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Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome. ispartof: NATURE MICROBIOLOGY vol:4 issue:10 pages:1727-1736 ispartof: location:England status: published

Bacterial motility is associated to a wide range of biological processes and it plays a key role in the virulence of many pathogens. Here we describe a method to distinguish the dynamic properties of bacteria by analyzing the statistical functions derived from the trajectories of a bacterium trapped by a single optical beam. The approach is based on the model of the rotation of a solid optically trapped sphere. The technique is easily implemented in a biological laboratory, since with only a small number of optical and electronic components a simple biological microscope can be converted into the required analyzer. To illustrate the functionality of this method, we probed several Salmonella enterica serovar Typhimurium mutants that differed from the wild-type with respect to their swimming patterns. In a further application, the motility dynamics of the S. Typhimurium cheV mutant were characterized.

El ferro és vital per a gairebé tots els organismes vius, atès el paper crucial que té en nombrosos processos biològics. Però, en concentracions inadequades, aquest element és un verí per a qualsevol cèl·lula. Per això, els éssers vius disposen de mecanismes que en controlen la concentració. Així, els vertebrats tenen sistemes que segresten ferro per evitar-ne l'efecte deleteri. A més, el segrest té un altre efecte positiu per a aquests animals, ja que el ferro no estarà disponible per al desenvolupament dels bacteris patògens que els puguin infectar. Com a resposta, els patògens bacterians han desenvolupat diverses estratègies de captura del ferro segrestat.

Non-typhoid Salmonella is the principal pathogen related to food-borne diseases throughout the world. Widespread antibiotic resistance has adversely affected human health and has encouraged the search for alternative antimicrobial agents. The advances in bacteriophage therapy highlight their use in controlling a broad spectrum of food-borne pathogens. One requirement for the use of bacteriophages as antibacterials is the characterization of their genomes. In this work, complete genome sequencing and molecular analyses were carried out for three new virulent Salmonella-specific bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) able to infect a broad range of Salmonella strains. Sequence analysis of the genomes of UAB_Phi20, UAB_Phi78, and UAB_Phi87 bacteriophages did not evidence the presence of known virulence-associated and antibiotic resistance genes, and potential immunoreactive food allergens. The UAB_Phi20 genome comprised 41,809 base pairs with 80 open reading frames (ORFs); 24 of them with assigned function. Genome sequence showed a high homology of UAB_Phi20 with Salmonella bacteriophage P22 and other P22likeviruses genus of the Podoviridae family, including ST64T and ST104. The DNA of UAB_Phi78 contained 44,110 bp including direct terminal repeats (DTR) of 179 bp and 58 putative ORFs were predicted and 20 were assigned function. This bacteriophage was assigned to the SP6likeviruses genus of the Podoviridae family based on its high similarity not only with SP6 but also with the K1-5, K1E, and K1F bacteriophages, all of which infect Escherichia coli. The UAB_Phi87 genome sequence consisted of 87,669 bp with terminal direct repeats of 608 bp; although 148 ORFs were identified, putative functions could be assigned to only 29 of them. Sequence comparisons revealed the mosaic structure of UAB_Phi87 and its high similarity with bacteriophages Felix O1 and wV8 of E. coli with respect to genetic content and functional organization. Phylogenetic analysis of large terminase subunits confirms their packaging strategies and grouping to the different phage genus type. All these studies are necessary for the development and the use of an efficient cocktail with commercial applications in bacteriophage therapy against Salmonella.

Trimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydropteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here, we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim-resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen Acinetobacter baumannii , and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologues in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. Hence, this work confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants.

2.AbstractTrimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydro-pteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim resistance genes (dfrA). We show that most of the dfrA genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance (sul) genes, we find evidence of recurrent mobilization in dfrA genes. Phylogenetic evidence allows us to identify novel dfrA genes in the emerging pathogen Acinetobacter baumannii, and we confirm their resistance phenotype in vitro. We also identify a cluster of dfrA homologs in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported dfrA genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. This work hence confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants.3.Impact statementAntibiotic resistance is a pressing and global phenomenon. It is well-established that resistance to conventional antibiotics emerged millions of years ago in either antibiotic-producing bacteria or their competitors. Resistance to synthetic chemotherapeutic agents cannot be explained by this paradigm, since these drugs are not naturally produced. Resistance is hence assumed to have evolved rapidly following the clinical introduction of these drugs. Recently we showed that resistance to one such drug, sulfonamide, evolved not recently, but millions of years ago, suggesting that the diversity of bacterial genomes may well contain genes conferring resistance to drugs yet to be developed. Here we analyze the origin of resistance to trimethoprim, another chemotherapeutic agent developed in the 1960’s. Using phylogenetic methods, we identify new variants of the trimethoprim resistance genes that had not previously been reported, and we trace the chromosomal origins for a number of already known resistance variants. Our results show that resistance to trimethoprim is very diverse and has originated both from recent mutations and from preexisting ancient variants. These results stress the importance of gene mobilization mechanisms as the main drivers of the current antibiotic resistance phenomenon.4.Data summary- The scripts used for data collection and analysis can be obtained at the GitHub ErillLab repository (https://github.com/ErillLab/).- The Bayesian phylogenetic tree can be visualitzed online on iTOL (https://itol.embl.de/tree/855674159451585133078) [1].- The authors confirm all other supporting data has been provided within the article or through supplementary data files.

