Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.?aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.?aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.?aeruginosa in the environment.     

The role of lipopolysaccharide as a receptor for some bacteriophages of Pseudomonas aeruginosa

Typing phages of the Colindale typing set for Pseudomonas aeruginosa have been tested for the use of lipopolysaccharide (LPS) as a receptor. Studies using the reference strains of the International Antigenic Typing Scheme for O-serotypes of P. aeruginosa supported earlier indications that none of the phages were O-specific. Studies of the adsorption of phages to LPS showed that typing phages 16, 44, F8, 68, 109, 352, and 1214 (as well as other phages 2 and H22) were LPS-specific, but were not consistently adsorbed by isolated LPS from all sensitive strains. Water-soluble fractions from LPS did not adsorb phages and did not inhibit their neutralization by whole LPS. No endoglycosidase activity against LPS was detected for any phage. The significance of these results for the roles of LPS in the adsorption process and phage sensitivity are discussed.     

Plaque morphology and antigenic relationship of the Salmonella weltevreden typing phages

The plaque morphology and antigenic relationship of the six typing phages of Salmonella weltevreden were studied. Under identical conditions of plating, the phages could be classified into three groups based on plaque morphology. Neutralization tests with anti-phage sera showed that typing phages phi I and phi II were antigenically similar. Phages phi III, phi IV and phi VI also showed antigenic similarity. Typing phage phi V was antigenically distinct from all other phages. Thus the phages could be classified into three groups on the basis of both plaque morphology on their respective indicator strains and velocities of neutralization by homologous/heterologous anti-sera.     

Lytic activity of Pseudomonas bacteriophages

None of the 24 Pseudomonas syringae bacteriophages were found to be identical in the spectrum of lytic action. The phages were subdivided into five groups according to the number of sensitive bacterial strains and their qualitative composition.     

High diversity and novel species of Pseudomonas aeruginosa bacteriophages

The diversity of Pseudomonas aeruginosa bacteriophages was investigated using a collection of 68 phages isolated from Central Mexico. Most of the phages carried double-stranded DNA (dsDNA) genomes and were classified into 12 species. Comparison of the genomes of selected archetypal phages with extant sequences in GenBank resulted in the identification of six novel species. This finding increased the group diversity by ~30%. The great diversity of phage species could be related to the ubiquitous nature of P. aeruginosa.     

Bacteriophage types represented by lactose-fermenting Salmonella agona strains

Bacteriophage typing was performed on 1911 S. agona, lactose-fermenting strains. These strains were isolated from hospitalised newborns and neonates patients. Out of 1911 strains 98.8% were typable by means of phage set prepared for strains differentiation of Salmonella agona showing typical biochemical properties. It was shown that in 16 provinces from which the strains were obtained in 1983-1985 type V (49.5%) and type XI (25.4%) prevailed. Subtypes VA and VB were distinguished within type V. Altogether 20.3% of strains were classified as belonging to these subtypes. Their lytic reaction was weaker with phages 3, 4, and 9 with the characteristic range of phage type V strains. Among tested strains types I, XIII, and XVI were also represented composing 2, 6, 0, 9, and 0.3% of total number of strains respectively. 1.5% of strains were nontypable and 0.2% showed lytic reactions different from that included in up to now used scheme of typing. It can be concluded that lactose-fermenting S. agona strains show susceptibility to lowered number of phages than typical for Salmonella species strains. It seems that differentiation of this atypical biochemical variant of S. agona with, the use of phage set used up to now may be also usefull in practice as it is the case in respect to strains with typical biochemical properties.     

Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.     

Evaluation of the usefulness of new international experimental phages for typing methicillin resistant Staphylococcus aureus (MRSA)

The aim of the study was to determine the usefulness of the set of experimental phages obtained from the Central Public Health Laboratory in London for typing of MRSA strains in Poland. The study was performed on 150 MRSA strains isolated from various clinical materials in various regions of the country. The set of 10 experimental phages and the international basic set of 23 phages were used for typing. The results of the study showed that 76.8% of MRSA strains were typing with the experimental set of phages. The frequency of inhibition reactions was 19.9%. Only 3.3% of the strains were nontypable with the new phages while nearly half of the studied strains were nontypable with the basic set of phages. The studied strains were divided into 19 phagotypes. There was a high frequency of typable strains among MRSA typable and nontypable strains and those inhibited by the basic set of phages (71.4%-85.7%). These data indicate that the set of 10 experimental phages is useful for typing of MRSA strains isolated in Poland except for phage M3 which failed to react with almost all the strains and should be excluded from the proposed set.     

