Bacteriophage typing of Salmonella weltevreden

Salmonella weltevreden has been found to be one of the commonest Salmonella serotypes isolated from diverse sources in India and has also been isolated in a number of other countries. A phage typing scheme was developed for this serotype using a set of six typing phages. These phages had been selected out of 146 phage strains isolated and purified from stool samples of man, laboratory animals and other animals, sewage and surface water sources, and the lytic mutants of temperate phages from S. weltevreden.The phage typing scheme was applied systematically to type the 946 strains from India isolated during 1958–1974 and 148 strains originating from Australia, Burma, England, Gan Island, Holland, Hong Kong, Malaysia, New Zealand, Papua New Guinea, The Philippines, Thailand, The United States and Vietnam during 1953–1971. The scheme was particularly studied to evaluate its utility in mapping the epidemiologically related strains from various sources.The S. weltevreden strains could be classified into ten phage types. Phage types 2 and 7 were found exclusively amongst Indian strains, type 6 from Vietnam and type 8 from Burma, Thailand and Vietnam. Phage types were found to be stable and consistent with the independent epidemiological data available.     

Biological characteristics ofSalmonella weltevreden typing phages

The important biological characteristics ofSalmonella weltevreden (3,10∶r∶z₆) typing phages were studied. On the basis of these, the phages could be classified into three groups: phages Φ I and Φ II, phages Φ III, Φ IV and Φ VI, and phage Φ V. Part of the work was done at the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, India.     

Typing ofSalmonella weltevreden strains by means of lysis patterns of symbiotic phages

A typing scheme forSalmonella weltevreden using the lysogenicity and lysis patterns of their carried phages is presented. Six strains ofS. weltevreden were selected for use as indicator strains for recognizing the lysis patterns of the carried phages. Two hundred and forty-five strains were examined and 207 were grouped in concurrence with the 15 lysis pattterns obtained out of 64 theoretically possible. Lysis pattern I (all 6 indicator strains lysed by the carried phage) included 24.5% of the strains. Thirty-eight strains (15.5%) were grouped as untypable because their lysates did not lyse any of the indicator strains. No correlation could be established between the lysis patterns of carried phages and the host and geographic distribution ofS. weltevreden.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Deoxyribonucleic acids were isolated from Brevibacterium lacto fermentum No. 2256 and its four representative phages belonging to different serological groups, i.e., P465 (group I), P 468 II (group II), Ap 85 III (group III) and P4 (group IV), by phenol extraction.

DNA’s isolated from the four phages contained only usual four bases, i.e., guanine, adenine, cytosine and thymine. The G-C content of the phage DNA was determined by thermal denaturation method (T_m); the values of P465, P 468 II, Ap 85 III and P4 were 54.0, 54.6, 56.6 and 55.3%, respectively. Sedimentation coefficient was measured by ultracentrifugal analysis using ultraviolet optics; s_20,w of the phage DNA’s were 15.5 to 31.8 s.

Unusual bases were not detected also in the host bacterial DNA. The G-C content of the bacterial DNA determined by paperchromatography was 55.1% which came very close to the G-C content derived from the T_m.

Morphological properties of the P- and Ap-series phages described in previous papers were examined by means of electron microscopy, analytical centrifugation, CsCl density-gradient centrifugation and ultrafiltration.

In view of the buoyant density of phage particles, fourteen Brevibacterium phages were classified into four groups, i.e., the phages under group I had buoyant density of 1.511 to 1.514g cm^?3, and those under groups II, III and IV, densities of 1.482, 1.492 and 1.508 g cm^?3, respectively; and above grouping corresponded to that by the serological chracteristics of the phages.

Electron microscopic observation by means of shadow-casting or negative staining recognized all the phages as tadpole-shaped; their particles having polyhedral head (40 ~ 70 mμ in diameter) and long tail (80 ~ 275 mμ in length).

In relation to particle sizes of the phages as estimated by all of above-mentioned methods, no significant differences were observed between the sizes calculated from ultrafiltration and those obtained directly from electron micrographs by negative staining.     

