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.     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

Preparation and storage of high-titer lactic streptococcus bacteriophages

Various techniques were employed for preparation of high-titer bacteriophage lysates of Streptococcus lactis, S. cremoris, and S. diacetilactis strains. Infection of a 4-h host culture in litmus milk at 30 C yielded the highest titers (2 × 10⁹ to 4 × 10¹¹ plaque-forming units/ml) for most phages. Host infection in lactose-containing broth produced similar virus numbers only when 0.1 M tris(hydroxymethyl)aminomethane buffer stabilized the pH. The pH at the time of infection as well as the inoculum phage titer were critical in obtaining high titers. Optimum conditions for infection in broth were coupled with a polyethylene glycol concentration procedure to routinely produce milligram quantities of phage from 1 liter of lysate. Neutralization of whey lysates, as a means of storage, offered no survival advantage over unneutralized samples. Storage of phage lysates in a 15% glycerol whey solution at -22 C yielded a high rate of survival in most cases, even with repeated freezing and thawing, over a period of 24 months.     

Analysis of Lactobacillus phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt.

Yogurt and acidophilus milk that contain Lactobacillus acidophilus could promote human health because L. acidophilus can inhibit enteric and food-borne microbial pathogens. To evaluate the stability of diary L. acidophilus cultures, we studied whether some diary lactobacilli could be inhibited by phages or bacteriocins released by other dairy lactobacilli. From 20 yogurts and two acidophilus milks purchased at local food markets, 38 Lactobacillus strains were isolated. Eight Lactobacillus type strains were used as controls. With mitomycin induction and agar spot assay, phages and bacteriocins were isolated from these strains and their activities were analyzed. Lactobacillus strains from 11 yogurts released phages, while the strains from most of the remaining products released bacteriocins. One phage, designated phi y8, was characterized. It was spontaneously released from its host strain L. acidophilus Y8, at a rate of about 10(4)/ml. This phage lysed nine other dairy Lactobacillus strains tested. It had a burst size of 100, an elongated prolate head of 39 by 130 nm, a long, flexible but noncontractile tail of 300 nm, and a 54.3-kb linear double-stranded DNA. DNA fingerprinting analysis indicated that L. acidophilus phages of nine yogurts in this study belonged to the same type as phi y8. Although they may be sensitive to bacteriocins, all lysogens resisted further phage attacks, whereas most nonlysogens were sensitive to both phages and bacteriocins. Therefore, Lacotbacillus cultures of some American yogurts and acidophilus milks may be unstable or unsafe because they can either be inhibited by phages or bacteriocins or release them to inhibit lactobacilli or other diary products.     

Lysogenic Strains of Group N Lactic Streptococci

A temperate bacteriophage, designated r(1)t, was inducible from the group N lactic streptococcus, Streptococcus cremoris R(1), by ultraviolet irradiation or mitomycin C treatment. Induced lysates produced plaques on lawns of three closely related S. cremoris strains, AM(1), SK(11), and US(3). Strain SK(11) was readily lysogenized. S. cremoris AM(1) was the most reliable indicator strain, although the age of the culture used for seeding plates was critical. Zones of lysis but no plaque formation were observed on lawns of nine additional S. cremoris strains. Phage r(1)t could not be detected in filtrates of stationary-phase R(1) cultures and was near the limits of detection in logarithmically growing cultures. Phage levels were still very low (1 plaque-forming unit on AM(1) per 10 induced cells) in induced lysates of R(1) cultures. These low levels of detectable phage may be attributable to an inadequate indicator, lysogenization of the indicator, adsorption of induced phage to cellular debris, concurrent induction of other undetectable phages, or the production of high proportions of defective phages. Electron micrographs of induced R(1) lysates revealed a high incidence of incomplete phage particles, fragments, and ghosts.     

Propagation of ribonucleic acid coliphages in gnotobiotic mice.

