On the Nature of nontypable Haemophilus influenzae

193 Haemophilus cultures, including 71 nontypable H. influenzae isolates, were examined with respect to phage HP1 sensitivity, lysogeny for this and for other phages and for excretion of bacteriocins. Fifty of the 71 nontypable cultures were sensitive to phage HP1 but only three produced plaques. The other 47 isolates were thus probably not non-encapsulated derivatives of H. influenzae serotypes a, b, d, and e, which have discrete and characteristic phage HP1 restriction and modification systems, or serotype c which appears to be restriction negative. They could be derivatives of serotype f which does not give plaques with phage HP1. The nontypable three cultures that plated phage HP1 efficiently could be non-encapsulated serotype c derivatives. Fourteen of the phage HP1 insentitive nontypable cultures were found to be defectively lysogenic for this phage. Five of these were genetically transformed to wild type lysogens. Their phage produced plaques efficiently only on Rc strains and on a restriction-negative mutant of serotype d. These lysogenic nontypable isolates are thus modification (and restriction) negative and they are thus probably not nonencapsulated derivatives of serotypes a, b, d, e, or f. Fifty three of 56 serotype b cultures were found to excrete a bacteriocin, to which all other nonproducing Haemophilus cultures were more or less sensitive. The three restriction-negative nontypable H. influenzae cultures also excreted this bacteriocin but the other cultures listed did not do this. The tentative conclusion from this study is that nontypable H. influenzae isolates are probably not derivatives of the six known encapsulated strains.     

Human lactoferrin receptor activity in non-encapsulated Haemophilus influenzae

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.     

Relation between Px phages and generalised transducing particles of phage P22

Summary P22 mutants with an altered ability to form generalised transducing particles were tested for their ability to form the hybrid Px phages during growth on Py-lysogenic strains of Salmonella typhimurium. The mutant which produces more (less) transducing particles produces also more (less) Px phages. It is suggested that Px particles might be regarded as self-replicating generalised transducing particles.     

Studies on Temperate Phages of Lactobacillus salivarius

Twenty-three temperate phages of Lactobacillus salivarius isolated from human feces were studied as to their morphological, biological, and serological properties. (1) Among 30 strains of L. salivarius tested, 23 strains were lysed by induction with mitomycin C (MC). In all these lysates, phage particles were detected by electron microscopic examination. (2) These phages were morphologically divided into three groups: particles with a regular hexagonal head and a long flexible tail; particles having a regular hexagonal head with or without a short tail-like structure; particles with an elongated head and a long noncontractile tail. (3) Only two, phage 223 having an elongated head and phage 227 with a regular hexagonal head and a long noncontractile tail, produced tiny and very turbid plaques on several host bacteria. Six phages could produce only inhibition zones, ranging from complete inhibition through partial inhibition to normal growth by a serial dilution spot test. (4) All these killer particles could also inhibit the growth of their producer cells. (5) A serological relationship was observed between temperate phages and killer particles, and this was somewhat consistent with the morphological groupings.     

Induction of competence in nonencapsulated and encapsulated strains ofHaemophilus influenzae

A simple procedure for induction of competence in nonencapsulated and encapsulated strains ofHaemophilus influenzae is described, which consists of growing cells without shaking in brain-heart infusion broth under aerobic conditions. Competence emerged at the end of the exponential phase and reached a peak at the stationary phase. InH. influenzae Rd competence was maintained for at least 6 h at 37°C, whereas in two encapsulated clinical isolates ofH. influenzae type b a decrease in competence was observed after 4 h. Competence was maintained for 24 h at 22°C and 4°C as well as by freezing the cells in 15% glycerol and storing them at –70°C. Transformation frequencies of three chromosomal markers—streptomycin, nalidixic acid, and erythromycin resistance—were 0.5% to 1% inH. influenzae Rd and about tenfold lower in the two encapsulated clinical isolates ofH. influenzae type b. The advantage of this procedure is that it is simpler than the previously described procedures and yields stable, highly transformable cells. Unlike the standard M IV method, the static aerobic procedure does not interfere with the capsule synthesis and can be used for testing transforming activity of encapsulated virulent isolates ofH. influenzae.     

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

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

Prevalence of <Emphasis Type="Italic">H. influenzae</Emphasis> type b &; f among unvaccinated patients with respiratory tract infection and meningitis

Haemophilus influenzae type b meningitis has been reported predominantly among infants who have not completed the primary immunization series, whereas infection by capsular f has been reported mostly in patients with significant underlying diseases. The present study analyses H. influenzae type b & f infection among 25 unvaccinated patients with respiratory tract infections and symptoms of meningitis by PCR. 5/7 isolates from CSF sample were capsular type b; whereas capsular type f was detected in 2/10 and 5/8 isolates from throat swab and sputum samples, respectively. Rest 13 strains were negative for both the capsular types. To conclude, in India where H. influenzae type b conjugate vaccine is not included in national immunization program, individuals with respiratory tract infections and meningitis are at a greater risk of developing infection with H. influenzae type b strains along with other capsular types and non-capsular types.     

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and Podoviridae.     

