Localization of transfer RNA genes on the physical map of Vibrio eltor phage e4 genome

Transfer RNAs were isolated from uninfected and phage e4-infected Vibrio eltor Mak 757 cells. These tRNAs were then aminoacylated with 3H-labeled amino acids and hybridized to DNA isolated from phage e4. Significant hybridization was observed only with tRNA isolated from phage e4-infected cells. Restriction enzyme digestion of phage e4 DNA followed by Southern blot using [32P]tRNA from infected cells revealed that tRNA genes were contained in a 3.4-kb Kpnl fragment. The tRNA genes were located on the physical map of the phage genome 19 kb from one of the termini.     

Epidemiological markers of Salmonella typhimurium isolates in Rome.

The phage type, antimicrobial resistance pattern, colicinogenic activity and DNA plasmid content of 172 strains of Salmonella typhimurium isolated in Rome from 1984 to 1986 were determined; 142 isolates were from patients with enteritis, 30 were from asymptomatic subjects. Most of the phage types identified were isolated during 2 or 3 of the study years; others, e.g., PT141, PT 204c and PT 194 were isolated during 1 year only, and only the last of these was isolated in large numbers. Phage typing proved most valuable in identifying epidemiologically related strains; DNA plasmid analysis was most useful in characterising further cultures of the same phage type, especially those isolated during suspected epidemics.     

Experimental infection of chickens with Salmonella enteritidis

Following oral inoculation of newly hatched commercial broilers, four phage types of Salmonella enteritidis showed considerable variation in their virulence. The percentage mortality varied from 96%, produced by a phage type 4 strain, to 20% (phage type 13a). When high mortality was observed, chickens that died showed a polyserositis with pericarditis similar to that observed in naturally occurring cases. Strains of phage types 4, 6 and 8 were more virulent than a phage type 13a strain when inoculated by the intramuscular route. Similar variations in virulence were observed in newly hatched Rhode Island Red chickens although the overall mortality observed was much reduced. Polyserositis was again observed. Despite similarly high viable numbers of all the phage types being present in the caecal contents, the strains of phage types 4 and 6 were isolated from the spleen at 12 h post-inoculation whereas phage types 8 and 13a were not. After 24 h the viable counts of phage type 4 in the spleen were ten times higher than those of type 13a. However, it is unclear whether this is the result of enhanced invasiveness only or whether an increased ability to multiply in the spleen is also involved. All strains were very invasive in cultured Vero cells; phage type 4 strains slightly more so than types 6, 8 and 13a. Strains of phage type 4 and 13a persisted in the faeces of chickens placed in-contact with orally inoculated birds longer than did strains of types 6 or 8.     

Lysogeny of Streptococcus pneumoniae with MM1 phage: improved adherence and other phenotypic changes

Pneumococcal prophages are extremely frequent, but no role in pathogenesis has so far been attributed to them. We isolated a variant of phage MM1, named MM1-1998, from a serotype 24 strain of Streptococcus pneumoniae. We created three isogenic strain pairs (serotypes 3, 4, and 24) that differed only by the lysogenic presence of the MM1-1998 phage and did a phenotypic comparison. Lysogeny led to improved adherence to inert surfaces and pharyngeal cells compared to that with the cured variants of the strains. We found that lysogeny with MM1-1998 coincided with a more transparent phenotype and phage curing with more opaque colonies in all strain pairs, and we discovered that transparency was associated with more successful and stable lysogeny. Since transparency alone was possibly responsible for the adherence difference, we further compared the TIGR4 lysogen with an equally transparent variant of TIGR4 in order to reassess the role of phage or transparency separately. The results revealed that improved adherence was independently associated with lysogeny with the MM1-1998 phage. Other phenotypic differences such as faster growth, increased autolysis, and decreased intracellular hemolytic activity were more likely due to transparency. By improving the adherence of pneumococci, this prophage may contribute to their fitness and possibly to their persistence in humans.     

Nontypeable bacteriophage patterns of methicillin-resistant Staphylococcus aureus involved in a hospital outbreak.

Of 93 strains of Staphylococcus aureus isolated from inpatient wards of Ismailia General Hospital, 48 (51%) were proven to be methicillin resistant (MR). Of these MR S. aureus strains, 44 were isolated from patients and 4 were isolated from healthy carriers, who were newly arrived interns working in the same wards. Bacteriophage patterns of MR S. aureus were identified by using routine test dilution (RTD) and 100-fold dilutions (100 RTD) of phages. Of these 48 strains, 37 (75%) (33 from patients and 4 from interns) were nontypeable when using RTD and 100 RTD of phages. Of the other 11 strains, 8 were nontypeable by RTD of phages, but 5 of them had the phage pattern D11/1136 when tested by 100 RTD. Three strains had the phage pattern 3A/3C/55/71, and three strains had different phage patterns, 29/81, 96, and 95/D11. The finding of colonization with virulent MR S. aureus strains in interns working on the wards in which these patients were located suggested that new strategies for control of MR S. aureus nosocomial infections must be considered and evaluated.     