To clarify the propagation cycle of bacteriophages in their natural habitats, we tested whether animals could support ribonucleic acid (RNA) phage propagation in their intestines, using germfree mice as the test animal. Propagation of four different antigenic types of RNA phages was tested. No detectable propagation or colonization of RNA phages was observed either in germfree mice or in gnotobiotic mice infected with the F- strain of Escherichia coli. Propagation or colonization was observed when RNA phages were orally introduced into gnotobiotic mice harboring the F+ or F' strain of E. coli. These results were consistent with data for in vitro propagation experiments. Fecal titers of phages were monitored over 24 to 98 days and were found to vary from 10(5) to 10(11) plaque-forming units per g of feces. Streptomycin administration gradually led to the disappearance of bacteria and, concomitantly, the RNA phages. Phages recovered from gnotobiotic mice feces included some of novel antigenic types. The bacterial isolates recovered from gnotobiotic mice harboring F+ bacteria included the original F+ strain, strains which had become F-, and some which had become inefficient hosts for the propagation of RNA phages.     

Propagation of ribonucleic acid coliphages in gnotobiotic mice.

To clarify the propagation cycle of bacteriophages in their natural habitats, we tested whether animals could support ribonucleic acid (RNA) phage propagation in their intestines, using germfree mice as the test animal. Propagation of four different antigenic types of RNA phages was tested. No detectable propagation or colonization of RNA phages was observed either in germfree mice or in gnotobiotic mice infected with the F- strain of Escherichia coli. Propagation or colonization was observed when RNA phages were orally introduced into gnotobiotic mice harboring the F+ or F' strain of E. coli. These results were consistent with data for in vitro propagation experiments. Fecal titers of phages were monitored over 24 to 98 days and were found to vary from 10(5) to 10(11) plaque-forming units per g of feces. Streptomycin administration gradually led to the disappearance of bacteria and, concomitantly, the RNA phages. Phages recovered from gnotobiotic mice feces included some of novel antigenic types. The bacterial isolates recovered from gnotobiotic mice harboring F+ bacteria included the original F+ strain, strains which had become F-, and some which had become inefficient hosts for the propagation of RNA phages.     

Molecular taxonomy of Lactobacillus phages

Forty-eight strains of lactobacilli used as starter strains in the dairy industry were examined for lysogeny after treatment with mitomycin C. Two strains of L. delbrueckii subsp. bulgaricus were able to produce active phages. These temperate phages as well as 4 virulent phages isolated during abnormal fermentations were compared to a previously characterized phage mv4 which is temperate. All these phages were shown to be partially homologous by DNA-DNA hybridization. Genes that code for viral proteins seem to be well conserved since 2 major virion polypeptides of 18 (or 19) kD and 34 kD could be detected in the protein composition of each phage. Immunoblotting studies of the 7 phages using serum raised against phage mv4 confirmed that the proteins of the different phages were related. All these phages can be classified in the previously constituted group a, which now comprises 4 temperate and 15 virulent phages. These results show that some virulent phages appearing during abnormal fermentations and some temperate phages isolated by appearing during abnormal fermentations and some temperate phages isolated by induction of starter strains can be closely related genetically. Five virulent phages of L. helveticus were also compared according to their restriction pattern and their DNA homology. They were shown to be related to one another, but unrelated to phages of other lactic acid bacteria species.     

Isolation of Psychrophilic Bacteriophage-Host Systems from Refrigerated Food Products

Thirty-eight bacteriophage-host systems were isolated from 22 of 45 refrigerated food products examined under psychrophilic conditions. Isolates were obtained from ground beef, pork sausage, chicken, raw skim milk, and oysters, whereas no isolations were made from liquid egg whites and processed meat products. Thirty of the 38 psychrophilic bacterial hosts were gram-negative rods, and 27 of these were classified within the genus Pseudomonas; three were members of the family Enterobacteriaceae. The remaining eight were gram-positive cocci, which were tentatively classified as Leuconostoc. Plate counts of psychrophilic bacteria were greater than 2.2 × 10⁵/ml (g) in all but one sample which contained phage, whereas phage titers ranged from less than 100 to 6.3 × 10⁶ plaque-forming units/ml (g). Phage isolates showed limited host ranges usually attacking only those hosts upon which they were isolated. Of eight phages tested against 13 cultures of known identity, one showed lytic action, and this was against strains of P. fragi.     

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.     