This study sheds light on the biodistribution of orally administered, liposome-encapsulated bacteriophages, and their transcytosis through intestinal cell layers. Fluorochrome-labeled bacteriophages were used together with a non-invasive imaging methodology in the in vivo visualization of bacteriophages in the stomach and intestinal tract of mice. In those studies, phage encapsulation resulted in a significant increase of the labeled phages in the mouse stomach, even 6 h after their oral administration, and without a decrease in their concentration. By contrast, the visualization of encapsulated and non-encapsulated phages in the intestine were similar. Our in vivo observations were corroborated by culture methods and ex vivo experiments, which also showed that the percentage of encapsulated phages in the stomach remained constant (50%) compared to the amount of initially administered product. However, the use of conventional microbiological methods, which employ bile salts to break down liposomes, prevented the detection of encapsulated phages in the intestine. The ex vivo data showed a higher concentration of non-encapsulated than encapsulated phages in liver, kidney, and even muscle up to 6 h post-administration. Encapsulated bacteriophages were able to reach the liver, spleen, and muscle, with values of 38% ± 6.3%, 68% ± 8.6%, and 47% ± 7.4%, respectively, which persisted over the course of the experiment. Confocal laser scanning microscopy of an in vitro co-culture of human Caco-2/HT29/Raji-B cells revealed that Vybrant-Dil-stained liposomes containing labeled bacteriophages were preferably embedded in cell membranes. No transcytosis of encapsulated phages was detected in this in vitro model, whereas SYBR-gold-labeled non-encapsulated bacteriophages were able to cross the membrane. Our work demonstrates the prolonged persistence of liposome-encapsulated phages in the stomach and their adherence to the intestinal membrane. These observations could explain the greater long-term efficacy of phage therapy using liposome-encapsulated phages., This work was supported by the Ministerio de Economía, Industria y Competitividad in Spain grant BIO2016–77011-R. JO and AG-R received predoctoral fellowships from the UAB. ICN2 is supported by the Severo Ochoa program from Spanish MINECO (Grant No. SEV-2017-0706).

Es presenta l’experiencia de la implementacio del projecte MicroMon a Catalunya. Es tracta d’una proposta de proveiment participatiu (crowdsourcing) que adopta una estrategia d’aprenentatge servei (ApS) que apropa la cultura cientifica i fomenta la vocacio investigadora d’alumnes de 4t d’ESO i 1r de Batxillerat, alhora que contribueix a millorar la informacio i la consciencia ciutadana envers a la greu amenaca que representa, per a la salut publica mundial, la manca creixent d’antibiotics efectius pel tractament de malalties infeccioses. En el projecte, estudiants de diferents graus del ambit de les Biociencies i de la Salut, acompanyats per professorat universitari especialista en Microbiologia, porten a les aules d’instituts i escoles un projecte de recerca real en el que es recullen i es sembren mostres de sol en condicions esterils per cercar, dins la diversitat bacteriana de la mostra, bacteris productors d'antibiotics. En el present article s’analitzen tambe les respostes a questionaris dels diferents actors involucrats en el projecte aixi com les observacions realitzades durant les sessions de treball per avaluar el funcionament d‘aquesta prova pilot.

Encapsulation methodologies allow the protection of bacteriophages for overcoming critical environmental conditions. Moreover, they improve the stability and the controlled delivery of bacteriophages which is of great innovative value in bacteriophage therapy. Here, two different encapsulation methodologies of bacteriophages are described using two biocompatible materials: a lipid cationic mixture and a combination of alginate with the antacid CaCO

Encapsulation methodologies allow the protection of bacteriophages for overcoming critical environmental conditions. Moreover, they improve the stability and the controlled delivery of bacteriophages which is of great innovative value in bacteriophage therapy. Here, two different encapsulation methodologies of bacteriophages are described using two biocompatible materials: a lipid cationic mixture and a combination of alginate with the antacid CaCO3. To perform bacteriophage encapsulation, a purified lysate highly concentrated (around 1010-1011 pfu/mL) is necessary, and to dispose of a specific equipment. Both methodologies have been successfully applied for encapsulating Salmonella bacteriophages with different morphologies. Also, the material employed does not modify the antibacterial action of bacteriophages. Moreover, both technologies can also be adapted to any bacteriophage and possibly to any delivery route for bacteriophage therapy.

The diversity of 55 Salmonella-specific bacteriophages isolated from 191 fecal samples of poultry and swine from farms located in diverse geographic areas of Spain was determined using lysis profiling, DNA restriction and random amplification of polymorphic DNA (RAPD-PCR). Among them, lysis profiling and RAPD-PCR exhibited 100% typeability and DNA restriction 96%, with discriminatory power of 0.978 (± 0.016), 0.938 (± 0.028) and 0.982 (± 0.013), respectively. The highest concordance (0.974) was that between RAPD-PCR and lysis profiling. None of the bacteriophages isolated from poultry and swine shared any DNA restriction or RAPD-PCR patterns and only two lysis profiles were common to bacteriophages isolated from poultry and swine. The major part of the lysis and RAPD-PCR profiles from the bacteriophages isolated from poultry included only one or two bacteriophages, while those obtained from swine contained more than two bacteriophages. Overall, our results provide evidence of the remarkable diversity exhibited by bacteriophages of Salmonella in farm animals. Moreover, they also show that RAPD-PCR may also be suitable for the pre-screening of the diversity of Salmonella bacteriophages for further use in biocontrol and therapeutic strategies.