Heterogeneity of a Pseudomonas bacteriophage population

Twenty-five isolates of virulent bacteriophages for Pseudomonas belonging to the morphological group C (Bradley, 1967) were obtained from different natural habitats. The phages of each isolate were found to differ from one another in at least one of the following characteristics: the sensitivity to an osmotic shock, to heating at 60 degrees C and to UV; the ability to cause lysis of 86 Pseudomonas strains; the reaction of neutralisation with antisera. At the same time, the phages were related by the existence of common antigens. These properties are responsible for the genetic stability of the bacteriophages in their complicated relationship with their host, Pseudomonas bacteria.     

Study of morphology and genome structure of Pseudomonas putida bacteriophages for their classification

A group of 27 bacteriophages specific for Pseudomonas putida strains PpG1 and PpN has been isolated. The phages were characterized and compared with the previously described virulent (pf 16, af, tf and PMW) and temperate (PP56 and PP71) phages. The new phages belong to B1 and C1 morphotypes, according to Ackerman's classification. Phage DNAs were digested with several endonucleases; the molecular weights and homology of the DNAs were determined. All phages of P. putida isolated up to now were distributed into 10 species (groups), on the basis of particle morphology, genome size and the results of homology studies. Recombination processes are believed to participate in formation of phages belonging to certain species.     

A new phage typing scheme forProteus mirabilis andProteus vulgaris strains

A new bacteriophage typing set, composed of 22 phages, was established as a tool for differentiation ofProteus strains. All the phages were tailed and included 4 morphological types (A1, A2, B1 and C1). They were classified into the familiesMyoviridae, Siphoviridae andPodoviridae. From the set, 19 phages had double-stranded DNA and 3 were single-stranded DNA phages.     

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.     

Isolation and characteristics of new nonlysogenic bacteriophages of Pseudomonas aeruginosa

A large group of nonlysogenic bacteriophages specific for Pseudomonas aeruginosa was studied. According to their absorption characteristics and serological properties, the phages were subdivided into four groups: luminal diameter k, luminal diameter m, luminal diameter mnP78 and luminal diameter mnF82. Within each of the groups, the phages were similar in the morphology of their particles and certain physiological characteristics. The luminal diameter m phages were similar to the P. aeruginosa bacteriophage E79 in their adsorption properties and antigenic specificity. The phages of the other groups differed in the above characteristics from the known P. aeruginosa bacteriophages. The effect of some plasmids on the growth of bacteriophages luminal diameter k, luminal diameter m, luminal diameter mnP78 and luminal diameter mnF82 was studied. The growth of new bacteriophages on certain plasmid-containing strains was inhibited in some cases.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Pyocine Typing of Clinical Strains of Pseudomonas aeruginosa

A total of 954 clinical isolates of Pseudomonas aeruginosa were typed by their ability to produce pyocines. The strains of Pseudomonas were isolated from urines, bloods, sputa, stools, and miscellaneous infectious exudates or tissue of patients of the Mayo Clinic and four associated hospitals. About 80% of the typable strains could be grouped into three major pyocine types: A (30.9%), B (34.8%), and D (14.1%). These large groups could be divided into subtypes by using additional indicator strains. There was no significant difference in the distribution of types by either institutional or specimen source, except that urine specimens yielded the highest percentage of one type. By this procedure, 93% of all isolates could be typed. Repeated typing of serially transferred strains indicated that the procedure has a high degree of reliability. Several strains exhibited extreme fluctuation in inhibition pattern. The procedure is a simple and reliable method to monitor the patterns of nosocomial infections due to P. aeruginosa.     

Phage-stable mutants of Pseudomonas putida: new types of stability

More than 170 phage-resistant mutants (PRM) of the first order of Pseudomonas putida strain PpG1 were obtained using newly isolated and previously described bacteriophages specific for this strain. According to the results of analysis of resistance of the mutants to each of 31 phages of PpG1 strain and 8 phages of the PpN strain, the PRM strains were distributed into 20 groups. In most cases, the reason for resistance is loss of absorption capacity of bacteria. However, no direct relation between the level of absorption and efficiency of phage plating was detected. It was shown that some of the PRM of P. putida PpG1 strains acquired the ability to maintain the growth of phages specific for the other P. putida strain, PpN. Frequencies of isolating mutants of various resistance types depend on the concrete phage used. In accordance with their absorption specificity, all phages were distributed into 23 groups, and a tridimensional formal scheme of receptor sites for these phages on the PpG1 strain was drawn. In the process of selection of the PpG1 clones resistant to non-lysogenizing mutant of temperate PP71 phage, a variant of this strain manifesting the phenomenon of "auto-plaquing" was found. These results support the mutational origin of this phenomenon in some cases.     