Molecular characterization and comparison of 38 virulent and temperate bacteriophages of Streptococcus lactis

Thirty-three virulent and five temperate phages of Streptococcus lactis and Streptococcus cremoris were differentiated into three groups by DNA homology. A complete lack of DNA homology was demonstrated between the phage groups. Within each group, large parts of the phage genomes were homologous except for a few phages. One group consisted of five temperate and two virulent phages suggesting that virulent phages isolated during abnormal fermentations and temperate phages isolated after induction from lactic streptococcal starter cultures may be related to one another. We observed a good correlation between the grouping of phages by DNA homology and by their protein composition since within the same DNA homology group, the protein composition of a phage exhibited some similarities with that of the other phages of the group. Therefore, the DNA homologies seemed to be located, at least, in the region coding for the structural proteins. By immunoblotting, we confirmed the relatedness between the proteins of the phages belonging to the same DNA homology group. The important host range factor in bacterium-phage interactions appeared to be an unreliable criterion in determining phage taxonomy.     

Inactivation of Nocardiophages φC and φEC by Extracts of Bacteriophage-Attachable Cells

Cultures of several species of Nocardia, including N. erythropolis Mat-Ce and Mat-cE mating strains, were extracted with solvents in an attempt to isolate an inactivating complex for nocardiophages phiC and phiEC. Ethanol was the only solvent found effective in solubilizing an inhibitory substance. Inactivating extracts were obtained from the cells of all species to which the phage were able to attach. After extraction of whole cells or cell wall preparations, the phage could not effectively attach to them. Both phages phiC and phiEC were inactivated by the same complex. However, phage phiEC inactivation was 10-fold greater than phiC inactivation. The velocity of inactivation was about 4.1 x 10(2) plaque-forming units per microgram per minute for phiC and 1.1 x 10(3) plaque-forming units per microgram per minute for phage phiEC. The cell extracts required divalent cations for phage inactivation. The inhibitory capacity of the cell extracts was reduced or lost by the activity of proteolytic enzymes, Tween 80, 2-mercaptoethanol, thymol, and sodium lauryl sulfate. Boiling the extract for 10 min did not alter its activity. The inactivating substance was postulated to be a lipoprotein of considerable complexity, unique in the ease with which it is solubilized from host cells by ethanol.     

Spread and variability of the integrase gene in Lactobacillus delbrueckii ssp. lactis strains and phages isolated from whey starter cultures

AIMS: To determine the presence, diffusion and variability of the integrase (int) gene in Lactobacillus delbrueckii ssp. lactis isolated from natural whey starters used for the production of Italian hard cheeses. METHODS AND RESULTS: A PCR-based protocol aimed to amplify an internal fragment of the int gene was optimized taking into account phage genome sequences available from public databases. Thirty-seven of the 39 strains tested showed the presence of the putative int gene. Southern blot hybridization experiments confirmed data obtained by PCR. The presence of the putative int gene was observed also in 20 of 23 Lact. delbrueckii ssp. lactis lytic phages isolated from the same starter cultures used to isolate strains. Phylogenetic analysis of partial int gene revealed a high similarity both within and between strain- and phage-derived sequences. Sixty per cent of the int-positive strains resulted inducible with mitomycin C, and two of them released active phage particles. CONCLUSIONS: Our preliminary findings seem to suggest that an important number of Lact. delbrueckii ssp. lactis strains associated with the whey starters are lysogenic. SIGNIFICANCE AND IMPACT OF THE STUDY: Further contribution to obtain a clearer picture of the complex relationship between thermophilic lactic acid bacteria phage and host in whey starters for Italian, hard-cooked cheeses.     

Resistance against Industrial Bacteriophages Conferred on Lactococci by Plasmid pAJ1106 and Related Plasmids

Plasmid pAJ1106 and its deletion derivative, plasmid pAJ2074, conferred lactose-fermenting ability (Lac) and bacteriophage resistance (Hsp) at 30°C to Lac⁻ proteinase (Prt)-negative Lactococcus lactis subsp. lactis and L. lactis subsp. lactis var. diacetylactis recipient strains. An additional plasmid, pAJ331, isolated from the original source strain of pAJ1106, retained Hsp and conjugative ability without Lac. pAJ331 was conjugally transferred to two L. lactis subsp. lactis and one L. lactis subsp. cremoris starter strains. The transconjugants from such crosses acquired resistance to the phages which propagated on the parent recipient strains. Of 10 transconjugant strains carrying pAJ1106 or one of the related plasmids, 8 remained insensitive to phages through five activity test cycles in which cultures were exposed to a large number of industrial phages at incubation temperatures used in lactic casein manufacture. Three of ten strains remained phage insensitive through five cycles of a cheesemaking activity test in which cultures were exposed to approximately 80 different phages through cheesemaking temperatures. Three phages which propagated on transconjugant strains during cheesemaking activity tests were studied in detail. Two were similar (prolate) in morphology and by DNA homology to phages which were shown to be sensitive to the plasmid-encoded phage resistance mechanism. The third phage was a long-tailed, small isometric phage of a type rarely found in New Zealand cheese wheys. The phage resistance mechanism was partially inactivated in most strains at 37°C.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Abstract Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.     