Bacteriophages are promising therapeutic agents that can be applied to different stages of the commercial food chain. In this sense, bacteriophages can be orally administered to farm animals to protect them against intestinal pathogens. However, the low pH of the stomach, the activities of bile and intestinal tract enzymes limit the efficacy of the phages. This study demonstrates the utility of an alginate/CaCO encapsulation method suitable for bacteriophages with different morphologies and to yield encapsulation efficacies of ∼100%. For the first time, a cocktail of three alginate/CaCO-encapsulated bacteriophages was administered as oral therapy to commercial broilers infected with Salmonella under farm-like conditions. Encapsulation protects the bacteriophages against their destruction by the gastric juice. Phage release from capsules incubated in simulated intestinal fluid was also demonstrated, whereas encapsulation ensured sufficient intestinal retention of the phages. Moreover, the small size of the capsules (125-150 μm) enables their use in oral therapy and other applications in phage therapy. This study evidenced that a cocktail of the three alginate/CaCO-encapsulated bacteriophages had a greater and more durable efficacy than a cocktail of the corresponding non-encapsulated phages in as therapy in broilers against Salmonella, one of the most common foodborne pathogen., This work was supported by grants from La Caixa and the Associació Catalana d’Universitats Públiques (2010ACUP00300), AGAUR-ACCIÓ-Generalitat de Catalunya (2010VALOR00114), AGAUR-Generalitat de Catalunya (2014SGR572) and of the Spanish MINECO (BIO2016–77011-R). ICN2 acknowledges the support of the Spanish MINECO through the Severo Ochoa Centres of Excellence Programme, under Grant SEV-2013-0295. We are grateful to the Servei de Granges i Camps Experimentals and the Servei de Microscòpia of the Universitat Autònoma de Barcelona (UAB) for their support. J.C. and J.O. received predoctoral fellowships from the UAB.

Rhenium complexes with the antibiotic ciprofloxacin have been prepared to be studied as models of technetium radiopharmaceuticals. With this aim, the new rhenium complexes 1 {[ReO(Cpf)2]Cl}, 2 {[ReO(CpfH)2]Cl3} and 3 {fac-[Re(CO)3(H2O)(Cpf)]} with ciprofloxacin (CpfH=ciprofloxacin; Cpf=conjugated base of ciprofloxacin) have been synthesised and characterised by elemental analyses, IR, NMR ((1)H, (19)F and (13)C CP-MAS) spectroscopy, as well as MS measurements. All spectroscopic data are consistent with the coordination of ciprofloxacin in all these complexes through the carbonyl and the carboxylate oxygen atoms with the formation of a six member chelate ring. The study of a Tc-ciprofloxacin solution by ESI-MS reveals the presence of [TcO(Cpf)2](+) cations, which agrees with the hypothesis that complexes 1 and 2 can be seen as model rhenium complexes of this radiopharmaceutical. Antimicrobial and DNA gyrase inhibition studies performed with complexes 2 and 3 have shown a very similar behaviour between complex 2 and the free antibiotic, whereas complex 3 exhibit a lower antimicrobial activity. Based on a joint analysis of the data reported in the literature and the chemical and biological results obtained in this study, a tentative proposal to explain some aspects of the behaviour of Tc-ciprofloxacin radiopharmaceutical has been made.

The use of lytic bacteriophages for the biocontrol of food-borne pathogens in food and in the food industry is gaining increasing acceptance. In this study, the effectiveness of a bacteriophage cocktail composed of three different lytic bacteriophages (UAB_Phi 20, UAB_Phi78, and UAB_Phi87) was determined in four different food matrices (pig skin, chicken breasts, fresh eggs, and packaged lettuce) experimentally contaminated with Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis. A significant bacterial reduction (> 4 and 2 log/cm 2 for S . Typhimurium and S . Enteritidis, respectively; p ≤ 0.005) was obtained in pig skin sprayed with the bacteriophage cocktail and then incubated at 33 °C for 6 h. Significant decreases in the concentration of S . Typhimurium and S . Enteritidis were also measured in chicken breasts dipped for 5 min in a solution containing the bacteriophage cocktail and then refrigerated at 4 °C for 7 days (2.2 and 0.9 log 10 cfu/g, respectively; p ≤ 0.0001) as well as in lettuce similarly treated for 60 min at room temperature (3.9 and 2.2 log 10 cfu/g, respectively; p ≤ 0.005). However, only a minor reduction of the bacterial concentration (0.9 log 10 cfu/cm 2 of S . Enteritidis and S . Typhimurium; p ≤ 0.005) was achieved in fresh eggs sprayed with the bacteriophage cocktail and then incubated at 25 °C for 2 h. These results show the potential effectiveness of this bacteriophage cocktail as a biocontrol agent of Salmonella in several food matrices under conditions similar to those used in their production.