Survey of Pseudomonas aeruginosa and its phages: de novo peptide sequencing as a novel tool to assess the diversity of worldwide collected viruses

A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales . Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.     

Evaluation of the international phage typing set and some experimental phages for typing of Listeria monocytogenes from poultry in Spain

AIMS: The validity of the international phage set and 13 experimental phages for subtyping Listeria monocytogenes strains isolated from poultry in Spain was investigated. METHODS AND RESULTS: Ninety-six L. monocytogenes strains (52 from serogroup 1/2 and 44 from serogroup 4) were phage-typed using the international phage set, 10 experimental phages for typing serogroup 1/2 strains (seven isolated in France: 1313, 9425, 1807, 351, 881, 717 and 586-, and three from Denmark: 5775, 12682 and 6223-) and three experimental phages isolated in France for typing serogroup 4 strains (2425 A, 4286 and 197). Percentages of serogroup 1/2, serogroup 4 and total phage-typeable strains were 57.7%, 52.3% and 55.2%, respectively. Important differences in the behaviour of the phages tested were found. The typeability rate, the specificity index and the percentage of strong reactions were greater in the phages of international set than in the experimental phages. The number of phage typeable strains and the number of phage types (42) were not modified by the use of experimental phages. CONCLUSIONS: The phage set used was not effective for typing L. monocytogenes strains from poultry in Spain, because a low typeability rate was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest the importance of the availability of new phages specific to a geographical area in order to improve the typeability of the system.     

Recharacterization of Pseudomonas fulva Iizuka and Komagata 1963, and proposals of Pseudomonas parafulva sp. nov. and Pseudomonas cremoricolorata sp. nov

Seven Pseudomonas fulva strains obtained from culture collections were taxonomically studied. The seven strains were separated into three clusters (Clusters I to III) on the basis of 16S rRNA gene sequences, and located phylogenetically in the genus Pseudomonas sensu stricto. Further, the strains were classified into 4 groups (Groups I to IV) on the basis of DNA-DNA similarity. As a result, Cluster I was split into Groups I and II. Group I included the type strain of P. fulva and two strains, and levels of DNA-DNA similarity ranged from 88 to 100% among the strains. Group II contained two strains, and the level between the two strains ranged from 91 to 100%. Group III consisted of one strain. Group IV included one strain, and this strain showed a high level of DNA-DNA similarity with the type strain of Pseudomonas straminea NRIC 0164(T). Clusters II and III corresponded to Groups III and IV, respectively. The four groups were separated from one another and from related Pseudomonas species at the level from 3 to 45% of DNA-DNA similarity. The strains of Groups I, II, and III had ubiquinone 9 as the major quinone. According to numerical analysis by the use of 133 phenotypic characteristics, the seven P. fulva strains were split into four phenons (Phenons I to IV). The groups by DNA-DNA similarity corresponded well with the phenons produced by numerical taxonomy, and differential characteristics were recognized. Consequently, Group I was regarded as P. fulva because the type strain (NRIC 0180(T)) of this species was included in this group. Strains in Group II were identified as a new species, Pseudomonas parafulva sp. nov., and the type strain is AJ 2129 (=IFO 16636=JCM 11244=NRIC 0501). NRIC 0181 in Group III was identified as a new species, Pseudomonas cremoricolorata sp. nov., and the type strain is NRIC 0181 (=IFO 16634=JCM 11246). NRIC 0182 in Group IV was identified as P. straminea on the basis of the high level of DNA-DNA similarity with the type strain of this species.     

A numerical taxonomic study of fluorescent Pseudomonas strains isolated from natural mineral waters

M. ELOMARI, L. COROLER, D. IZARD AND H. LECLERC. 1995. Forty-six strains of fluorescent pseudomonads, isolated from natural mineral waters, together with 12 strains from clinical material and 44 reference strains, were phenotypically classified by 281 characteristics. The data were processed by the Dice similarity coefficient and unweighted pair group algorithm with arithmetic averages. Eight clusters were defined at the 62% similarity level. Clusters I, II and IV were further divided into nine subclusters. Virtually all the mineral water strains fall into three groups: Ib (eight strains), Ha (14 strains) and V (16 strains). Subclusters Ib and IIa included natural mineral water strains only. Cluster V contained 13 mineral water strains and three culture collection strains of Pseudomonas fluorescens biovar III. DNA/DNA hybridization studies are needed to define the taxonomic status of these three groups within the genus Pseudomonas.