The NRRL B-24224 genome sequence was submitted to GenBank and annotated with NCBI’s Prokaryotic Genome Annotation Pipeline (11), which identified 3,264 protein-coding genes, 45 tRNA genes, and 2 rRNA operons. A search with PHASTER (12) revealed no intact prophages, such that neither prophage-encoded superinfection immunity nor prophage-mediated heterotypic defense systems should constrain the types of phages isolated on this strain (13). The genome does contain an intact type II VapBC toxin-antitoxin system but no apparent restriction-modification or CRISPR system. These findings suggest that NRRL B-24224 has many useful attributes as a host for exploring the diversity and evolution of the bacteriophage population.

A single colony of NRRL B-24224 was picked and grown to saturation in a peptone yeast calcium (PYCa) medium. Genomic DNA was isolated by lysing the cultures of NRRL B-24224 in a 3110BX Mini-BeadBeater for 45 s, treating them with RNase, and performing a phenol-chloroform extraction. A barcoded library for Illumina sequencing was prepared with an NEB Ultra II FS kit and run on an Illumina MiSeq system, which produced just over 3 million 150-base single-end reads. From the same DNA sample, a second library for Oxford Nanopore sequencing was prepared with a rapid barcoding kit (SQK-RBK004) and run on a MinION sequencer with a FLO-MIN6 (R9.4) flow cell for 6 h, which produced ∼30,000 reads with an average length of ∼5.7 kb. These 2 sets of reads were assembled with Unicycler v0.4.4 (9), with the -s flag for the Illumina reads and the -l flag for the Nanopore reads and default settings. Unicycler was used to assemble the reads into a single circular bacterial contig, with 126-fold Illumina coverage and 47-fold Nanopore coverage, which was evaluated and adjusted for accuracy and completeness using Consed v29 and custom scripts as previously described (10).

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During the middle Viséan stage, relative sea-level rise and reduced siliciclastic influx promoted increased carbonate productivity and creation of carbonate factory on the shelf. This situation led to deposition of the lower member (the Ras Samra Member) of the Um Bogma Formation (Fig. 3). During the initial stage of transgression, due to slow rise of relative sea-level, rate of carbonate sediment production and accumulation were able to keep up with the rate of creation of accommodation space. This resulted in deposition of the carbonate sediments of the lower member in intertidal to shallow subtidal environments in the initial stage of the transgressive system tract. The consequence higher rate of relative sea-level rise during transgression, resulted flooding of the carbonate shelf and deposition of the highly fossiliferous middle member (the El Qor Member) in open marine environment (Fig. 3). Deposition of the calcareous mudstone and marl in this member indicates higher rate of creation of accommodation space than carbonate accumulation. At this time, the marine transgression resulted in the shoreline stepping landward, toward the southeast of the Sinai (Kora et al., 1994, Sobhy and Ezaki, 2005). Therefore, the lower member and the middle member of the Um Bogma Formation make up the transgressive system tract that are consist of retrogradational parasequence sets deposited in intertidal to shallow subtidal to open marine environments. At the Um Bogma region, the transgressive system tract on lapped directly onto the sequence boundary, and thus the sequence boundary coincided with the transgression surface.

Depositional environment and sequence stratigraphy of the Um Bogma Formation were interpreted from two outcrop successions at the studied localities in the Um Bogma district, Sinai, by detailed field work. Forty five samples represented various depositional facies, and system tracts and key sequence stratigraphic surfaces were collected from the successions.Optical microscopic study of the samples was performed on all of the forty five thin sections, which were prepared after impregnation with blue epoxy under vacuum. Cathodoluminescence (CL) petrography was carried out on seven collected thin sections using a Technosyn 8200 MK II cold luminoscope, connected to a petrographic microscope in order to determine chemical zoning and different generations of dolomite crystals. Twenty five bulk dolostone samples were coated with a thin layer of gold and examined by a Jeol JSM-T330 scanning electron microscope (SEM) equipped with a digital imaging system to investigate crystal habits and paragenetic relationship of diagenetic components. A Cameca SX50 electron microprobe (EMP) equipped with three WD spectrometers and a backscattered electron (BSE) detector was used on seventeen polished, carbon-coated thin sections for determining chemical composition of the dolomite crystals.Stable carbon and oxygen isotope analyses were carried out on seven dolostone samples in order to determine the geochemical conditions of dolomite precipitation including sources of dissolved carbon, pore-water composition and temperature of precipitation of the dolomites. The isotope data are reported as delta (δ) notation, in per mil (‰), relative to the V-PDB (Vienna PeeDee Belemnite) and V-SMOW (Vienna Standard Mean Ocean Water) standard.

The age of the Um Bogma Formation, according to its macro- and microfaunal content, is considered to be the Middle Viséan stage of Early Carboniferous Period (El Shahat et al., 1994, Kora et al., 1994). The faunal associations are consistent with deposition in a warm, shallow marine environment (El Shahat et al., 1994, Kora et al., 1994). Paleomagnetic and paleogeographic studies show that the Um Bogma carbonate shelf occurred at latitude 10° south of the equator in a subtropical epicontinental sea with a northeast-southwest shoreline and an open sea toward the northwest (Kora et al., 1994, Tawadros, 2001, Sobhy and Ezaki, 2005).