This paper addresses the use of bacteriophages immobilized on magnetic particles for the biorecognition of the pathogenic bacteria, followed by electrochemical magneto-genosensing of the bacteria. The P22 bacteriophage specific to Salmonella (serotypes A, B, and D1) is used as a model. The bacteria are captured and preconcentrated by the bacteriophage-modified magnetic particles through the host interaction with high specificity and efficiency. DNA amplification of the captured bacteria is then performed by double-tagging polymerase chain reaction (PCR). Further detection of the double-tagged amplicon is achieved by electrochemical magneto-genosensing. The strategy is able to detect in 4 h as low as 3 CFU mL(-1) of Salmonella in Luria-Bertani (LB) media. This approach is compared with conventional culture methods and PCR-based assay, as well as with immunological screening assays for bacteria detection, highlighting the outstanding stability and cost-efficient and animal-free production of bacteriophages as biorecognition element in biosensing devices.

Bacteriophages are promising therapeutic agents that can be applied to different stages of the commercial food chain. In this sense, bacteriophages can be orally administered to farm animals to protect them against intestinal pathogens. However, the low pH of the stomach, the activities of bile and intestinal tract enzymes limit the efficacy of the phages. This study demonstrates the utility of an alginate/CaCO3 encapsulation method suitable for bacteriophages with different morphologies and to yield encapsulation efficacies of ~100%. For the first time, a cocktail of three alginate/CaCO3-encapsulated bacteriophages was administered as oral therapy to commercial broilers infected with Salmonella under farm-like conditions. Encapsulation protects the bacteriophages against their destruction by the gastric juice. Phage release from capsules incubated in simulated intestinal fluid was also demonstrated, whereas encapsulation ensured sufficient intestinal retention of the phages. Moreover, the small size of the capsules (125–150 μm) enables their use in oral therapy and other applications in phage therapy. This study evidenced that a cocktail of the three alginate/CaCO3-encapsulated bacteriophages had a greater and more durable efficacy than a cocktail of the corresponding non-encapsulated phages in as therapy in broilers against Salmonella, one of the most common foodborne pathogen.

Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella , is required to achieve effective bacterial reduction over time.

Many cell surface-associated, divalent cation-regulated proteins are immunogenic, and some of them confer protection against the bacterial species from which they are derived. In this work, two Streptococcus suis divalent cation uptake regulator genes controlling zinc/manganese and iron uptake (adcR and fur, respectively) were inactivated in order to study the protective capacities of their cell surface-associated proteins. The results obtained showed overexpression of a set of immunogenic proteins (including members of the pneumococcal histidine triad family previously reported to confer protection against streptococcal pathogens) in S. suis adcR mutant cell surface extracts. Likewise, genes encoding zinc transporters, putative virulence factors and a ribosomal protein paralogue related to zinc starvation appeared to be derepressed in this mutant strain. Moreover, protection assays in mice showed that although neither adcR- nor fur-regulated cell surface-associated proteins were sufficient to confer protection in mice, the combination of both adcR- and fur-regulated cell surface-associated proteins is able to confer significant protection (50 %, P=0.038) against a challenge to mice vaccinated with them.

Objectives: Having shown that the Xeral-Calde Hospital in Lugo (Spain) has been concerned by Escherichia coli clone O25:H4-ST131 producing CTX-M-15 (Nicolas-Chanoine et al. J Antimicrob Chemother 2008; 61: 273-81), the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum β-lactamase (ESBL)-producing E. coli isolates and also to mole cularly characterize the E. coli isolates producing ESBL other than CTX-M-15. Methods: In the first part of this study, 105 ESBL-producing E. coli isolates (February 2006 to March 2007) were characterized with regard to ESBL enzymes, serotypes, virulence genes, phylogenetic groups, multilocus sequence typing (MLST) and PFGE. In the second part of this study, 249 ESBL- producing E. coli isolates (April 2007 to May 2008) were investigated only for the detection of clone O25b:H4-ST131 producing CTX-M-15 using a triplex PCR developed in this study and based on the detection of the new operon afa FM955459 and the targets rfbO25b and 3' end of the bla CTX-M-15 gene. Results: Of the 105 ESBL-producing E. coli isolates, 60 (57.1 %) were positive for CTX-M-14, 23 (21.9%) for CTX-M-15, 10 (9.5%) for SHV-12 and 7 (6.7%) for CTX-M-32. Serotypes, virulence genes, phyloge netic groups and molecular typing by PFGE demonstrated high homogeneity within those producing CTX-M-15 and high diversity within E. coli producing CTX-M-14 and other ESBLs. By PFGE, CTX-M-15- producing E. coli isolates O25b:H4 belonging to the phylogenetic group B2 and MLST profile ST131 were grouped in the same cluster. The epidemic strain of clone O25b:H4-ST131 represented 23.1%, 22.5% and 20.0% of all ESBL-producing E. coli isolated in 2006, 2007 and 2008, respectively. Conclusions: CTX-M-type ESBLs, primarily CTX-M-14 and CTX-M-15, have emerged as the predominant types of ESBL produced by E. coli isolates in Lugo. In view of the reported findings, long-term care facilities for elderly people may represent a significant reservoir for E. coli clone O25b:H4-ST131 pro ducing CTX-M-15. The triplex PCR developed in this work will be useful for rapid and simple detection of this clone.