Single phage-typing set for differentiating salmonellae.

A phage-typing system is described for characterizing commonly isolated salmonellae. Fifty-eight serovars representative of groups A, B, C1, C2, D, E1, E2, E3, and E4 were delineated by using a single set of 50 phages isolated from sewage. All of the 735 cultures used in this effort were typable and were distinguished and differentiated on the basis of the 347 phage patterns observed. All results were reproducible. Characteristic phage patterns were produced by a variety of Salmonella serovars isolated from a campus incident and a number of hospital and family outbreaks to indicate an existing epidemiological relationship.     

The activity in the chicken alimentary tract of bacteriophages lytic for Salmonella typhimurium

Bacteriophages lytic for Salmonella typhimurium were isolated in considerable numbers from chickens experimentally infected with S. typhimurium, and in much lower numbers from the chicken feed. Lytic phages were also regularly isolated from human sewerage systems. One of these was used to inoculate S. typhimurium--infected two day-old chickens orally and via the feed. The phage took longer to establish in the caeca than did the Salmonella and it disappeared when the caecal S. typhimurium counts fell to 10(6) CFU/ml. No neutralizing antibodies to the phage were detected in the serum of these chickens. In a second experiment, five of 30 chickens similarly infected with S. typhimurium were inoculated with the phage. Within 3 days, the phage was isolated from 72% of the "in-contact" birds. A second phage, isolated from sewage, when inoculated into newly-hatched chickens simultaneously with any of 3 strains of S. typhimurium, produced a considerable reduction in mortality in the birds. This effect was only produced by inoculation of high concentrations of phage (greater than 10(10) PFU/ml). The phage produced reductions in the viable numbers of S. typhimurium in the crop, small intestine and caeca for up to 12 h after inoculation, with smaller reductions in bacterial numbers in the liver at 24 and 48 h after infection.     

Lysogenicity of methicillin-resistant strains of Staphylococcus aureus

The lysogenic status of 23 strains of methicillin-resistant Staphylococcus aureus, isolated at the Royal Prince Alfred Hospital, Sydney, since 1980, was studied. Twenty strains, belonging to the four predominant phage types isolated in this hospital, carried the same lysogenic phage which we have designated C. Three other phages were isolated from five strains belonging to phage type 84/85/90. The presence of phage C had little effect on the phage-typing pattern of the strains. Similarly, lysogenization with the other three phages did not result in a significant change in phage-typing patterns. However, when strain 1489, isolated in 1969, was lysogenized with these three phages, there was a change in phage-typing pattern. Lysogenization of this strain with phage 47T resulted in a marked loss of sensitivity to both group-I and group-III phages. The lysogenic status of these methicillin-resistant strains of S. aureus was compared with that of strains isolated between 1967 and 1970. There was no evidence that the strains isolated recently were either related to, or derived from, the earlier ones.     

Salmonella phage PSP3, another member of the P2-like phage group

Freshly isolated DNA of phage PSP3, whose morphology closely resembles that of phage P2, contained both circular and linear molecules about 31 kb in length. Linear PSP3 DNA molecules possess single-stranded cohesive termini (cos). Sequencing of the fragment anticipated to contain cos revealed a 19-base sequence identical to cos of phage 186. Of the 107 bp to the right of cos, 94 were identical in 186 DNA (88% similarity), and of the 370 bp to the left, 229 were identical (62% similarity). Cos flanking sequences in both P2 and P4 were also highly conserved in PSP3. A number of restriction sites were at similar locations on the two phage DNAs. The parasitic phage P4 propagated on PSP3 lysogens. PSP3 integrates into the Escherichia coli chromosome at 27 min.     

Characterization of a shiga toxin 2e-converting bacteriophage from an Escherichia coli strain of human origin

An infectious Shiga toxin (Stx) 2e-converting bacteriophage (phiP27) was isolated from Stx2e-producing Escherichia coli ONT:H(-) isolate 2771/97 originating from a patient with diarrhea. The phage could be transduced to E. coli laboratory strain DH5alpha, and we could show that lysogens were able to produce biologically active toxin in a recA-dependent manner. By DNA sequence analysis of a 6,388-bp HindIII restriction fragment of phiP27, we demonstrated that the stx(2e) gene was located directly downstream of ileZ and argO tRNA genes. Although no analogue of an antiterminator Q encoding gene was present on this fragment, a lysis cassette comprising two holin genes which are related to the holin genes of Pseudomonas aeruginosa phage phiCTX and a gene homologous to the endolysin gene gp19 of phage PS3 were detected. The results of our study demonstrated for the first time that Stx2e can be encoded in the genome of an infectious bacteriophage.     