The occurrence and taxonomic value of PBS X-like defective phages in the genus Bacillus

72 strains of 24 Bacillus species were induced with mitomycin C. The lysates were examined for the presence of defective phages resembling PBS X in morphology. All strains tested of B. amyloliquefaciens. B, licheniformis, B. pumilus and B. subtilis contained such phages. Five morphological types of defective, PBS X-like phage could be distinguished, differing in their tail lengths and in the number of cross-striations on the tail. The quaternary structure of the tail, the molecular weight of the main tail protein and the antigenic properties of the phages were identical. The killing ranges of the defective phages have been determined and their possible use in taxonomy discussed.     

In vivo morphogenesis and growth characteristics of phages CM (Myoviridae) virulent forRhizobium meliloti

FourRhizobium meliloti bacteriophages belonging to theMyoviridae family of tailed phages were studied. Burst sizes (50–100 virulent particles per infected cell), adsorption rates (2.6–4.1×10^–9 ml/min), and latent periods (2–4 h) made this group heterogeneous. However, these characteristics indicate an important infection ability compared with other rhizobiophages. In vivo morphogenesis of phage CM1, studied by electron microscopy, seems to have steps similar to those of some other tailed phages such as coliphages.     

Antigenic relationship betweenHaemophilus influenzae andHaemophilus isolated from porcine pleuropneumonia

Comparative double immunodiffusion techniques were used to study capsular and O antigenic relationships betweenHaemophilus influenzae types a-fandH. pleuropneumoniae types 1–5 and a strain (202) closely related toH. pleuropneumoniae. Culture fluids or culture supernatants were used as antigens and rabbit antisera were produced against cell suspensions of the strains tested. A reaction of identity was obtained between the capsular precipitate ofH. influenzae c and a precipitate formed by strain 202, when developed with anti-H. influenzae c serum or the serum produced against strain 202. Mutual cross-absorption of capsular antibodies was also demonstrable. No other capsular or O antigenic cross-reactivity was demonstrable between the strains tested.     

New shuttle vectors for Haemophilus influenzae and Escherichia coli: P15A-derived plasmids replicate in H. influenzae Rd

With the aim of identifying new plasmids useful for molecular cloning in Haemophilus influenzae, several P15A-derived plasmids were tested for their ability to transform H. influenzae Rd. The four plasmids tested, pACYC177, pACYC184, pSU2718, and pSU2719 were all able to establish in H. influenzae Rd. The plasmids were stable, could be purified by standard protocols, and were compatible with a plasmid carrying the RSF0885 origin of replication.     

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.     

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.     

牛源无乳链球菌长尾噬菌体的特性

【目的】分离鉴定噬菌体,对其生物学特性进行研究,并筛选候选毒株为防控牛源无乳链球菌的感染提供依据。【方法】分别采用从牛奶或环境中分离、溶原菌诱导两种方法分离鉴定无乳链球菌噬菌体,利用双层琼脂平板法纯化。将新分离鉴定毒株与前期已分离鉴定的源自乳腺炎牛奶的无乳链球菌噬菌体JX01进行分析和比较,包括噬菌体透射电镜形态观察、对55株无乳链球菌和其他细菌的宿主谱鉴定、噬菌体基因Eco R I、Sal I、Xba I或Pst I的酶切图谱、最适MOI、吸附曲线和一步生长曲线、不同保存条件下的稳定性等。【结果】分离鉴定的3株噬菌体LYGO9、HZ04和p A11(诱导自牛源菌株HAJL2011070601)与JX01比对分析,结果显示,4株噬菌体均为长尾噬菌体;Eco R I、Sal I、Xba I、Pst I的酶切图谱分获4、3、3或2种带型,显示4株噬菌体为不同毒株;均特异性裂解牛源无乳链球菌,对42株牛源无乳链球菌的裂解率如下:LYGO9为28.6%(12/42)、p A11为31%(13/42)、HZ04为47.6%(20/42)、JX01为54.8%(23/42);同时,LYGO9与p A11、HZ04和JX01分别有共同宿主11、12和11株;HZ04与JX01有共同宿主18株,提示它们具有同源性。LYGO9感染宿主的潜伏期短,仅5 min,平均裂解量为30。分离株在SM液中4°C至少可保存1个月。【结论】分离鉴定的3株牛源无乳链球菌噬菌体均为长尾噬菌体,其中LYGO9潜伏期短、裂解量较大。     

Bacillus polymyxa bacteriophages from Brazilian soils

Ten phages of Bacillus polymyxa were isolated from four different Brazilian soils. All were dsDNA-containing phages belonging to Bradley types A and B. Data obtained from electron microscopy and tests of resistance against physical and chemical agents showed that the isolates could be distributed among six different groups. Host range data were in agreement with this classification. When tested against 88 strains of 18 Bacillus species, these phages only infected B. polymyxa strains, thus revealing specificity for this species. Three phage groups lysed all 42 available B. polymyxa strains and are suggested for use in rapid identification of this species.This work was sponsored by the National Research Council of Brasil (CNPq) and CAPES.     

Bacteriophages of Erwinia amylovora

Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario. Forty-two phages survived the isolation, purification, and storage processes. The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms. Only five phages were isolated from infected aerial tissue in pear and apple orchards. To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes. Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered. Ten phage isolates were related to the previously characterized E. amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites. A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E. amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans. Representatives from the six molecular groups were studied by electron microscopy to determine their morphology. The phages exhibited distinct morphologies when examined by an electron microscope. Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages. Based on morphotypes, the bacteriophages of E. amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:     

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.     

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.