Matched-pair dosimetric comparison of cardiac radiation exposure between deep-inspiration breath-hold whole-breast radiation therapy with Active Breathing Coordinator and interstitial multicatheter high-dose-rate brachytherapy as accelerated partial breast irradiation in adjuvant treatment of left-sided breast cancer after breast-conserving surgery.

BACKGROUND AND PURPOSE: To compare dosimetrically the radiation exposure to heart, left ventricle (LV), and left anterior descending artery (LAD) between whole-breast radiotherapy (WBRT) with Active Breathing Coordinator (ABC; ABC-WBRT) and interstitial multicatheter high-dose-rate (HDR) brachytherapy as accelerated partial breast irradiation (ABPI; imHDR-APBI) for left-sided breast cancer (BCA) after breast-conserving surgery (BCS). MATERIALS AND METHODS: Between January 2016 and December 2019, 32 and 20 patients were treated with ABC-WBRT (63 Gy/2.25 Gy) and imHDR-APBI (32 Gy/4 Gy), respectively. Among them a matched-pair analysis was performed according to tumor location (clock position) before BCS as well as planning target volume of imHDR-APBI and boost volume of ABC-WBRT. This yielded 17 pairs of patients for whom dosimetric parameters for heart, LV, and LAD were evaluated. The Mann-Whitney test was used for comparison after adjusting for equivalent dose in 2­Gy fractions (EQD2). In addition, a second analysis of ABC-WBRT to 40.05 Gy in 15 fractions was performed in order to account for the EQD2 difference between the 63-Gy ABC-WBRT and the imHDR-APBI protocol. RESULTS: Tumor location for the 17 pairs of patients relative to breast quadrant was as follows: upper outer 8, lower outer 5, upper inner 3, and lower inner 1. There was no difference regarding mean heart dose (MHD) and V5, whereas D25%, D45%, V10, and V25 significantly favored imHDR-APBI. Likewise, mean dose- and V5-LV did not differ, while Dmax- and V23-LV were significantly higher for ABC-WBRT. For LAD, Dmax, D25%, and V30 significantly favored imHDR-APBI without differences for mean dose and V40. When comparing imHDR-APBI with the 40.05 Gy ABC-WBRT schedule, MHD and mean dose LV were significantly lower in favor of ABC-WBRT. CONCLUSION: ABC-WBRT and imHDR-APBI yield similar low heart and LV exposure for left-sided BCA after BCS, whereas LAD can be better spared with imHDR-APBI.

The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions.

Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are radically altered (melting temperature (Tm), buoyant density and susceptibility to restriction endonucleases). Using 454 pyrosequencing technology, we sequenced the genomes of both viruses. Phage ΦW-14 possesses a 157-kb genome (56.3% GC) specifying 236 proteins, while SP-15 is larger at 222 kb (38.6 mol % G + C) and encodes 318 proteins. In both cases, the phages can be considered genomic singletons since they do not possess BLASTn homologs. While no obvious genes were identified as being responsible for the modified base in ΦW-14, SP-15 contains a cluster of genes obviously involved in carbohydrate metabolism.

Purse-string closure technique reduces the incidence of incisional hernias following the reversal of temporary ileostomy.

PURPOSE: The incidence of incisional hernia (IH) at ileostomy closure site has not been sufficiently evaluated. Temporary loop ileostomy is routinely used in patients after low anterior resection for rectal cancer. The goal of this study was to compare the IH rates of standard suture skin closure and purse-string skin closure techniques. PATIENTS AND METHODS: Patients undergoing ileostomy reversal and follow-up CT scan at the University Hospital Frankfurt between January 2009 and December 2015 were retrospectively analyzed regarding IH and associated risk factors. Patients received either direct stitch skin closure (group DC) or purse-string skin closure (group PS). RESULTS: In total, 111 patients underwent ileostomy reversal in the aforementioned period. In 88 patients, a CT scan was performed 12-24 months after ileostomy reversal for cancer follow-up. Median follow-up was 12 months. Median time interval between ileostoma formation and closure was 12 (± 4 SD) weeks. In 19 of 88 patients (21.5%), an IH was detected. The incidence of IH detected by CT scan was significantly lower in the PS group (n = 7, 12.9%) compared to the DC group (n = 12, 35.2%, p = 0.017). CONCLUSIONS: This retrospective study shows an advantage of the purse-string skin closure technique in ileostomy reversals. The use of this technique for skin closure following ileostomy reversals is recommended to reduce the IH rates. Randomized controlled trials are needed to confirm these findings.

Essential Steps in Characterizing Bacteriophages: Biology, Taxonomy, and Genome Analysis.

