Morphology of the Bacteriophages of Lactic Streptococci

Electron microscope studies have been made of a number of phages of lactic streptococci, seven of which were phages of Streptococcus lactis C10. Two of the phages are thought to be identical; five have been classified by the method of Tikhonenko as belonging to group IV (phages with noncontractile tails) with type III tail plates; one belongs to group V (phages with tails possessing a contractile sheath). Both prolate polyhedral heads and isometric polyhedral heads are represented among the group IV phages. The phage drc3 of S. diacetilactis DRC3 has been shown to have similar structure to the group IV phages of S. lactis C10 with prolate polyhedral heads. The phages ml1, hp, c11, and z8 of the S. cremoris strains ML1, HP, C11, and Z8, respectively, were shown to belong to the group IV phages with type III tail plates by the method of Tikhonenko. All had octahedral heads and tended to be larger than most of the other phages studied.     

Morphology of Bacteriophages of Lactobacillus bulgaricus, L. lactis and L. helveticus

Eleven virulent phages isolated from cheese or yoghurt factories and active on thermophilic lactobacilli used as starters were examined by electron microscopy. Five phages active on Lactobacillus bulgaricus belonged to Bradley's group B and can be divided into two groups with different host specificity. The three phages of the first group (two isolated in France, one in the USA) differ in size; the heads are either icosahedrons or octahedrons and the tails end in short-pronged base plates. The two phages of the second group, isolated in the USA. appear very similar. These are similar in length, have collars and octahedral heads. The non-contractile tails end in clusters of short fibres. An L. lactis phage, isolated in Finland, belongs to the same morphological group. It is similar in overall appearance to the two latter phages of the second group of L. bulgaricus , and there were numerous ghosts with polytails. Five phages. active on L. helveticus belong to Bradley's group A and may be divided into two groups with different host specificity. The first group contains a phage isolated in Finland. The second group is composed of four similar phages isolated in France. The Finnish phage is the largest, but the five phages show similar morphology. They have octahedral heads, firmly attached to the tails by connector devices, and they possess necks. The contractile sheaths have a helicoidal arrangement of hollow tubular subunits. They appear contracted either in a distal or a cervical position, revealing axial cores. Short tail fibres are probably present at the tail tip.     

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk "Viili"

"Viili," a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Ultrastructure and Host Specificity of Bacteriophages of Streptococcus cremoris, Streptococcus lactis subsp. diacetylactis, and Leuconostoc cremoris from Finnish Fermented Milk “Viili”

“Viili,” a fermented milk product, has a firm but viscous consistency. It is produced with traditional mesophilic mixed-strain starters, which have various stabilities in dairy practice. Thirteen morphologically different types of phages were found in 90 viili samples studied by electron microscopy. Ten of the phage types had isometric heads with long, noncontractile tails, two had elongated heads with long, noncontractile tails, and one had a unique, very long elongated head with a short tail. Further morphological differences were found in the tail size and in the presence or absence of a collar, a baseplate, and a tail fiber. To find hosts for the industrially significant phages, we examined the sensitivities of 500 bacterial isolates from starters of the viili. Seven of the phages attacked Streptococcus cremoris strains, three attacked S. lactis subsp. diacetylactis strains, and four attacked Leuconostoc cremoris strains. Some phages differed only in their host specificity. Hosts were not found for 4 of the 13 morphological types of phages.     

Morphology and General Characteristics of Phages Specific for Astragalus cicer Rhizobia

Three newly isolated phages, K1, K2, and C1, specific for A. cicer rhizobia were characterized by their morphology, host range, rate of adsorption, restriction endonuclease patterns, and DNA molecular weights. All three phages were classified to the morphological group B of Bradley's (Siphoviridae family) on the basis of presence of hexagonal in outline heads and long noncontractile tails. Phages K1, K2, and C1 are related by host range and restriction endonuclease patterns. The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from 64.6 kb to 68.5 kb. Received: 21 July 1999 / Accepted: 25 August 1999     

Direct electron microscopy study on the morphological diversity of bacteriophage populations in lake plusssee

Direct electron microscopy of bacteriophages adsorbed to a carbon film without prior enrichment by specific host strains or concentration by physical or chemical methods was used to study the morphological diversity of natural bacteriophage assemblages in a North German lake. All samples contained a mixture of morphologically different tailed viruses, which were regarded as bacteriophages. Most of them had isometric heads and long noncontractile tails, belonging to morphotype B1 (Siphoviridae). In addition, members of morphotypes A1 (Myoviridae), B2 (Siphoviridae with elongated heads), and C1 (Podoviridae) were present in lower numbers. Only one cubic virus was detected, while no filamentous or pleomorphic phages were found. Up to 11 different phages per sample, and a total of 39 phages when all samples were considered together, could be distinguished by morphological criteria. The total number of phages was estimated to be on the order of 10/ml.     

Structural aberrations in group A Staphylococcus bacteriophages.

