Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures.

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inducible bacteriophages of Actinobacillus actinomycetemcomitans

Doses of 0.1 to 1.0 g/ml of mitomycin C induced cell lysis of six of eight strains of Actinobacillus actinomycetemcomitans tested. Infectious phages were induced from ATCC strains 43717, 29524, 33384, and 43719; non-plaque-forming, possibly defective phages were induced from ATCC strains 29522 and 29523. No phages were detected in strain FDC 651 or ATCC strain 43718. No correlation between lysogeny and leukotoxin production or serotype of the strains could be established. Gel electrophoresis of phage DNAs indicated that the induced phages were of three types, based on size. By electron microscopy, the phages were found to belong to either morphotype A1 or morphotype B1; no other morphotypes were observed. Curing experiments led to the isolation of nonlysogenic derivatives of two strains, which supported plaque formation by the phages they originally carried. On the basis of our results, lysogeny appears to be widespread in A. actinomycetemcomitans.     

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.     

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.     

Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines

Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery. Bacteriophages were not found in samples of 16 other wines. The morphology of the phages was examined by electron microscopy. The phages did not lyse all strains of L. oenos, and susceptibility correlated to some extent with the colony morphology of the strain. Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite. Phage did not affect the growth of a sensitive strain of L. oenos in filter-sterilized wine.     

Occurrence and properties of bacteriophages of Leuconostoc oenos in Australian wines.

Bacteriophages specific for Leuconostoc oenos were isolated from four red wines undergoing malolactic fermentation in one winery. Bacteriophages were not found in samples of 16 other wines. The morphology of the phages was examined by electron microscopy. The phages did not lyse all strains of L. oenos, and susceptibility correlated to some extent with the colony morphology of the strain. Phage survived in wines at pH values greater than 3.5 but was inactivated in wines of lower pH and by the addition of sulfur dioxide or bentonite. Phage did not affect the growth of a sensitive strain of L. oenos in filter-sterilized wine.     

Isolation and Partial Characterization of Two Aeromonas hydrophila Bacteriophages

Two Aeromonas hydrophila bacteriophages, Aeh1 and Aeh2, were isolated from sewage. Both phages showed binal symmetry. The dimensions of A. hydrophila phages Aeh1 and Aeh2 differed from those of the other Aeromonas phages. Also, phage Aeh2 was the largest Aeromonas phage studied to date. Phage Aeh1 formed small, clear plaques, and phage Aeh2 formed turbid plaques with clear centers. Both phages were sensitive to chloroform treatment, being totally inactivated after treatment for 1 h at 60°C at pH 3 and 11. However, the infectivity of Aeh1 phage stocks increased by approximately fivefold after they were treated at pH 10 for 1 h at 22°C. Phages Aeh1 and Aeh2 were serologically unrelated and had latent periods of 39 and 52 min, respectively. The average burst sizes of phages Aeh1 and Aeh2 were 17 and 92 PFU per cell, respectively. Phage Aeh1 infected 13 of 22 A. hydrophila strains tested, whereas phage Aeh2 infected only its original host. Phage Aeh1 infected some A. hydrophila strains only at or below 37°C. Neither phage infected the two A. (Plesiomonas) shigelloides strains used in this study.     

Bacteriophage types of Salmonella enteritidis occurring in Poland in 1986-1995

The Lalko phages collection was used to phage type a total of 517 Salmonella Enteritidis strains isolated from food-poisoning outbreaks (312 strains) and other common sources (205 strains) in Poland, during the years 1986-1995. Above 99.0% of all strains tested were recognized as belonging to definitive phage type. Phage types 1, 6 and 7 were predominant. The strains of type 1 and 7 were most numerous. Of the 517 examined strains 312 were isolated from 46 food-poisoning outbreaks. Most of them came from the one phage type outbreaks; 8 mixed outbreaks were noted. The greatest number of the food-poisoning outbreaks was caused by Salmonella Enteritidis phage types 1, 6 and 7. Phage type 16 was isolated from persons for the first time.     

Temperate phages of Clostridium perfringens type C1.

Four phages isolated from carrier strains of Clostridium perfringens type C belong to two classes. The three phages of class I, c1, c3, and c4, and homoimmune and serologically closely related. The phage of class II, c5, is heteroimmune to the class I phages and not related to them serologically. Transduction experiments with several of the phages were negative. Mutants of the indicator strain with surface alterations occurred spontaneously in stock cultures. Electron micrographs show the phages of each class to be distinct yet similar, having polyhedral heads of about the same diameter 55 nm, and long, flexible tails without sheaths or collars. Phages c4 and c5 were characterized for their lysogenic properties. Phage c4 was inducible with mitomycin C. Both c4 and c5 were temperate viruses by the test of stability of their respective lysogens to phage-specific antisera.     