Because of the rise in antimicrobial resistance there has been a significant increase in interest in phages for therapeutic use. Furthermore, the cost of sequencing phage genomes has decreased to the point where it is being used as a teaching tool for genomics. Unfortunately, the quality of the descriptions of the phage and its annotation frequently are substandard. The following chapter is designed to help people working on phages, particularly those new to the field, to accurately describe their newly isolated viruses.

Comparative Analysis of 37 Acinetobacter Bacteriophages.

Members of the genus Acinetobacter are ubiquitous in the environment and the multiple-drug resistant species A. baumannii is of significant clinical concern. This clinical relevance is currently driving research on bacterial viruses infecting A. baumannii, in an effort to implement phage therapy and phage-derived antimicrobials. Initially, a total of 42 Acinetobacter phage genome sequences were available in the international nucleotide sequence databases, corresponding to a total of 2.87 Mbp of sequence information and representing all three families of the order Caudovirales and a single member of the Leviviridae. A comparative bioinformatics analysis of 37 Acinetobacter phages revealed that they form six discrete clusters and two singletons based on genomic organisation and nucleotide sequence identity. The assignment of these phages to clusters was further supported by proteomic relationships established using OrthoMCL. The 4067 proteins encoded by the 37 phage genomes formed 737 groups and 974 orphans. Notably, over half of the proteins encoded by the Acinetobacter phages are of unknown function. The comparative analysis and clustering presented enables an updated taxonomic framing of these clades.

The lambda - P22 problem.

Lambda and P22 are members of 2 families of tailed phages and have limited genomic relationships. Both form hybrids with many phages. P22 appears as a hybrid of mixed ancestry. Despite their similarities, lambda and P22 and their relatives form 2 distinct lineages and must be classified separately.

Taxonomic reassessment of N4-like viruses using comparative genomics and proteomics suggests a new subfamily - "Enquartavirinae".

The GenBank database currently contains sequence data for 33 N4-like viruses, with only one, Escherichia phage N4, being formally recognized by the ICTV. The genus N4likevirus is uniquely characterized by that fact that its members possess an extremely large, virion-associated RNA polymerase. Using a variety of proteomic, genomic and phylogenetic tools, we have demonstrated that the N4-like phages are not monophyletic and that N4 is actually a genomic orphan. We propose to create four new genera: "G7cvirus" (consisting of phages G7C, IME11, KBNP21, vB_EcoP_PhAPEC5, vB_EcoP_PhAPEC7, Bp4, EC1-UPM and pSb-1), "Lit1virus" (LIT1, PA26 and vB_PaeP_C2-10_Ab09), "Sp58virus" (SP058 and SP076), and "Dss3virus" (DSS3φ2 and EE36φ1). We propose that coliphage N4, the members of "G7cvirus", Erwinia phage Ea9-2, and Achromobacter phage JWAlpha should be considered members of the same subfamily, which we tentatively call the "Enquartavirinae".

Feces of feedlot cattle contain a diversity of bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli.

This study aimed to isolate and characterize bacteriophages that lyse non-O157 Shiga toxin-producing Escherichia coli (STEC) from cattle feces. Of 37 non-O157 STEC-infecting phages isolated, those targeting O26 (AXO26A, AYO26A, AYO26B), O103 (AXO103A, AYO103A), O111 (AXO111A, AYO111A), O121 (AXO121A, AXO121B), and O145 (AYO145A, AYO145B) were further characterized. Transmission electron microscopy showed that the 11 isolates belonged to 3 families and 6 genera: the families Myoviridae (types rV5, T4, ViI, O1), Siphoviridae (type T5), and Podoviridae (type T7). Genome size of the phages as determined by pulsed-field gel electrophoresis ranged from 38 to 177 kb. Excluding phages AXO26A, AYO103A, AYO145A, and AYO145B, all other phages were capable of lysing more than 1 clinically important strain from serogroups of O26, O91, O103, O111, O113, O121, and O128, but none exhibited infectivity across all serogroups. Moreover, phages AYO26A, AXO121A, and AXO121B were also able to lyse 4 common phage types of STEC O157:H7. Our findings show that a diversity of non-O157 STEC-infecting phages are harbored in bovine feces. Phages AYO26A, AYO26B, AXO103A, AXO111A, AYO111A, AXO121A, and AXO121B exhibited a broad host range against a number of serogroups of STEC and have potential for the biocontrol of STEC in the environment.

A proposed new bacteriophage subfamily: "Jerseyvirinae".

Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named "Jerseyvirinae" and consisting of three genera, "Jerseylikevirus", "Sp3unalikevirus" and "K1glikevirus". To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phages Jersey, vB_SenS_AG11, vB_SenS-Ent1, vB_SenS-Ent2, vB_SenS-Ent3, FSL SP-101, SETP3, SETP7, SETP13, SE2, SS3e and wksl3 form the proposed genus "Jerseylikevirus". The proposed genus "K1glikevirus" consists of Escherichia phages K1G, K1H, K1ind1, K1ind2 and K1ind3. The proposed genus "Sp3unalikevirus" contains one member so far. Jersey-like phages appear to be widely distributed, as the above phages were isolated in the UK, Canada, the USA and South Korea between 1970 and the present day. The distinguishing features of this subfamily include a distinct siphovirus morphotype, genomes of 40.7-43.6 kb (49.6-51.4 mol % G+C), a syntenic genome organisation, and a high degree of nucleotide sequence identity and shared proteins. All known members of the proposed subfamily are strictly lytic.

Characterization of two temperate Lactobacillus paracasei bacteriophages: morphology, kinetics and adsorption.

BACKGROUND/AIMS: Adsorption and kinetic parameters, latent period, burst size and burst time, are characteristics of phage/host systems and can be affected by several environmental factors. As only few studies have focused on temperate dairy phages, we characterized these parameters on temperate Lactobacillus paracasei phages Φ iLp84 and Φ iLp1308, infective for probiotic strains. METHODS: Phages were characterized by transmission electron microscopy and genomic DNA restriction. Adsorption under different environmental conditions, phage kinetics and efficiency of plating (EOP) were determined using the double-layer titration method. RESULTS: Phages Φ iLp84 and Φ iLp1308 belong to the Siphoviridae family and have genome sizes of 38 and 34 kbp, respectively. Adsorption was affected by calcium concentration, pH, temperature and host viability, and reached a limit at very high multiplicity of infection. Latency, burst time and burst size were of 85 min, 131 min and 46 for Φ iLp84, and 51 min, 92 min and 28 for Φ iLp1308, respectively, at 37°C. A clear influence of temperature on phage kinetics was observed. Regarding EOP, Φ iLp84 produced plaques on only 1 out of 8 strains tested. CONCLUSION: Noticeable differences in adsorption, kinetics and EOP were found for two morphologically identical temperate L. paracasei phages of similar origin.

Integration of genomic and proteomic analyses in the classification of the Siphoviridae family.

Using a variety of genomic (BLASTN, ClustalW) and proteomic (Phage Proteomic Tree, CoreGenes) tools we have tackled the taxonomic status of members of the largest bacteriophage family, the Siphoviridae. In all over 400 phages were examined and we were able to propose 39 new genera, comprising 216 phage species, and add 62 species to two previously defined genera (Phic3unalikevirus; L5likevirus) grouping, in total, 390 fully sequenced phage isolates. Many of the remainders are orphans which the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) chooses not to ascribe genus status at the time being.

Characterization of Staphylococcus epidermidis phage vB_SepS_SEP9 - a unique member of the Siphoviridae family.

Relatively few phages (<10) of coagulase negative staphylococci (CoNS) have been described. Staphylococcus epidermidis phage vB_SepS_SEP9 is a siphovirus with a unique morphology as a staphylococcal phage, possessing a very long tail. Its genome is unique and unrelated to any phage genomes deposited in public databases. It appears to encode a nonfunctional integrase. Due to the not having a recognizable lysogeny module, the phage is unable lysogenize. The genome comprises 129 coding sequences (CDS), 46 of which have an assigned function and 59 are unique. Its unique morphology and genome led to the proposal of the establishment of a new Siphoviridae genus named "Sep9likevirus".

Supersize me: Cronobacter sakazakii phage GAP32.

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), which possesses the second largest sequenced phage genome (358,663bp). A total of 571 genes including 545 protein coding sequences and 26 tRNAs were identified, thus more genes than in the smallest bacterium, Mycoplasma genitalium G37. BLASTP and HHpred searches, together with proteomic analyses reveal that only 23.9% of the putative proteins have defined functions. Some of the unique features of this phage include: a chromosome condensation protein, two copies of the large subunit terminase, a predicted signal-arrest-release lysin; and an RpoD-like protein, which is possibly involved in the switch from immediate early to delayed early transcription. Its closest relatives are all extremely large myoviruses, namely coliphage PBECO4 and Klebsiella phage vB_KleM-RaK2, with whom it shares approximately 44% homologous proteins. Since the homologs are not evenly distributed, we propose that these three phages belong to a new subfamily.