Six related Staphylococcus phages spontaneously produced various abnormal head and tail structures: (i) giant capsids which were tailed and apparently contained nucleic acid; (ii) regular and irregular smooth polyheads; (iii) heads and polyheads with wavy outlines; (iv) mottled heads and polyheads; (v) abnormally long and short tails; and (vi) "double capsids" connected by a small bridge. Some of these structures are rare, or have not yet been reported. The frequency os specific aberrant particles varied from one phage to another. Length distribution of smooth irregular polyheads and of abnormal tails indicated that these structures assemble at random from protein synthesized in excess. These phages represent an interesting model for genetic and morphogentic studies.     

Electron microscopy of virulent phages for Streptococcus lactis.

Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern.     

Morphology of Staphylococcus saprophyticus bacteriophages

The morphology of 6 bacteriophages isolated from Staphylococcus saprophyticus was studied by electron microscopy.Phages had isometric heads and non-contractile tails, thus belonging to the Siphoviridae family. They were subdivided into two morphological groups based on head diameter and tail length. Type I, represented by phages 1154A and 1405, and characterized by an unusually small capsid, may be a new species.     

Direct Electron Microscopy Study on the Morphological Diversity of Bacteriophage Populations in Lake Plußsee

Direct electron microscopy of bacteriophages adsorbed to a carbon film without prior enrichment by specific host strains or concentration by physical or chemical methods was used to study the morphological diversity of natural bacteriophage assemblages in a North German lake. All samples contained a mixture of morphologically different tailed viruses, which were regarded as bacteriophages. Most of them had isometric heads and long noncontractile tails, belonging to morphotype B1 (Siphoviridae). In addition, members of morphotypes A1 (Myoviridae), B2 (Siphoviridae with elongated heads), and C1 (Podoviridae) were present in lower numbers. Only one cubic virus was detected, while no filamentous or pleomorphic phages were found. Up to 11 different phages per sample, and a total of 39 phages when all samples were considered together, could be distinguished by morphological criteria. The total number of phages was estimated to be on the order of 10⁸/ml.     

Large Pseudomonas phages isolated from barley rhizosphere

Abstract: Five bacteriophages infecting common fluorescent pseudomonads ( Pseudomonas fluorescens and Pseudomonas putida ) were isolated from barley rhizosphere soil. Morphological and molecular characteristics of the phages are described together with selected phage-host interactions. All phages belonged to the Myoviridae family with isometrical heads on contractile tails; 4 of them were unusually large and had complex protein and DNA profiles. The large phages had estimated genome sizes of 200 kb or more. Restriction enzyme analyses and DNA-DNA hybridizations showed that all isolates represented different phage species. None of the isolates were observed to establish lysogeny with the main host strain, P. putida MM1. The large phages multiplied slowly on their hosts, producing very small plaques; one-step growth experiments with one of the large phages (Psp 4) hence demonstrated a long latent period (2.5 h) and a very small burst size (10 particles). One of the large phages (Psp 3) was abundant in the rhizosphere (approx. 10⁴ pfu g⁻¹ soil) and had a particularly broad host range which extended to both fluorescent ( Pseudomonas aeruginosa, P. fluorescens, P. putida and Pseudomonas chlororaphis ) and non-fluorescent (Pseudomonas stutzeri) Pseudomonas spp. occurring in soil. The ecological importance of the large Pseudomonas phages must be further studied, but their slow multiplication rates suggested a possible mechanism of balanced phage-host co-existence in the rhizosphere.     

Incidence and properties of temperate bacteriophages induced from lactic streptococci.

Sixty-three strains of lactic streptococci isolated from commercial lactic streptococcal starter cultures were examined for lysogeny by treatment with ultraviolet light or mitomycin C. After treatment with the inducing agent, all strains, whether or not they lysed, were examined for evidence of phage release by electron microscopy. Thirty-eight strains yielded intact phages or phage particles of varying morphology. All the temperate phages had isometric heads and noncontractile tails; some had collars and structurally distinctive baseplates. Indicator host strains were found for phages induced from seven different strains. Three strains that released phages spontaneously yielded titers of 10(3) to 10(4) plaque-forming units per ml. When strains that spontaneously released phages were grown in mixed culture with indicator strains, increased phage titers of 10(6) to 10(7) plaque-forming units per ml were observed. These findings indicate that lysogenic lactic streptococcal strains may serve as a reservoir for phages that attack sensitive strains in mixed- or multiple-strain lactic starter cultures.     

Incidence and properties of temperate bacteriophages induced from lactic streptococci.

Sixty-three strains of lactic streptococci isolated from commercial lactic streptococcal starter cultures were examined for lysogeny by treatment with ultraviolet light or mitomycin C. After treatment with the inducing agent, all strains, whether or not they lysed, were examined for evidence of phage release by electron microscopy. Thirty-eight strains yielded intact phages or phage particles of varying morphology. All the temperate phages had isometric heads and noncontractile tails; some had collars and structurally distinctive baseplates. Indicator host strains were found for phages induced from seven different strains. Three strains that released phages spontaneously yielded titers of 10(3) to 10(4) plaque-forming units per ml. When strains that spontaneously released phages were grown in mixed culture with indicator strains, increased phage titers of 10(6) to 10(7) plaque-forming units per ml were observed. These findings indicate that lysogenic lactic streptococcal strains may serve as a reservoir for phages that attack sensitive strains in mixed- or multiple-strain lactic starter cultures.     