The Um Bogma Formation is subdivided into three lithostratigraphic members (Kora et al., 1994; Fig. 2). The Ras Samra Member at the base, (ca 18 m thick), mainly consists of grayish sandy dolostone in which the sand content decreases upward concomitant with an increase in dolomite. This member is interpreted to have been deposited in intertidal to shallow subtidal environments (El Shahat et al., 1994, Sobhy and Ezaki, 2005). The El Qor Member at the middle, (ca 6 m thick), is composed of cycles of fine- to medium-grained, locally coarse- to very coarse-grained, poorly-sorted, yellow, sandy dolostone overlain by calcareous, silty mudstone and marl. The cycles tend to be thinner at the top of the member. This member is interpreted as open-marine carbonate deposits (El Shahat et al., 1994, Kora et al., 1994). The middle member is highly fossiliferous and rich in gastropods, echinoids, ostracods, corals, brachiopods, crinoids and trilobites. The Um Shebba Member at the top, (ca. 8 m thick) consists of medium- to thick bedded, orange-brown to gray, sandy dolostones, similar to those in the lower member, but richer in sand at the top. The upper member was deposited in a shallow subtidal environment (El Shahat et al., 1994, Sobhy and Ezaki, 2005).

The Um Bogma Formation unconformably overlies Cambrian-Ordovician fluvial and subtidal deposits of the Adedia Formation (uppermost formation of the Lower Sandstone Unit) and is conformably overlain by the Early Carboniferous subtidal-shoreface to fluvial deposits of the Lower Abu Thora Formation (lowermost formation of the Upper Sandstone Unit) (Barron, 1907, Kora et al., 1994; Fig. 2).

The study area is located in the Um Bogma district, on the eastern coast of the Gulf of Suez, Sinai, where the Um Bogma Formation is well exposed (Fig. 1). The Outcrop studied successions are situated in the valleys which are called Wadi Nasib and Wadi El-Shallal (Fig. 1). In this region, the Paleozoic sedimentary rocks

In this study we provide evidences suggesting that formation of dolomite and distribution of dolostone beds in the Early Carboniferous Um Bogma Formation in southwestern Sinai, Egypt, was associated with marine transgressions within sequence stratigraphic framework.2. Geologic setting and field de

In spite of extensive studies and the many models available, conditions and mechanism of formation of dolomite is still controversial and enigmatic (e.g. Warren, 2000; Machel, 2004; Sanchez Roman et al., 2008a, Sanchez-Roman et al., 2008b; Krause et al., 2012; Gregg et al., 2015). Recently some diagenetic studies have demonstrated that distribution of diagenetic alterations and their effects on reservoir quality can be determine and even be predicted within sequence stratigraphic framework (e.g. Morad et al., 2000, Morad et al., 2012; Taylor et al., 2000; Ketzer et al., 2002; Al-Ramadan et al., 2012).

This exercise was scheduled to be completed within a 15-minute period of a two-hour microbiology laboratory with a class size of 120 students. Students were instructed to record phage typing results of all S. aureus strains/wards as outlined in Table 1 (Appendix 1). Although many plaques are generally round, they can be variety of shapes and sizes (1). A plaque is indicated by a zone of no growth (clear plaque) in the lawn of S. aureus. If phages 1, 5, and 7 gave a positive result (clear plaques), the phage type would be recorded as 1/5/7.The results were discussed by groups of students, focusing on answering the question outlined in Table 2 (Appendix 1), aiming for engagement of students in active learning about the relationship between bacteria and viruses, as well as to encourage teamwork and the development of critical thinking skills. Students were also given the opportunity to question the teaching assistants if required and to learn how to both convey and receive constructive criticism/feedback from their peers as well as their instructors.

Groups of four students were provided with three culture plates labeled “ward #1,” “ward #3,” and “ward #6,” showing phage typing (Fig. 1) of S. aureus strains isolated from three different hospital wards. Preparation of culture plates showing phage typing activity and detailed instructor notes are available in Appendix 1. Student instructions were provided in each student’s laboratory manual (Appendix 3). Clearly labeled biohazard bags were available for waste disposal, and disinfectant was available to disinfect benches.

Students had already received adequate training about laboratory safety and been introduced to all basic microbiology laboratory techniques such as aseptic technique, culture transfer, staining, bacterial isolation and purification, making a lawn plate, biochemical identification, and safe handling of BSL1 and BSL2 microorganisms, etc. during their previous microbiology laboratory sessions and lectures before performing this practical exercise (please see prerequisite student knowledge, Appendix 1). The bacterial strain (Staphylococcus aureus subsp. aureus ROSENBACH ATCC 35556) used for this laboratory exercise is classified as a BSL2 organism, and students therefore handled all bacterial lawn plates according to ASM biosafety BSL2 level guidelines (https://www.asm.org/images/asm_biosafety_guidelines-FINAL.pdf). Students performed this activity with appropriate personal protective equipment (i.e., lab coat, closed-toed shoes, gloves, and safety glasses). After completing this practical exercise, students disinfected lab-bench surfaces, all biohazard wastes were disposed of according to the university biohazard waste disposal guideline, and finally, students thoroughly washed their hands in compliance with ASM guidelines.CONCLUSION

This exercise is suitable for students with undergraduate experience in microbiology, virology, or associated laboratory techniques whose prior lectures have covered introductory aspects of microbiology, viruses, and bacteriophages along with safety measures taught during the course and during the laboratory exercise.