Differentiation of methicillin-resistant strains of Staphylococcus aureus by prophage specificity]

By inducing with mitomycin C the following phages were isolated from all the tested 32 methicillin resistant strains of S. aureus: the serogroup B phage was isolated from 2 strains, the serogroup B and F phages were isolated from 5 strains and the serogroup F phage was isolated from 25 strains. The phages were divided into 5 groups by the antiphage immunity. In group 1 of the phages 4 additional phages were specified. By the specificity of the prophages in the cultures all the strains were divided into 5 groups. Group 1 of the cultures was divided into 5 subgroups (A, B, C, D and E).     

Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli

Aims: The objective of this study was to isolate, identify and characterize a collection of lytic bacteriophages capable of infecting enterohaemorrhagic Escherichia coli (EHEC) serotypes. Methods and Results: Phages were isolated from dairy and cattle feedlot manure using E. coli O157, O26 and O111 strains as hosts. Phages were enriched from faecal slurries by culture in 10× trypticase soy broth at 37°C overnight. Phage plaques were obtained by mixing the filtered culture supernatant with molten tryptone agar containing the phage E. coli host strain, pouring the inoculated agar on top of cooled TS agar and incubating the culture overnight. Phages were purified from plaques and screened against additional E. coli and EHEC strains by the efficiency of plating method (EOP). Phage CEV2, and five other phages previously isolated, were able to lyse all of the 15 O157 strains tested with EOP values consistently above 0·001. Two phages were found to be highly effective against strains of E. coli O157 through EOP tests and against O26 strains through spot tests, but not against the O serogroup 111 strains. A cocktail of eight phage that lyse E. coli O157 strains resulted in >5 log CFU ml^?1 reductions at 37°C. Multiplex‐PCR revealed that none of these eight phages carried stx1, stx2, hlyA or eaeA genes. Conclusions: A cocktail of bacteriophages was capable of lysing most strains of two EHEC serotypes. Significance and Impact of the Study: This collection of phages can be combined and potentially used as an antimicrobial cocktail to inactivate E. coli strains from O serogroups 157 and 26 and reduce their incidence in the food chain.     

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.     

Characterisation of major biological properties of Bordetella bacteriophages

The main biological properties (morphology of negative colonies, parameters of adsorption and single development cycle) of B. pertussis and B. bronchiseptica phages, isolated spontaneously and by induction with mitomycin C, were studied. To compare these characteristics, one B. parapertussis indicator strain was used, and the experiments were carried out under identical conditions. Highly active sera were obtained with the use of complete Freund's adjuvant. B. pertussis phages isolated from the strains of different serovars were serologically related, but not identical, and differed in their constant characterizing their rate of neutralization with homologous antisera. The adsorption of the phages on homologous strains was more intensive than on the cells of B. parapertussis indicator strain. However, the authors failed to observe the further development of the phages in the host cells.     

Multiple-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains

We used multilocus sequence typing (MLST) to characterize phylogenetic relationships for a collection of Bacillus cereus group strains isolated from forest soil in the Paris area during a mild winter. This collection contains multiple strains isolated from the same soil sample and strains isolated from samples from different sites. We characterized 115 strains of this collection and 19 other strains based on the sequences of the clpC, dinB, gdpD, panC, purF, and yhfL loci. The number of alleles ranged from 36 to 53, and a total of 93 allelic profiles or sequence types were distinguished. We identified three major strain clusters-C, T, and W-based on the comparison of individual gene sequences or concatenated sequences. Some less representative clusters and subclusters were also distinguished. Analysis of the MLST data using the concept of clonal complexes led to the identification of two, five, and three such groups in clusters C, T, and W, respectively. Some of the forest isolates were closely related to independently isolated psychrotrophic strains. Systematic testing of the strains of this collection showed that almost all the strains that were able to grow at a low temperature (6 degrees C) belonged to cluster W. Most of these strains, including three independently isolated strains, belong to two clonal complexes and are therefore very closely related genetically. These clonal complexes represent strains corresponding to the previously identified species Bacillus weihenstephanensis. Most of the other strains of our collection, including some from the W cluster, are not psychrotrophic. B. weihenstephanensis (cluster W) strains appear to comprise an effectively sexual population, whereas Bacillus thuringiensis (cluster T) and B. cereus (cluster C) have clonal population structures.     

Regulation of the glucose phosphotransferase system in Brochothrix thermosphacta by membrane energization.