Enterotoxigenicity and invasiveness of Salmonella species

A large number of enterotoxigenic strains was encountered in a group 56 Salmonella cultures belonging to 8 species viz., S. alachua, S. anatum, S. dublin, S. enteritidis, S. hindmarsh, S. newport, S. typhimurium, S. weltevreden, and 5 serotypes of S. arizona (16:z₄:-; 48:1,v:z₅₆; 53:z₅₂:z₅₃; 60:r:z; 61:i:z₅₃). These cultures were isolated mainly from humans and animals suffering from gasteroenteritis. The enterotoxigenic (diarrhoeagenic) Salmonella cultures possess capacities for both skin permeation and epithelial penetration (invasiveness). Preliminary characterization revealed that Salmonella enterotoxin is a heatlabile protein of high molecular weight. It is suggested that enterotoxigenic and invasive propeties play a vital role in the pathogenesis of Salmonella diarrhoea.     

Bacteriophage Typing of Salmonella. I. Isolation and Host Range Study of Bacteriophages

A series of bacteriophages, lytic for bacteria belonging to the genera Escherichia and Salmonella, were isolated. The phages were isolated from fecal samples, intestinal contents of turkey poults, and carrier cultures of S. typhimurium, S. typhimurium var copenhagen, S. heidelberg, and E. coli. The feasibility of using different habitats as sources of Salmonella phages was evaluated. The carrier cultures were the most promising source for phages active on the serotypes for which the phages were sought. A host range study of the isolated phages was made. Eight phages were selected to develop a phage typing scheme for S. typhimurium, S. typhimurium var copenhagen, and S. heidelberg.     

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.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

The growth characteristics of phages were investigated with the four phages, active on Brevibacterium lactofermentum, which were selected from the respective serological groups, namely, P465 (group I), P468II (group II), Ap85III (group III) and P4 (group IV).

The adsorption rate of the phages, P465 and P468II, on the host bacteria was low, whereas that of the phages, Ap85III and P4, was higher. The adsorption rate constants for the four phages were respectively calculated at 2.02 × 10^?10, 1.87 × 10^?10, 4.32 × 10^?10 and 3.15 × 10^?10 cm³ per minute, at 30°C in G₅B₂ medium. With reference to the ionic environment for adsorption, the phages, P465 and Ap85III, specifically required either for Ca⁺⁺ or Mg⁺⁺; the phage P468II, for both; and the phage P4, for neither.

The growth characteristics of these phages were examined by the one-step growth experiment. The latent periods of the phages were 50, 53, 57 and 47 minutes, respectively; and the corresponding average burst sizes were about 98, 31, 145 and 126. The growth of the phage P4 was completely suppressed at above 34°C, although the host bacteria and the other three phages were capable of the full growth at that temperature.     

Phage types of Staphylococcus aureus strains and their antibiotic resistance in carriers of medical students population

The phage types of 78 S. aureus strains isolated from nose swabs obtained from a medical students in 2005 -2006 was determined and antibiotic resistance of the phage types was analysed. 680 students were tested in order to obtain the strains and 11.5% of them were carriers of S. aureus. Phage typing was performed using basic set of23 phages and 3 additional phages: 88, 89 and 187. Drug resistance was determined by the disc-diffusion method. The most frequent in studied population were the group III (21.8%) and strains lysed by phages belonging to varied groups (21.8%). Highly different phage patterns were observed among strains belonging to each of the group. Strains belonging to the group III as the strains lysed by phages from varied groups were most frequently resistant only to penicillin (52,9% respectively). Resistance to penicillin was also most often observed in the strains belonging to another groups and phage types. Usefulness of the additional phages 88,89 and 187 was in the investigations as no more than 51% of strains was lysed by this phages.     