This low-cost exercise effectively facilitated student– student and student–instructor interactions, engaging students to link theory to practical exercises in order to interpret phage typing results and to discuss the answer to the question provided in the students’ laboratory manual. This created an effective collaborative learning situation in the classroom for teaching students about bacteriophages without the use of real phages.

To mimic viral plaques, we used a commercially available laboratory disinfectant, TRIGENE (diluted at a 1:100 ratio), which is capable of clearing bacterial growth on the agar plate, resulting in a visual effect similar to that of lytic phages on bacterial lawns. The teaching strategy involved analyzing phage typing of three different culture plates (with different configurations of artificial plaques, Fig. 1)

We have designed a simple laboratory exercise in a single practical class for a second-year biomedical science course to demonstrate the plaque assay and to use phage typing to differentiate three bacterial strains of Staphylococcus aureus.

The assay can also be used for the purposes of phage typing, whereby phages are used to identify pathogenic bacteria in diagnostic laboratories. Finally, there is renewed interest in the therapeutic use of phages to treat antibiotic-resistant pathogenic bacterial infections (5).

One of the most common detection methods used is a plaque assay, whereby bacteria and bacteriophages are mixed on an agar plate and the plaques are observed to assess whether lytic viruses specific for the bacterial hosts are present (2).

Bacteriophages are viruses that infect specific bacterial species, hijacking the infected bacterium’s machinery to multiply and eventually burst its prey, resulting in zones of clearing on the culture plate known as plaques (1).

Bacteriophages (phages) are a category of viruses capable of infecting bacteria. Phages were first documented in 1915 and were named bacteriophages in 1917 [1], [2]. Attempts to use phages to treat infectious diseases have been made but were generally abandoned after the 1940s owing to their difficulty in use, poor efficacy and the increasing use of antibiotics [3]. However, with the emergence of antimicrobial resistance, phage therapy has become a promising therapeutic option for combating these ‘superbugs’. Numerous successful cases and clinical trials on phage therapy have been published in the last few years, including: studies on conventional monophage therapy; phage-derived enzymes; synergistic effects of phages, antibiotics and the immune response; and bioengineered phages [4]. However, a lack of randomised controlled trials (RCTs) demonstrating the safety and efficacy of phage therapy, as well as several regulatory issues such as production and marketing authorisation, pose obstacles to its practical use [5].

Patients in intensive care unit (ICU) settings often suffer from ventilator associated pneumonia (VAP) infections caused by MDR Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, Acinetobacter

The classifications are based upon the evaluation of diverse phage properties such as genome composition, morphology, host range, sequence similarity and pathogenicity (Table 1).

Phage therapy is an age old practice of pre-antibiotic era in which bacteriophages are harnessed as bio-agents against bacterial infections.

In 1896 Ernest Hanbury Hankin discovered bacteriophages displaying antibacterial properties against Vibrio cholerae from the water of Indian rivers [6]. Phages were discovered in 1915 and 1917 by Fredrick William Twort and Felix d’Herelle respectively. It was Felix d’Herelle French Canadian microbiologist who coined the name “Bacteriophage” (viruses that kill bacteria) for the first time. After his discovery he suggested that phages could serve as a therapeutic tool. He used phage preparations to successfully treat Shigella dysentery patients in France and Cholera outbreak in India. Since then phage therapy was considered as a significant therapeutic tool and the exclusive treatment for bacterial infections [4].

Bacteriophages are viruses, the most abundant organisms and the natural predators of bacteria. They are self-replicating, obligatory intracellular parasites and inert biochemically in extracellular environment. They control the biosynthetic machinery of bacterial host and behest them to produce different viral proteins. They are considered as particles outside the host cell containing nucleic acid (DNA or RNA) which encode necessary information required for their replication. They are primordial ubiquitous organisms found in diverse environment such as soil, water, feces etc [4,5]. Typically, bacteriophage morphology exhibit well defined three-dimensional structure. The genetic material is enclosed in an icosahedral protein capsid head, a tail (spiral contractile sheath surrounding a core pipe and a baseplate with tail fibers) and surface receptor proteins responsible to recognize specific surface molecules on the host bacterium [5].

Bacteriophages are antibacterial agents ubiquitous in nature. With increase in antibiotic resistance, use of bacteriophages as therapeutics has become resurgent in recent times. This review focuses on the recent developments in phage therapy and its applications with respect to human infections, animal, food and environment. Moreover, use of phage proteins, bioengineered bacteriophages, and phage derived vaccines is also highlighted.