institute of Epidemiology and Microbiology in Moscow (M). In this study made with the use of both the phages of the International Diagnostic Set and phages L in the standard typing dose of 1 TP about 6% of the cultures under study proved to be sensitive. When the typing dose was increased to 100 TP the phages of the international diagnostic set lyzed 75.5% of the cultures. The typed strains were found to belong to phage types 77 (71.7%), 77/84/85 (19.6%) and 94/96 (6.5%). At a concentration of 100 TP phages L lyzed 83.7% of the cultures, but the dominating phage types could not be determined due to a great variety of phage markers. In contrast to the two preceding collections, the third phage collection M was composed in such a way that in the study of the investigated culture the specificity of its restriction modification was primarily evaluated and only then the presence of antiphage immunity was determined. This latter collection was used in the evaluation of 93.1% of the cultures. By the specificity of their restriction specification system the majority of them were classified with two new groups, heretofore not described. Only this collection M made it possible to differentiate epidemic and sporadic strains and to evaluate the epidemic situation in all 6 hospitals.     

Bacteriophages of lactobacilli

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.     

Bacteriophages ofBacillus subtilis: Comparison of different isolation techniques and possible use for classification ofBacillus subtilis strains

When different techniques are used for the isolation of bacteriophages ofBacillus subtilis a number of different phages may be obtained. Furthermore defective phages are found in old cultures of all strains ofB. subtilis tested so far. The possible use of the phages and the defective phages for classifyingB. subtilis strains into a number of groups according to their susceptibility to different phages and according to the presence of certain defective phages in the cells is discussed.     

Specific phages used for differentiation of the entomopathogenic bacterium Bacillus thuringiensis]

The sensitivity to specific phages and morphological, physiological, and antigenic properties were compared among several strains of Bacillus thuringiensis isolated from insects inhabiting various geographical zones. All 43 cultures assigned to Bac. thuringiensis var. sotto and 198 among 170 cultures classed as Bac. thuringiensis var. dendrolimus were found to belong to Bac. thuringiensis var. dendrolimus. None of these cultures was resistant to its specific phage. The same was true of 22 studied cultures of Bac. thuringiensis var. galleriae. Only two among studied 45 cultures of Bac. thuringiensis var. thuringiensis were resistant to phages specific for this variety. Therefore, the abundance of variants resistant to specific phages in natural conditions differs among the varieties of Bac. thuringiensis. In most cases, cultures of the same variety of Bac. thuringiensis isolated from various insects inhabiting different geographical zones are identical by their sensitivity to specific phages and by other important characteristics.