Molecular typing of Salmonella weltevreden and Salmonella chincol by pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)

Genomic DNA of Salmonella weltevreden (10 isolates from poultry, two isolates each from raw vegetables and river water) and S. chincol (15 isolates from poultry) were characterized by pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis. These isolates originated from a single location in Kajang, Selangor. The results of the PFGE and ERIC-PCR were analysed and comparisons were made using GelCompar software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the S. weltevreden into five clusters and two single isolates and S. chincol into two clusters and two single isolates at a similarity level of 80%, respectively. PFGE produced a single cluster and eight single isolates for S. weltevreden, and one cluster and 11 single isolates for S. chincol at a similarity level of 80% after digestion with the restriction enzyme XbaI, respectively. These results demonstrate that both PFGE and ERIC-PCR are suitable tools for molecular typing of the isolates examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phage susceptibility and plasmid profile analysis of Sinorhizobium fredii

This is the first report identifying bacteriophages and documenting megaplasmids of Sinorhizobium fredii. Plasmid DNA content and bacteriophage typing of eighteen strains of S. fredii were determined. S. fredii strains fell into ten plasmid profile groups containing 1 to 6 plasmids, some evidently larger than 1000 MDa. Twenty-three S. fredii lytic phages were isolated from soil, and they lysed six different S. fredii strains. The host range and plaque morphology of these phages were studied. Susceptibility to S. fredii phages was examined for S. meliloti; Rhizobium leguminosarum bvs. viceae, trifolii and Phaseoli; R. loti; Bradyrhizobium japonicum; B. elkanii and Bradyrhizobium sp. (Arachis). Several phages that originally lysed S. fredii strain USDA 206 also lysed strains of all three S. fredii serogroups described originally by Sadowsky et al. Phages that infected S. fredii strains USDA 191 and USDA 257 were highly specific and lysed only serogroup 193 strains. S. meliloti strains L5-30 and USDA 1005 were lysed by three of the phages that lysed S. fredii strain USDA 217. No other Rhizobium or Bradyrhizobium strain tested was susceptible to lysis by any of the S. fredii phages. The present investigation indicates that phage susceptibility in conjunction with plasmid profile analysis may provide a rapid method for identification and characterization of strains of S. fredii.     

The use of <Emphasis Type="Italic">spa</Emphasis> and phage typing for characterization of clinical isolates of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in the University Clinical Center in Gdańsk,Poland

The emergence of spa types and spa–clonal complexes (CC) among clinical methicillin-resistant Staphylococcus aureus isolates collected from the University Clinical Center in Gdańsk between 2008 and 2009 were investigated. Phage typing was used as the initial screening in the study. The basic set of phages and the additional set of phages were used. Most of the isolates (56 %) belonged to the phage group III. With the additional set of phages, eight types were found, with predominant one MR8 (50 %). Sixteen distinct spa types were observed. The most frequent were t003 (22 %), t151 (16 %), and t008 (12 %). The spa types were clustered into two spa-CC and eight singletons. The predominant CC010 (50 %) consisted of six types, with the most common t003 (36.7 %) and t151(26.7 %), and in 80 % was identified as staphylococcal chromosomal casette mec (SCCmec) type II. The second cluster has no founder (12 %) with only two spa types: t037 belonging to SCCmec type III and t029. In the most frequent singleton, spa type t008 alone was clustered in 12 % of the isolates. All singletons correspond to SCCmec type IV. The CC010 was distributed in most of the hospital wards, corresponded to Multilocus sequence typing type ST5/ST225 and was constantly present throughout the observed period. The isolates of CC010 generally belonged to the phage group III, and most of them (53.3 %) were resistant to erythromycin, clindamycin, and ciprofloxacin. The concordance between spa-clone and phage type was very high, but the same phage type MR8 was observed within different spa types of the predominant clone.     

Simplex and multiplex real‐time PCR assays for the detection of flagellar (H‐antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7

Aims: To develop real‐time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7. Methods and Results: Primers and probes specific to fliC_H2, fliC_H7, fliC_H8, fliC_H11, fliC_H28, eae‐β1, eae‐γ1, eae‐ε and eae‐θ were combined in simplex and multiplex 5′‐nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5 CFU per 25 g after overnight enrichment using the PCR assays. Conclusions: The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. Significance and Impact of the Study: Application of real‐time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Specific Phages with Broad Activity Spectra