Aims: We addressed the process of immigration of Bacillus thuringiensis from soil to leaves and its capacity to grow on bean diffusate medium (BDM), a medium designed to simulate the nutrient composition of the phylloplane. Methods and Results: Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (

The ability of several Bacillus thuringiensis strains to colonize plant surfaces was assessed and compared with that of more common epiphytic bacteria. While all B. thuringiensis strains multiplied to some extent after inoculation on bean plants, their maximum epiphytic population sizes of 10(6) cfu/g of leaf were always much less than that achieved by other resident epiphytic bacteria or an epiphytically fit Pseudomonas fluorescens strain, which attained population sizes of about 10(7) cfu/g of leaf. However B. thuringiensis strains exhibited much less decline in culturable populations upon imposition of desiccation stress than did other resident bacteria or an inoculated P. fluorescens strain, and most cells were in a spore form soon after inoculation onto plants. B. thuringiensis strains produced commercially for insect control were not less epiphytically fit than strains recently isolated from leaf surfaces. The growth of B. thuringiensis was not affected by the presence of Pseudomonas syringae when co-inoculated, and vice versa. B. thuringiensis strains harboring a green fluorescent protein marker gene did not form large cell aggregates, were not associated with other epiphytic bacteria, and were not found associated with leaf structures, such as stomata, trichomes, or veins when directly observed on bean leaves by epifluorescent microscopy. Thus, B. thuringiensis appears unable to grow extensively on leaves and its common isolation from plants may reflect immigration from more abundant reservoirs elsewhere.

Bacteriophages UAB_Phi20, UAB_Phi78, and UAB_Phi87 were encapsulated in liposomes, and their efficacy in reducing Salmonella in poultry was then studied. The encapsulated phages had a mean diameter of 309 to 326 nm and a positive charge between +31.6 and +35.1 mV (pH 6.1). In simulated gastric fluid (pH 2.8), the titer of nonencapsulated phages decreased by 5.7 to 7.8 log units, whereas encapsulated phages were significantly more stable, with losses of 3.7 to 5.4 log units. The liposome coating also improved the retention of bacteriophages in the chicken intestinal tract. When cocktails of the encapsulated and nonencapsulated phages were administered to broilers, after 72 h the encapsulated phages were detected in 38.1% of the animals, whereas the nonencapsulated phages were present in only 9.5%. The difference was significant. In addition, in an in vitro experiment, the cecal contents of broilers promoted the release of the phages from the liposomes. In broilers experimentally infected with Salmonella, the daily administration of the two cocktails for 6 days postinfection conferred similar levels of protection against Salmonella colonization. However, once treatment was stopped, protection by the nonencapsulated phages disappeared, whereas that provided by the encapsulated phages persisted for at least 1 week, showing the enhanced efficacy of the encapsulated phages in protecting poultry against Salmonella over time. The methodology described here allows the liposome encapsulation of phages of different morphologies. The preparations can be stored for at least 3 months at 4°C and could be added to the drinking water and feed of animals., This work was supported by grants from La Caixa and the Associació Catalana d’Universitats Públiques (2010ACUP00300), AGAUR-ACCIÓ-Generalitat de Catalunya (2010VALOR00114), and AGAUR-Generalitat de Catalunya (2014SGR572). We are grateful to the Servei de Granges i Camps Experimentals and the Servei de Microscòpia of the Universitat Autònoma de Barcelona (UAB) for their support. J.C. and J.O. received predoctoral fellowships from the UAB.

This study aims to determine the presence of extended-spectrum (ESBL) and plasmidic class C beta-lactamase-producing Enterobacteriaceae in poultry, pig and rabbit farms of Catalonia (Spain). PFGE typing showed a low clonal relationship among strains carrying these mechanisms of resistance. Ninety-three percent of them were resistant to two or more of the non-beta-lactam antimicrobials tested and harboured ESBL and plasmidic class C beta-lactamases. Greater diversity of these enzymes was found in strains from poultry farms, the CTX-M-9 family, especially CTX-M-14, with CMY-2 being the most frequent. The isolation of TEM-52 and SHV-2-producing Escherichia coli strains from these animal farms is noteworthy. In contrast, 73% of the strains from pig farms had CTX-M-1, and neither the CMY-type nor CTX-M-9 family enzyme was found. Likewise, it is the first time that CTX-M-1 and SHV-5 encoding strains have been isolated in pigs. On the other hand, in rabbit farms CTX-M-9 family was also the most frequent, being detected in three of a total of four strains. The last one showed a CMY-2, for the first time detected in these animals, too. In conclusion, commensal E. coli strains of food-producing animal farms are a reservoir of ESBL and plasmidic class C beta-lactamases.

By complementation of an Escherichia coli fur mutant, the Haemophilus parasuis fur gene has been isolated from a genomic library of this organism. The H. parasuis fur gene is the distal one of a three-gene operon. Two genes placed upstream of the H. parasuis fur open-reading frame encode for a hypothetical protein and a flavodoxin, respectively. Attempts performed to isolate an H. parasuis fur-defective mutant either through manganese-resistance selection or exchange markers were unsuccessful. Likewise, anaerobic growth conditions do not enable the attainment of H. parasuis fur-defective mutants either. Nevertheless, H. parasuis clones carrying a knockout mutation in the chromosomal fur gene by insertion of a KmR cassette were obtained when a stable plasmid, containing an additional copy of the transcriptional unit to which the fur gene belongs, was present. Likewise, the presence of a plasmid in which the H. parasuis fur gene is under the control of the Escherichia coli tac promoter allows for the isolation of fur::Km mutants of this organism. Nonetheless, no fur-defective mutants may be isolated from H. parasuis cells harbouring a stable plasmid in which only the single fur gene is contained. These data clearly indicate that H. parasuis cell viability requires the presence of a wild-type fur gene.