Characterization of the phage-specific transfer RNA molecules coded by cholera phage phi 149

Aminoacylation of tRNA isolated from choleraphage phi 149-infected cells with individual 3H-labeled L-amino acids followed by hybridization with phage DNA revealed that the phage encodes tRNAs specific for arginine, proline, glycine, isoleucine, serine, valine, tyrosine, histidine, lysine, leucine, tryptophan, and aspartic acid. Aminoacylation of phage-coded tRNAs isolated from phage DNA-RNA hybrids also confirmed this observation except for tryptophan.     

A beta-related corynebacteriophage which lacks a tox allele but can acquire it by recombination with converting phage.

Corynebacteriophage 782, a phage highly related to the beta family of corynebacteriophages but lacking a tox allele, was isolated from a nontoxinogenic clinical isolate of Corynebacterium diphtheriae. Phage 782 exhibits beta immunity but has a wider host range than beta, forming plaques on strains of C. ulcerans and C. pseudotuberculosis as well as on C. diphtheriae. Phage 782 and beta differed in their DNA mass and in their restriction endonuclease digest patterns, but were similar in possessing cos (cohesive) and attP (phage attachment) sites. Moreover, all the BamHI fragments of 782 and beta except one hybridized with a DNA probe of the other. The exception in both cases was the attP-containing fragment, which in beta also carries the tox gene. Recombinants between phage 782 and pi phage, a tox+ beta-related phage, were isolated which contained ca. 70% of phage 782 DNA but carried the attP-tox-bearing fragment of pi and were thus now converting phages. The recombinants had lost the wide-host-range phenotype of 782 and had the narrower host range of pi. The significance of the tox-less, beta-related phages to the natural history of diphtheria is discussed.     

A comparative study of randomly amplified polymorphic DNA analysis and conventional phage typing for epidemiological studies of Listeria monocytogenes isolates.

The analysis of RAPD profiles generated by PCR with a single 10-mer, HLWL74, was compared to bacteriophage susceptibility data for epidemiological typing of Listeria monocytogenes strains. A total of 104 L. monocytogenes strains was screened, all from serogroup 1 or serotype 4b. Of these, 53 had been isolated during 6 different listeriosis outbreaks. The remaining 51 strains were chosen randomly from our collection. A total of 38 RAPD types were observed, although each epidemic group of strains isolated during one of these outbreaks displayed a specific RAPD profile. For 98% of the strains isolated during outbreaks, the correlation between RAPD typing and phage typing was complete. Only one strain, typed as epidemic by phage typing, was clearly distinguishable from the others by RAPD analysis. Among the 51 strains not related to an outbreak, 12 were linked to epidemic groups by RAPD analysis. Two of these rearrangements were supported by phage typing. The remaining 10 strains could be excluded by phage typing from any of the epidemic groups studies. Considering all 104 isolates, the decision to relate a strain to a particular epidemic group or to exclude a strain from any epidemic group was the same for 92 isolates, using either phage typing or RAPD analysis. The RAPD analysis, which is quick, simple and suited for automation, is proposed as an attractive alternative for phage typing in epidemiological studies of listeriosis.     

Genomic analysis of bacteriophage ESP2949-1, which is virulent for Cronobacter sakazakii

Virulent phage ESP2949-1, which was isolated from sewage, has an icosahedral head, a contractile tail, and a double-stranded DNA genome with a length of 49,116 bp with 50.09% G+C content. Phage ESP2949-1 showed 3% similarity to enterobacteria phage TLS. Bioinformatics analysis of the phage genome revealed 43 putative open reading frames (ORFs). Predicted protein products of the ORFs were determined and described. Based on its morphology, phage ESP2949-1 can be classified as a member of the family Myoviridae. To our knowledge, this is the first report of the genomic sequence and characterization of phage ESP2949-1 isolated from sewage.     

T4 phages against Escherichia coli diarrhea: Potential and problems

A combination of in vitro and in vivo experiments with comparative phage genomics was used for the rational design of a phage cocktail against E. coli diarrhea. Orally applied T4 coliphages representing three different subgroups (T4-, RB49- and JS98-like phages) had no negative impact on the murine gut microbiota. T4 phages were found with high titers in the cecum and colon and lower titers in the small intestine, but were not detected in the blood, liver or spleen. No adverse effects were observed after one-month exposure to phage nor were serum anti-T4 antibodies detected. T4 phages belonging to the same subgroup showed closely related genomes that differed by 12 (phage JS10 vs. JS98 reference) to 17 (phage JSE vs. RB49 reference) insertion/deletions mostly representing single small ORFs. Bioinformatic analysis did not reveal undesired genes in the T4 genomes. Sequence variability was seen over the tail fibre genes, but the variability did not correlate with phage host range. The investigated T4 phages were not only species- but also strain-specific, necessitating the use of phage cocktails consisting of 10 and 16 T4 phage isolates to cover half to two thirds of E. coli strains representing the five main pathotypes isolated from diarrhea patients.     