The aim of the study was to screen various kinds of samples for Pseudomonas aeruginosa specific phages and to isolate and partially characterize those with broad activity spectra. The Pseudomonas specific phages were isolated using an enrichment procedure with single strains or the cocktail of P. aeruginosa strains as hosts. Using the described procedure, phages were successfully isolated only from water samples, while in soil and feces no Pseudomonas specific phages were detected. The lytic spectra of isolated phages were determined by spot method on lawns of 33 P. aeruginosa strains and five species belonging to family Enterobacteriaceae. The results showed that among isolated phages, 001A, δ, and I possessed the broad activity spectra, as were able to plaque on more than 50% of tested P. aeruginosa strains, while none of the phages were able to lyse the other tested species. Significant differences in phage activity spectra were not observed when P. aeruginosa cocktail was applied for sample enrichment. The most of the phages examined by electron microscopy belonged to family Siphoviridae, while the broad activity spectra isolates, except for 001A, possessed morphological characteristics of family Podoviridae. Digested DNA of the phages δ and I showed similar patterns, indicating the prevalence and success of this phage type in the environment.     

Genotypic and Phenotypic Diversity among Induced, stx2-Carrying Bacteriophages from Environmental Escherichia coli Strains

Shiga toxin 2 (stx₂) gene-carrying bacteriophages have been shown to convert Escherichia coli strains to Shiga toxin-producing Escherichia coli (STEC). In this study, 79 E. coli strains belonging to 35 serotypes isolated from wastewaters of both human and animal origin were examined for the presence of stx₂-carrying bacteriophages in their genomes. The lytic cycle of the bacteriophages was induced by mitomycin, and the bacteriophage fraction was isolated and used for morphological and genetic characterization. The induced bacteriophages showed morphological diversity, as well as restriction fragment length polymorphism variation, in the different strains belonging to different serotypes. The ability to infect new hosts was highly variable, although most of the induced phages infected Shigella sonnei host strain 866. In summary, in spite of carrying either the same or different stx₂ variants and in spite of the fact that they were isolated from strains belonging to the same or different serotypes, the induced bacteriophages were highly variable. The high level of diversity and the great infectious capacity of these phages could enhance the spread of the stx₂ gene and variants of this gene among different bacterial populations in environments to which humans may be exposed.     

Comprehensive analysis of <Emphasis Type="BoldItalic">Salmonella</Emphasis> sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes

Salmonella is a major cause of food-borne disease, and Salmonella enterica subspecies I includes the most clinically relevant serotypes. Salmonella serotype determination is important for the disease etiology assessment and contamination source tracking. This task will be facilitated by the disclosure of Salmonella serotype sequence polymorphisms, here annotated in seven genes (sefA, safA, safC, bigA, invA, fimA, and phsB) from 139 S. enterica strains, of which 109 belonging to 44 serotypes of subsp. I. One hundred nineteen polymorphic sites were scored and associated to single serotypes or to serotype groups belonging to S. enterica subsp. I. A diagnostic tool was constructed based on the Ligation Detection Reaction—Universal Array (LDR-UA) for the detection of polymorphic sites uniquely associated to serotypes of primary interest (Salmonella Hadar, Salmonella Infantis, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Virchow, and Salmonella Paratyphi B). The implementation of promiscuous probes allowed the diagnosis of ten further serotypes that could be associated to a unique hybridization pattern. Finally, the sensitivity and applicability of the tool was tested on target DNA dilutions and with controlled meat contamination, allowing the detection of one Salmonella CFU in 25 g of meat.     

Specificity patterns of Agrobacterium tumefaciens phages

Summary Lysogeny was not detected in 10 strains of A. tumefaciens by plating techniques or ultra-violet induction. Fifteen phages were isolated from raw sewage against 13 cultures of A. tumefaciens and purified by single-plaque selections. No phage lysed all of the strains of A. tumefaciens tested; one phage lysed only a single strain; 2 other phages attacked 7 strains. Ten of the 15 phages lysed no more than 3 strains. Three host strains showed identical phage susceptibilities. No relationship was noted between susceptibility to phage and ability of a strain to incite crown galls.Thirteen phages lysed at least 1 of 4 strains of A. radiobacter, but none attacked single strains of A. rubi or A. pseudotsugae. Eleven phages lysed the one strain of A. rhizogenes used. None of the phages had identical host ranges with respect to all the Agrobacterium spp. tested. Similarly none of 5 selected phages attacked any one of 59 strains of bacteria from 12 different genera including 35 strains of rhizobia. Within the limits of this study the phages used were genus-specific.Published with approval of the Director, Wisconsin Agricultural Experiment Station, Madison, Wisconsin, U.S.A. 53706.     