Comparative genomic and morphological analyses of Listeria phages isolated from farm environments.

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large genome (~135-kb) myoviruses belonging to the genus "Twort-like viruses," three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (~66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (~56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.

Leuconostoc bacteriophages from blue cheese manufacture: long-term survival, resistance to thermal treatments, high pressure homogenization and chemical biocides of industrial application.

Nine Leuconostoc mesenteroides phages were isolated during blue cheese manufacture yielding faulty products with reduced eye formation. Their morphologies, restriction profiles, host ranges and long-term survival rates (25°C, 8°C, -20°C and -80°C) were analysed. Based on restriction analysis, six of them were further examined regarding resistance to physical (heat and high pressure homogenization, HPH) and chemical treatments (ethanol, sodium hypochlorite, peracetic acid, biocides A, C, E and F). According to their morphology, L. mesenteroides phages studied in the present work belonged to the Caudovirales order and Siphoviridae family. Six distinct restriction patterns were obtained with EcoRV, HindIII, ClaI and XhoI enzymes, revealing interesting phage diversity in the dairy environment. No significant reductions in phage counts were observed after ten months of storage at -20°C and -80°C, while slightly and moderate decrease in phage numbers were noticed at 8°C and 25°C, respectively. The phages subjected to heat treatments generally showed high resistance at 63°C and moderate resistance at 72°C. However, 80°C for 30 min and 90°C for 2 min led to complete inactivation of viral particles. In general, the best ethanol concentration tested was 75%, as complete inactivation for most Leuconostoc phages within 30 min of incubation was achieved. Peracetic acid, and biocides A, C, E and F were highly effective when used at the same or at a moderately lower concentration as recommended by the producer. Usually, moderate or high concentrations (600-1,600 ppm) of sodium hypochlorite were necessary to completely inactivate phage particles. Leuconostoc phages were partially inactivated by HPH treatments as remaining viral particles were found even after 8 passes at 100 MPa. This is the first report of L. mesenteroides phages isolated from an Argentinean dairy cheese plant. The results of this work could be useful for establishing the most effective physical and chemical treatments for inactivating phages in industrial plants and laboratory environments.

The genome and proteome of Serratia bacteriophage η which forms unstable lysogens.

BACKGROUND: Serratia marcescens phage η is a temperate unclassified member of the Siphoviridae which had been reported as containing hypermodified guanine residues. METHODS: The DNA was characterized by enzymatic digestion followed by HPLC analysis of the nucleoside composition, and by DNA sequencing and proteomic analysis. Its ability to form stable lysogens and integrate was also investigated. RESULTS: Enzymatic digestion and HPLC analysis revealed phage η DNA did not contain modified bases. The genome sequence of this virus, determined using pyrosequencing, is 42,724 nucleotides in length with a mol% GC of 49.9 and is circularly permuted. Sixty-nine putative CDSs were identified of which 19 encode novel proteins. While seven close genetic relatives were identified, they shared sequence similarity with only genes 40 to 69 of the phage η genome, while gp1 to gp39 shared no conserved relationship. The structural proteome, determined by SDS-PAGE and mass spectrometry, revealed seven unique proteins. This phage forms very unstable lysogens with its host S. marcescens.

A giant Pseudomonas phage from Poland.

A novel giant phage of the family Myoviridae is described. Pseudomonas phage PA5oct was isolated from a sewage sample from an irrigated field near Wroclaw, Poland. The virion morphology indicates that PA5oct differs from known giant phages. The phage has a head of about 131 nm in diameter and a tail of 136 × 19 nm. Phage PA5oct contains a genome of approximately 375 kbp and differs in size from any tailed phages known. PA5oct was further characterized by determination of its latent period and burst size and its sensitivity to heating, chloroform, and pH.

Isolation and characterization of a new Staphylococcus epidermidis broad-spectrum bacteriophage.

Staphylococcus epidermidis is considered an important nosocomial pathogen, being very tolerant to the host immune system and antibiotherapy, particularly when in biofilms. Due to its high resistance, alternative antimicrobial strategies are under development. The use of bacteriophages is seen as an important strategy to combat pathogenic organisms. In this study, a S. epidermidis myovirus, SEP1, was isolated and characterized. The genome of this phage was sequenced and shown to be related peripherally to the genus Twortlikevirus. However, when compared with other phages of this genus, it showed DNA sequence identities no greater than 58.2 %. As opposed to other polyvalent viruses of the genus Twortlikevirus, SEP1 is highly specific to S. epidermidis strains. The good infectivity shown by this phage as well as its high lytic spectrum suggested that it might be a good candidate for therapeutic studies.