Two bacteriophages of Clostridium difficile.

Two temperate bacteriophages of differing morphology and host range were isolated by screening 94 isolates of Clostridium difficile. Phage 41 had a 300-nm flexible tail, whereas phage 56 had a shorter tail with a contractile sheath. Electron microscopy of phage 56 lysates exposed to elevated magnesium concentrations showed small virus-like particles which were 21 nm in diameter. The addition of MgCl2 to semisolid agar overlays enhanced both the titer and plaque size of phage 56. Phage 56 was more temperature labile than phage 41 and demonstrated unusual lability in buffer at pH 7.0. One-step growth and adsorption experiments revealed that both phages had latent periods of about 60 min, but phage 56 adsorbed to its indicator strain more efficiently. Phage 56, which was obtained from a toxigenic strain of C. difficile, was used to lysogenize its nontoxigenic indicator strain, but no conversion to toxigenicity was observed in this strain.     

Reduced set of phages for typing salmonellae.

A set composed of 27 phages is described for differentiating Salmonella spp. representative of groups A, B, C1, C2, D1, D2, E1, E2, E3, E4, G1, K, and N. All of the 1,245 cultures used in this effort were typable and were differentiated on the basis of the 420 phage patterns observed. All results were reproducible. Characteristic phage patterns were produced by a variety of Salmonella serovars isolated from campus incidents and a number of hospital, family, restaurant, and processing plant outbreaks to indicate an existing epidemiological relationship.     

Exfoliative toxin plasmids of bacteriophage group 2 Staphylococcus aureus: sequence homology.

The plasmid contents of seven exfoliative toxin-producing strains of phage group 2 Staphylococcus aureus were analyzed by agarose gel electrophoresis and deoxyribonucleic acid-deoxyribonucleic acid hybridization. All strains were found to contain a large plasmid with a molecular weight of 27 X 10(6) except for strain RW1005. A comparison of the restriction endonuclease cleavage products by agarose gel electrophoresis showed that the number and size distribution of the fragments of all these Tox plasmids were similar, except for pRW002, which appeared to contain two deletions. Deoxyribonucleic acid-deoxyribonucleic acid hybridization studies confirmed that these plasmids were related to a plasmid which carried the genes for exfoliative toxin B and bacteriocin R1 biosynthesis and that they shared some sequence homology with the penicillinase plasmid pI258 isolated from a phage group 3 S. aureus.     

The lysogenic cycle of the filamentous phage Cflt from Xanthomonas campestris pv. citri

A phage, Cflt, forming turbid plaques, was isolated from Xanthomonas campestris pv. citri. After infection, infected sensitive cells become immune to Cflt and produce very few phages. These properties were genetically rather stable. The phage was purified and shown to be filamentous with a size of 1157 +/- 73 nm. The genome size is about 7.62 kb. The phage does not affect the growth of host bacteria. Under natural cultivation conditions Cflt-lysogenized cells could be induced spontaneously to give high phage yields, or cured to give phage-free cells. The integration of Cflt DNA into host DNA was proved by Southern blot hybridization. The lysogenic phage was genetically stable in log phase cells and persisted in stationary phase cells through many cell generations in the absence of extracellular phage reinfection.     

Single-chain Fv antibody with specificity for Listeria monocytogenes

Single chain antibodies (scFv) exhibiting specific binding to Listeria monocytogenes strains were isolated from a pool of random scFvs expressed on the surface of filamentous bacteriophages. Positive selection (panning) using L. monocytogenes was used to enrich for phage clones with the desired binding affinity, and negative selection using L. innocua and L. ivanovii was used to remove phages expressing cross-reactive antibody fragments. A single phage clone, P4:A8, was selected using two independent panning schemes. A rapid assay was devised to determine phage antibody binding specificity and was used to develop a selectivity profile for individual phage clones. The P4:A8 clone was screened against a panel of bacteria consisting of eight strains of L. monocytogenes, one each of the other six species of Listeria and nine other relevant bacterial species. A collection of individual clones from the penultimate panning was also screened against a subset of the panel of bacteria. The selectivity profiles indicate that multiple clones, including P4:A8, exhibit binding to one or more strains of L. monocytogenes without cross-reactivity toward any other species in the panel. This is the first report of a species-specific antibody for viable cells of L. monocytogenes (i.e., the ability to bind to L. monocytogenes without cross-reactivity toward any other species of Listeria).