The utilization of phosphorus from muds by the phytoplankter,Scenedesmus dimorphus,and the significance of these findings to the practice of pond fertilization

Muds from 12 types of soils were used as the only source of phosphorus in cultures of Scenedesmus dimorphus. Some muds supported as much algal growth as was obtained with 0.075 to 0.5 mg/1 of phosphorus, while little or no growth occurred in cultures which contained other muds as a source of phosphorus. Algal growth was correlated with the fractions of soil phosphorus which were extracted with the following solutions; I — the phosphorus — free nutrient solution, II — 0.05N HCl plus 0.025N H₂SO₄, III — 0.002N H₂SO₄ plus 3 g/l of K₂SO₄, and IV — 0.1N HCl plus 0.03N/NH₄F. Additions of phosphorus to the soils prior to their use as muds increased the suitability of some as sources of phosphorus, but for others the added phosphorus was so tightly bound to the soil that little or none was available to S. dimorphus. The findings indicate that the type of soil in a pond will likely have a large influence on the efficiency of fertilization with phosphate fertilizers.

     

Plutonium(IV) Reduction by the Metal-Reducing Bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1

The bacterial reduction of actinides has been suggested as a possible remedial strategy for actinide-contaminated environments, and the bacterial reduction of Pu(VI/V) has the potential to produce highly insoluble Pu(IV) solid phases. However, the behavior of plutonium with regard to bacterial reduction is more complex than for other actinides because it is possible for Pu(IV) to be further reduced to Pu(III), which is relatively more soluble than Pu(IV). This work investigates the ability of the metal-reducing bacteria Geobacter metallireducens GS15 and Shewanella oneidensis MR1 to enzymatically reduce freshly precipitated amorphous Pu(IV) (OH)₄ [Pu(IV)(OH)_4(am)] and soluble Pu(IV)(EDTA). In cell suspensions without added complexing ligands, minor Pu(III) production was observed in cultures containing S. oneidensis, but little or no Pu(III) production was observed in cultures containing G. metallireducens. In the presence of EDTA, most of the Pu(IV)(OH)_4(am) present was reduced to Pu(III) and remained soluble in cell suspensions of both S. oneidensis and G. metallireducens. When soluble Pu(IV)(EDTA) was provided as the terminal electron acceptor, cell suspensions of both S. oneidensis and G. metallireducens rapidly reduced Pu(IV)(EDTA) to Pu(III)(EDTA) with nearly complete reduction within 20 to 40 min, depending on the initial concentration. Neither bacterium was able to use Pu(IV) (in any of the forms used) as a terminal electron acceptor to support growth. These results have significant implications for the potential remediation of plutonium and suggest that strongly reducing environments where complexing ligands are present may produce soluble forms of reduced Pu species.     

Bacteriophage typing of Listeria species.

A bacteriophage typing scheme for differentiating Listeria isolates from dairy products and various other foodstuffs was developed. Sixteen selected phages isolated from both environmental sources and lysogenic strains were used for typing and, according to their lytic spectra, divided into four groups. Thus far, 41 distinct patterns of lysis were seen when this set was used in typing 57 defined reference strains, representing all five confirmed species and 16 serotypes in addition to 454 Listeria isolates of primarily foodborne origin. Overall, typability was 84.5%; i.e., a strain was lysed by at least one phage at 100x routine test dilution. Strains belonging to serovar 3 were mostly resistant to lysis by the phages employed. The results were highly reproducible, as determined in retyping trials several weeks later. Some phages isolated from environmental sources showed a wider lytic spectrum than did those isolated from lysogenic strains. In accordance with this, the phages were found in different clusters within a computer-generated linkage map. Species specificity and serovar specificity of the lytic reaction were not found. None of the phages was able to lyse strains of Listeria grayi, Listeria murrayi or Jonesia denitrificans. This phage typing system may provide important information for a means of recognizing and eliminating sources of contamination by Listeria spp. within dairy plant equipment.