Morphology and General Characteristics of Bacteriophages Infectious to Robinia pseudoacacia Mesorhizobia

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley’s classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10–25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100–120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 μg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from ~39 kb for group C phages to ~80 kb for B2.     

Characterization of 4 T1-like lytic bacteriophages that lyse Shiga-toxin Escherichia coli O157:H7

Bacteriophages are associated with reduced fecal shedding of Shiga-toxin-producing Escherichia coli O157:H7 (STEC O157:H7) in cattle. Four phages exhibiting activity against 12 of 14 STEC O157:H7 strains, representing 11 common phage types, were isolated. Phages did not lyse non-O157 E. coli, with 11 of the 12 STEC strains exhibiting extreme susceptibility (average multiplicity of infection (MOI) = 0.0003-0.0007). All phages had icosahedral heads with tapered, noncontractile tails, a morphology indicative of T1-like Siphoviridae. Genome size of all phages was ～44 kb, but EcoR? or HindIII digestion profiles differed among phages. Based on restriction enzyme digestion profiles, phages AHP24, AHS24, and AHP42 were more related (66.7%-82.4%) to each other than to AKS96, while AHP24 and AHS24, isolated from the same feedlot pen, exhibited the highest identity (88.9%-92.3%). Phages AHP24 and AHS24 exhibited the broadest host range and strongest lytic activity against STEC O157:H7, making them strong candidates for biocontrol of this bacterium in cattle.     

Morphology of Brochothrix thermosphacta phages

Twenty-one Brochothrix thermosphacta phages were examined by electron microscopy. They had isometric heads and contractile or long and noncontractile tails, and were grouped into three species belonging to the Myoviridae (species A19) or Siphoviridae (species NF5 and BL3) families of tailed phages. Species A19 has highly characteristic morphological features and resembles well-known phages of Bacillus, Lactobacillus, Staphylococcus and Streptococcus, indicating phylogenetic relationships between these phages as well as their hosts.     

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.     

Psychrotrophic Brocothrix thermosphacta bacteriophages isolated from beef.

A total of 15 wild-type Brocothrix thermosphacta strains isolated from beef and the type strain, B. thermosphacta ATCC 11509, were used as hosts for the isolation of bacteriophages under psychrotrophic conditions (7 degrees C). A total of 21 virulent, psychrotrophic phages were successfully isolated and purified from aqueous extracts of spoiled rib steaks. Phage plaque size and plating efficiency significantly increased as incubation temperature was reduced from 25 to 1 degree C. Electron microscopy of two homologous B. thermosphacta phages showed the virions to consist of hexagonal heads and tails, with terminal appendages clearly visible on one of the phages. On the basis of culture and biochemical data, the wild-type B. thermosphacta strains had characteristics identical to those of strain ATCC 11509. However, specific differences in the pattern of susceptibilities to the phages revealed the presence of 14 distinct phage lysotypes. Phage typing may provide a rapid and sensitive means of differentiating B. thermosphacta strains.     

Prevalence of Stx Phages in Environments of a Pig Farm and Lysogenic Infection of the Field <Emphasis Type="Italic">E. coli</Emphasis> O157 Isolates with a Recombinant Converting Phage

The prevalence and nature of Shiga toxin (Stx)-producing Escherichia coli (STEC) and Stx phage were investigated in 720 swine fecal samples randomly collected from a commercial breeding pig farm in China over a 1-year surveillance period. Eight STEC O157 (1.1%), 33 STEC non-O157 (4.6%), and two stx-negative O157 (0.3%) isolates were identified. Fecal filtrates were screened directly for Stx phages using E. coli K-12 derivative strains MC1061 as indicator, yielding 15 Stx1 and 57 Stx2 phages. One Stx1 and eight Stx2 phages were obtained following norfloxacin induction of the eight field STEC O157 isolates. All Stx1 phages had hexagonal heads with long tails, while Stx2 phages had three different morphologies. Notably, most of field STEC O157 isolates released more free phages and Stx toxin after induction with ciprofloxacin. Furthermore, upon infection with the recombinant phage ΦMin27(Δstx::cat), E. coli laboratory strains produced both lysogenic and lytic phage, whereas two of the eight O157 STEC isolates produced only lysogens. The lysogens from laboratory strains produced infectious particles similar to ΦMin27. Similarly, the lysogens from the STEC O157 isolates released Stx phage too, although free ΦMin27(Δstx::cat) particles were not detected. Collectively, our results reveal that breeding pig farms could be important reservoirs for Stx phages and that residual antibacterial agents may enhance the release of Stx phages and the expression of Stx.