A temperate bacteriophage (F108) has been isolated through mitomycin C induction of a Pasteurella multocida serogroup A strain. F108 has a typical morphology of the family Myoviridae , presenting a hexagonal head and a long contractile tail. F108 is able to infect all P. multocida serogroup A strains tested but not those belonging to other serotypes. Bacteriophage F108, the first P. multocida phage sequenced so far, presents a 30,505-bp double-stranded DNA genome with cohesive ends (CTTCCTCCCC cos site). The F108 genome shows the highest homology with those of Haemophilus influenzae HP1 and HP2 phages. Furthermore, an F108 prophage attachment site in the P. multocida chromosome has been established to be inside a gene encoding tRNA Leu . By using several chromosomal markers that are spread along the P. multocida chromosome, it has been demonstrated that F108 is able to perform generalized transduction. This fact, together with the absence of pathogenic genes in the F108 genome, makes this bacteriophage a valuable tool for P. multocida genetic manipulation.

The aim of this work is to investigate the usefulness of a modified protocol of the SOS Chromotest to detect antigenotoxicity activities against gamma-rays of plant extracts with proven antioxidant activity, and to elucidate the antigenotoxic mechanisms involved in radioprotection using this system.The methodology developed was assayed with amifostine, the most studied radioprotector, and with Phyllanthus orbicularis HBK, Cymbopogon citratus (DC) Stapf and Pinus caribaea Morelet extracts, using pre- and post-treatment procedures.The P. caribaea and C. citratus extracts were antigenotoxic against gamma-rays when the cells were pre-treated with both extracts, suggesting a possible antigenotoxic action through a free radical scavenging mechanisms. Amifostine and the P. orbicularis extract were also antigenotoxic under pre- and post-treatment conditions, indicating that several antimutagenic components of this plant extract may also operate by some intracellular mechanism, unlike its antioxidant activity.The results have demonstrated the usefulness of the modified SOS Chromotest assay in the screening of phytochemical radioprotectors as well as in the study of their antimutagenic mechanisms.

Phyllanthus orbicularis HBK (Euphorbiaceae) is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antimutagenic effects against hydrogen peroxide and some promutagenic aromatic amines (AAs), in addition to its antiviral properties. In this paper, antimutagenesis of this extract against two carcinogenic AAs, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP) has been studied. Liver microsomal fractions from both induced rats and humans were used to metabolise both procarcinogenic compounds in the Salmonella assay. The plant extract was effective in reducing the mutagenesis of these AAs, activated by both kinds of fractions. The optimal antimutagenic effect was obtained when both AAs were metabolised by human enzymes, with an almost total reduction of 4-ABP mutagenesis and a decrease of about 75% of PhIP mutagenicity. Mutagenicity of both AAs, activated by induced rat fraction, was only decreased by about 50%. Inhibition by plant extract of alkoxyresorufin O-dealkylation activities, dependent on CYP1A, of both fractions was determined. In accordance with the results obtained, the inhibition or modulation of CYP1A subfamily activities, and possibly of CYP1A2, is thought to be the main mechanism of antimutagenesis of the aqueous extract of Phyllanthus orbicularis against 4-ABP and PhIP.

Summary Myxococcus xanthus, a member of the Proteobacteria delta-class, has two independent recA genes, recA1 and recA2, but only recA2 is DNA damage-inducible. The lexA gene has been isolated from M. xanthus by PCR amplification with oligonucleotides designed after sequence identification by tblastn analysis of its genome at the Cereon Microbial Sequence Database. The M. xanthus purified LexA protein is shown to bind specifically to the consensus sequence CTRHAMRYBYGTTCAGS present upstream of lexA and recA2. A degenerate copy of this motif but with important differences can be identified in the region upstream of the recA1 gene. A knock-out lexA(Def) mutant that has been generated does not differ significantly from wild type in morphology, growth rate, light-induced carotenogenesis or development. Using transcriptional lacZ fusions and quantitative RT-PCR analysis, it has been demonstrated that expression of both lexA and recA2 genes is constitutive in the lexA(Def) mutant, whereas the transcription of the DNA damage non-inducible recA1 gene is not affected in this strain. recN and ssb, whose expression in Escherichia coli are LexA-regulated, are induced by DNA damage in the M. xanthus lexA(Def) mutant. These data reveal the existence of different regulatory mechanisms for DNA damage-inducible genes in bacteria belonging to different phyla.

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties and antimutagenic activities against aromatic amines and hydrogen peroxide. In addition, this plant extract presents antioxidant activity. In this paper, using the Salmonella assay with the experimental approaches of co-incubation, pre- and post-treatments, it is shown that the P. orbicularis extract protects bacterial cells from oxidative damage and mutation by some intracellular mechanism, irrespective of its antioxidant activity.

Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m -phenylenediamine ( m -PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium ( S. typhimurium ) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m -PDA>2-AA>2-AF>9-AP>1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m -PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m -PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m -PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds.