Uptake of 2-deoxyglucose, alpha-methylglucopyranoside, and glucose into intact cells of Brochothrix thermosphacta (formerly Microbacterium thermosphactum, ATCC 11509) was stimulated by KCN or CCCP. The glucose analogs were recovered almost totally as the sugar phosphates. Membrane vesicles were isolated from protoplasts and shown to be right side out by freeze fracturing and by using ATPase as a marker for the cytoplasmic membrane surface. Uptake of glucose into vesicles was dependent on the presence of phosphoenolpyruvate. NADH oxidation, K+ -diffusion gradients, and externally directed lactate gradients (pH greater than 7 initially) were used to generate transmembrane potentials across membrane vesicles. Above a threshold value of about -50 mV, uptake of glucose into membrane vesicles was reduced. Likewise, the maximum uptake of glucose and its two analogs into cells occurred when the protonmotive force was less than about -50 mV.     

阪崎肠杆菌噬菌体的分离及其生物学特性

[目的]以阪崎肠杆菌模式菌株及分离菌株为指示菌,从污水中分离出该菌噬菌体,并对其基本生物学特性进行研究.[方法]以双层琼脂法从污水中分离噬菌体,通过同属和同科参考菌株测定噬菌体的特异性和宿主谱;电镜观察噬菌体颗粒形态;随机扩增多态性DNA(RAPD)实验分析噬菌体的分子生物学特性.[结果]从污水中分离得到5株噬菌体,表现出较窄的宿主范围,仅裂解阪崎肠杆菌,以ATCC 51329分离的噬菌体SK2可裂解27株阪崎肠杆菌中的24株(89%),负染经电镜观察,5株噬菌体都是由多面体头部和尾部组成;随机引物(5′-GAAACGGGTG-3′)扩增DNA分析,5株噬菌体DNA明显不同.[结论]分离出的5株噬菌体仅对阪崎肠杆菌敏感,在阪崎肠杆菌的分型、预防、治疗、以及生态环境的净化等方面具有潜在用途.     

驯化噬菌体提高噬菌体对碳青霉烯类耐药肺炎克雷伯菌的杀菌能力

【目的】本研究旨在通过驯化提高噬菌体的裂解能力并降低其宿主菌耐受性产生的速度,从而提高对重要病原菌-碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae, CRKp)的杀菌效果。【方法】以临床CRKp菌株Kp2092为宿主菌,利用双层琼脂平板法从污水中分离噬菌体并分析其裂解谱;对其中的广谱强裂解性噬菌体通过透射电镜观察其形态特征并进行全基因组测序;通过噬菌体-宿主连续培养进行噬菌体驯化,并比较驯化前后噬菌体生物学特性的差异。【结果】分离得到的9株肺炎克雷伯菌噬菌体中,噬菌体P55anc裂解能力强且裂解谱广,透射电镜观察发现其为短尾噬菌体。P55anc基因组全长40 301 bp,包含51个编码序列,其中27个具有已知功能,主要涉及核酸代谢、噬菌体结构蛋白、DNA包装和细胞裂解等。噬菌体P55anc经9 d的驯化后,得到3株驯化噬菌体。驯化后噬菌体杀菌能力增强,主要表现为细菌生长曲线显著下降、噬菌体暴发量增多、裂解谱扩大,且宿主菌对其产生抗性的概率显著降低。与此同时,驯化后的噬菌体在热处理、紫外暴露以及血清等环境下保持较好的稳定性。【结论】利用噬菌体-宿主连续培养的方法可对噬菌体进行驯化和筛选,驯化后的噬菌体杀菌效果更强,且在不同压力处理下的稳定性良好,而细菌产生噬菌体抗性的概率也降低。     

Phage typing of indigenous soybean-rhizobia and relationship of a phage group strains for their asymbiotic and symbiotic nitrogen fixation

A total of 354 indigenous bradyrhizobia were isolated from soybean nodules collected from five major crop grown regions. Host-specific 12 phages, each active on particular strains were selected. Factors, which influence the interaction between the host and phage, were examined. Four different types of plaques were detected. Nearly 17% of isolates were found resistant to all phages. Phage sensitivity patterns revealed a total of 32 distinct phage genotype groups. Different set of phage combinations expressed variation in specificity for parasitizing against particular group of rhizobia. Distributions of isolates in each phage types differed markedly between regions. Interestingly, nine strains belonging to phage group 16 exhibited high ex planta nitrogenase activity in culture. However, no correlation could be established between high ex planta nitrogenase activity and their symbiotic effectiveness with soybean cultivars. Soybean cv. JS335 showed relatively superior performance than Bragg and Lee with indigenous bradyrhizobial strains. Phage typing revealed the existence of large genetic diversity among native rhizobia and selection of the superior bradyrhizobial strains can also be possible for a given soil-climate-cultivar complex.