DNA-DNA Homology Between Lactic Streptococci and Their Temperate and Lytic Phages

Temperate phages were induced from Streptococcus cremoris R₁, BK₅, and 134. DNA from the three induced phages was shown to be homologous with prophage DNA in the bacterial chromosomes of their lysogenic hosts by the Southern blot hybridization technique. ³²P-labeled DNA from 11 lytic phages which had been isolated on cheese starters was similarly hybridized with DNA from 36 strains of lactic streptococci. No significant homology was detected between the phage and bacterial DNA. Phages and lactic streptococci used included phages isolated in a recently opened cheese plant and all the starter strains used in the plant since it commenced operation. The three temperate phages were compared by DNA-DNA hybridizations with 25 lytic phages isolated on cheese starters. Little or no homology was found between DNA from the temperate and lytic phages. In contrast, temperate phages showed a partial relationship with one another. Temperate phage DNA also showed partial homology with DNA from a number of strains of lactic streptococci, many of which have been shown to be lysogenic. This suggests that many temperate phages in lactic streptococci may be related to one another and therefore may be homoimmune with one another. These findings indicate that the release of temperate phages from starter cells currently in use is unlikely to be the predominant source of lytic phages in cheese plants.     

Phage resistance in lactic acid bacteria

The interactions between lactic acid bacteria and their phages are commercially significant. Current research has focused on the elucidation of the mechanisms and genetics of phage resistance. Phage resistance genes have been linked to plasmid DNA for Streptococcus lactis and Streptococcus cremoris, and preliminary studies suggest the operation of mechanisms such as the prevention of phage adsorption, restriction/modification, and abortive infection. Some phage resistance plasmids can be conjugally transferred, providing a means of dissemination among phage-sensitive strains for the construction of phage-resistant starter cultures.     

Method for detecting small numbers of Vibrio cholerae in very polluted substrates.

A method is presented for the indirect detection of Vibrio cholerae by the multiplication of two specific bacteriophages: phiH74/64 for El-Tor vibrios, and phage group IV (Mukerjee) for classical vibrios. The product to be examined is seeded in alkaline tryptone water for enrichment, as in the classical method, and is then incubated for 6 h at 37 C. Thereafter, a loopful is transferred to each of two nutrient broth (pH 9) tubes. One of these receives a drop of phage phiH74/64; the other receives a drop of phage group IV. The stock phages are diluted so as to contain about 3,800 plaque-forming units in one drop; this is the maximum amount which, when added to 10 ml of broth, will not be detected in a loopful of 1 mm diameter. The tubes containing phage phiH74/64 are incubated at 42 C; those with phage group IV are incubated at 37 C. After 18 h the cultures are killed by agitation with chloroform, and a 1-mm loopful is deposited on a layer seeded with the detector strains: Makassar 757 for El-Tor phage and V. cholerae 154 for classical cholera phage. After 4 to 5 h at 37 C, lysis appears on the spot areas if there has been phage multiplication in the respective broth tubes. With experimentally contaminated sewage water, vegetables, or stools, 1 to 10 cholera vibrios were detected in every sample. In rare cases, false-positive results were obtained by multiplication of the phage on non-cholera vibrios.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Temperate bacteriophages and lysogeny in lactic acid bacteria

Lysogeny is widespread in the lactic acid bacteria. The majority of lysogens can be induced by UV irradiation or treatment with mitomycin C, but indicator strains which allow lytic growth of the induced phage are often not easy to identify. A few temperate phages have been shown to transduce chromosomal and/or plasmid markers. Information about the molecular biology of the temperate phages from lactic acid bacteria is sparse and needs significant supplementation in order that these potentially valuable phages might be utilized more efficiently as tools for improving existing starter strains in dairy fermentations.