This work explores the use of the phage P22 in a phagomagnetic immunoassay for the rapid detection of Salmonella. The covalent attachment of wild-type phages was performed on two different magnetic carriers: carboxyl-activated magnetic nanoparticles (300 nm) and tosyl-activated magnetic microparticles (2.8 μm). The bacteria were captured and preconcentrated by the phage-modified magnetic particles, followed by the detection using specific anti-Salmonella antibodies conjugated to horseradish peroxidase as an optical reporter. Outstanding selectivity and sensitivity was obtained with this approach, achieving detection limits of 19 CFU mL(-1) in 2.5 h without any pre-enrichment, in milk samples. Moreover, if the samples were pre-enriched for 6 h, the method was able to detect as low as 1.4 CFU in 25 mL of milk. Therefore, the proposed strategy based on the combined use of phagomagnetic separation with immunological labeling is promising as a rapid and simple method for food safety.

Plant activation of three isomers of phenylenediamine, o-, m- and p-phenylenediamine, has been studied. Two in vitro plant systems have been used: Persea americana S117 with mixed-function oxidase (MFO) and peroxidase activities, and Zea mays S9 which contains only peroxidase activity. As genetic endpoint, the classical Salmonella tester strains, TA98 and TA100, their derivatives with high O-acetyltransferase levels (YG1024 and YG1029, respectively) and TA98/1,8-DNP6, deficient in this enzyme, have been assayed. Of the three isomers studied, only m-PDA was activated to mutagenic product(s) by both plant systems. This activation required the bacterial O-acetyltransferase activity to give frameshift mutagenic product(s), detected in TA98 and YG1024 strains. In all the assays the P. americana system was more potent than the Z. mays system in activating m-PDA. A slight increase of the number of YG1029 revertants was detected when m-PDA was activated by P. americana, suggesting that this compound can be also converted into ultimate mutagenic product(s) that induce base-pair substitutions. m-PDA activation by Z. mays was dependent on the peroxidase activity of this system, but the activation produced by P. americana was totally dependent on MFOs, because a total inhibition of the mutagenic response was found when these activities were inhibited. In addition, the P. americana system was more potent in generating proximal mutagenic forms from m-PDA than S9 from non-induced rat liver, although S9 from Aroclor 1254-induced Sprague-Dawley male rats was the most potent system in the m-PDA activation. These results indicate that the P. americana system can be useful in determining the role of mixed-function oxidases in plant activation of xenobiotics.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.

The role of MucAB and Escherichia coli UmuDC proteins in mutagenesis by 4-quinolone (4-Q) compared to that in UV mutagenesis has been studied in hisG428 Salmonella typhimurium strains. A low-copy plasmid carrying mucAB genes, but not umuDC, promotes reversion of the hisG428 mutation by the 4-Q ciprofloxacin. In contrast, a umuDC plasmid mediates the reversion of hisG428 by UV, although less efficiently than a mucAB one. In addition, a unique copy of mucAB genes is enough to promote UV mutagenesis, whereas, several copies of them are required to detect ciprofloxacin mutagenesis. Therefore, the mutagenic repair of quinolone damage by MucAB proteins is not a very efficient process. The presence of an umuD'C plasmid but not a mucA'B one, slightly increases the reversion of the hisG428 mutation by ciprofloxacin and this finding is further discussed. In contrast, MucA'B are still more active than UmuD'C proteins in UV mutagenesis. These results suggest that the enhanced processing of MucA compared to UmuD would not explain all functional differences between MucAB and UmuDC proteins in the error-prone DNA repair.

The mutagenic events induced by ciprofloxacin, a potent antimicrobial agent, have been characterized. For this, a battery of His− mutants of Salmonella typhimurium (hisG428, hisG46, hisC9070, and hisG1775 targets) that detects the six possible transitions and transversions [Levin and Ames (1986): Environ Mutagen 8:9–28] and two additional His− strains (hisC3076 and hisD3052 targets) carrying frameshift mutations have been used. Our results indicate that GCTA transversions are the major base-pair substitution induced by ciprofloxacin and that GCAT transitions are also produced, but to a lesser degree. However, we cannot discard the fact that ATTA transversions are also induced. In addition, the data indicate that the mutational specificity of ciprofloxacin depends on the location of the target. Intragenic base-pair substitutions are the most frequent mutations at the hisG428 target when it is on the chromosome, whereas 3 or 6 base-pair deletions are the major mutagenic events when this target is on the plasmid pAQ1. We have shown that ciprofloxacin also induces deletions/insertions at the hisC3076 and hisD3052 frameshift targets. Therefore, this inhibitor of DNA gyrase promotes a wide pattern of mutations including different kinds of base-pair substitutions, 3 or 6 base-pair deletions, and insertions/deletions resulting in frameshifts. All of these mutagenic events require the MucAB proteins involved in the error-prone repair, with the exception of base-pair insertions/deletions at the hisD3052 target, which are independent of the presence of plasmid pKM101. © 1996 Wiley-Liss, Inc.

Microsomal fractions from different tissues of various plants (potato, cauliflower, aubergine, avocado pear, courgette, cucumber, banana, kiwi and strawberry) were prepared and their content of cytochrome P-450 (cyt-P450) determined. A S117 fraction from Persea americana (avocado pear) presented the highest content of cyt-P450. As a consequence of these data, we have developed and characterized this fraction as a plant metabolic activation system. The P. americana S117, used in this work, contains 0.75 +/- 0.04 mg of protein per ml, 0.788 +/- 0.078 nmol of cyt-P450 per mg of protein and has a peroxidase activity of 0.036 +/- 0.005 (nmol tetraguaiacol/micrograms protein/min). The P. americana cyt-P450 remained stable during at least 60 days, stored at -80 degrees C. This fraction activated 2-aminofluorene to a mutagenic product in S. typhimurium TA98, while it had no effect on the benzo[a]pyrene activation. The treatment of the P. americana S117 with CO, the addition of diethyldithiocarbamate (DETC) or the absence of a NADPH-generating system in the activation mix, produced a partial inhibition of the 2-aminofluorene activation. Both peroxidase activity and a cyt-P450-dependent activity are assumed to be involved in the activation of this chemical mediated by P. americana S117.

Summary Four new platinum compounds containing different pyrrolidines as ligands are reported. In vitro tests show damage to bacterial DNA and participation of RecA protein in the repair system. In vitro tests using leukemia L1210 cells show growth inhibition by the new compounds within the range of cisplatin and carboplatin. In vivo tests using leukemia L1210 cells on male BDF1 show that these compounds are significantly active.

The Streptococcus suis 103 gene product is an immunogenic and protective lipoprotein that is a component of an ATP-binding cassette transporter implicated in zinc uptake. Belonging to the same transcriptional unit and downstream of the 103 gene is a gene that encodes a homologue of the pneumococcal histidine triad (Pht) protein Pht309. In an intraperitoneal mouse model the virulence of a mutant lacking the 103 gene was more than 50 times lower than that of the wild-type (WT) parent strain, S. suis serotype 2 strain P1/7. In addition, the immunogenicity of this mutant was dramatically decreased. In striking contrast, the virulence and immunogenicity of a P1/7 mutant lacking the Pht309 gene were similar to those of the parent strain. These results demonstrate that the 103 lipoprotein is strongly involved in S. suis virulence and support the hypothesis that this lipoprotein might be an excellent candidate for vaccines aiming to achieve broad protection against streptococci.

To ascertain whether on animal farms there reside extended-spectrum β-lactamase (ESBL) and plasmidic class C β-lactamase-producing Escherichia coli isolates potentially pathogenic for humans, phylogenetic analyses, pulsed-field gel electrophoresis (PFGE) typing, serotyping, and virulence genotyping were performed for 86 isolates from poultry (57 isolates) and pig (29 isolates) farms. E. coli isolates from poultry farms carried genes encoding enzymes of the CTX-M-9 group as well as CMY-2, whereas those from pig farms mainly carried genes encoding CTX-M-1 enzymes. Poultry and pig isolates differed significantly in their phylogenetic group assignments, with phylogroup A predominating in pig isolates and phylogroup D predominating in avian isolates. Among the 86 farm isolates, 23 (26.7%) carried two or more virulence genes typical of extraintestinal pathogenic E. coli (ExPEC). Of these, 20 were isolated from poultry farms and only 3 from pig farms. Ten of the 23 isolates belonged to the classic human ExPEC serotypes O2:H6, O2:HNM, O2:H7, O15:H1, and O25:H4. Despite the high diversity of serotypes and pulsotypes detected among the 86 farm isolates, 13 PFGE clusters were identified. Four of these clusters contained isolates with two or more virulence genes, and two clusters exhibited the classic human ExPEC serotypes O2:HNM (ST10) and O2:H6 (ST115). Although O2:HNM and O2:H6 isolates of human and animal origins differed with respect to their virulence genes and PFGE pulsotypes, the O2:HNM isolates from pigs showed the same sequence type (ST10) as those from humans. The single avian O15:H1 isolate was compared with human clinical isolates of this serotype. Although all were found to belong to phylogroup D and shared the same virulence gene profile, they differed in their sequence types (ST362-avian and ST393-human) and PFGE pulsotypes. Noteworthy was the detection, for the first time, in poultry farms of the clonal groups O25b:H4-ST131-B2, producing CTX-M-9, and O25a-ST648-D, producing CTX-M-32. The virulence genes and PFGE profiles of these two groups were very similar to those of clinical human isolates. While further studies are required to determine the true zoonotic potential of these clonal groups, our results emphasize the zoonotic risk posed especially by poultry farms, but also by pig farms, as reservoirs of ESBL- and CMY-2-encoding E. coli .

Three new malonato platinum(II) complexes containing P and S ligands are reported. In in vitro tests, all three compounds showed damage to bacterial DNA and participation of RecA protein in the repair system. Results from a preliminary study in vivo using leukemia L1210° cells on mice BDF, show that two of these compounds are potentially active.

A new virulent bacteriophage, designated RZ1, was isolated from a local pond on the facultative phototrophic bacteriumRhodobacter sphaeroides ZZ101. Electron microscopic studies revealed that, in general morphology, phage RZ1 resembles the λ bacteriophage ofEscherichia coli. The host range of phage RZ1 is limited to some strains ofR. sphaeroides. The phage genome consists of double-stranded DNA of about 44 kb lacking cohesive ends and seems to present terminal redundancy and cyclic permutation. RZ1 phage may carry out a lytic cycle only in recombination-defective mutants ofR. sphaeroides. Nevertheless, a derivative of the RZ1 phage, termed RW1, able to grow in recombination-proficient strains ofR. sphaeroides, has also been obtained. In vitro restriction analysis of both RZ1 and RW1 phages shows the presence of a rearrangement in their DNA. Generalized transduction of Strr and Rifr chromosomal markers has not been detected with either RZ1 or RW1 phages.