Characterization of Virulent T4-Like Acinetobacter baumannii Bacteriophages DLP1 and DLP2.

The world is currently facing a global health crisis due to the rapid increase in antimicrobial-resistant bacterial infections. One of the most concerning pathogens is Acinetobacter baumannii, which is listed as a Priority 1 pathogen by the World Health Organization. This Gram-negative bacterium has many intrinsic antibiotic resistance mechanisms and the ability to quickly acquire new resistance determinants from its environment. A limited number of effective antibiotics against this pathogen complicates the treatment of A. baumannii infections. A potential treatment option that is rapidly gaining interest is "phage therapy", or the clinical application of bacteriophages to selectively kill bacteria. The myoviruses DLP1 and DLP2 (vB_AbaM-DLP_1 and vB_AbaM-DLP_2, respectively) were isolated from sewage samples using a capsule minus variant of A. baumannii strain AB5075. Host range analysis of these phages against 107 A. baumannii strains shows a limited host range, infecting 15 and 21 for phages DLP1 and DLP2, respectively. Phage DLP1 has a large burst size of 239 PFU/cell, a latency period of 20 min, and virulence index of 0.93. In contrast, DLP2 has a smaller burst size of 24 PFU/cell, a latency period of 20 min, and virulence index of 0.86. Both phages show potential for use as therapeutics to combat A. baumannii infections.

Bacteriophage Isolation, Purification, and Characterization Techniques Against Ubiquitous Opportunistic Pathogens.

Healthcare-associated infection with "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) is a global health crisis due to their extensive intrinsic antibiotic resistance and the ability to quickly acquire resistance determinants. Alternative treatment options are required to combat this crisis, and one possibility is the use of bacteriophages, or viruses that strictly infect the pathogenic bacteria. Currently, there is a renaissance in research and development into the use of phages to target multi-, extensively, and pan-resistant bacterial infections in humans, known as phage therapy. Using A. baumannii as an example, this article describes the isolation and purification of bacteriophages from sewage and soil samples, as well as general methods used in phage research such as precipitation of phages using polyethylene glycol, host range analysis, single-cell burst size determination, DNA extraction, and restriction fragment length polymorphism analysis. © 2022 National Research Council Canada. Current Protocols © 2022 Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Industry. Basic Protocol 1: Isolation of bacteriophages against A. baumannii from sewage samples Alternate Protocol 1: Isolation of bacteriophages against A. baumannii from soil samples Support Protocol 1: Titering a bacteriophage stock Basic Protocol 2: Purification of phage to an axenic working stock Support Protocol 2: Liquid propagation of bacteriophage Basic Protocol 3: Host range analysis using the spot plate method Basic Protocol 4: Single burst size analysis Alternate Protocol 2: One-step growth curve Basic Protocol 5: Precipitation of bacteriophage using PEG 6000 Basic Protocol 6: DNA extraction from dsDNA bacteriophages Basic Protocol 7: Restriction fragment length polymorphism analysis of novel phage genomes.

The Isolation and Characterization of a Broad Host Range Bcep22-like Podovirus JC1.

Bacteriophage JC1 is a Podoviridae phage with a C1 morphotype, isolated on host strain Burkholderia cenocepacia Van1. Phage JC1 is capable of infecting an expansive range of Burkholderia cepacia complex (Bcc) species. The JC1 genome exhibits significant similarity and synteny to Bcep22-like phages and to many Ralstonia phages. The genome of JC1 was determined to be 61,182 bp in length with a 65.4% G + C content and is predicted to encode 76 proteins and 1 tRNA gene. Unlike the other Lessieviruses, JC1 encodes a putative helicase gene in its replication module, and it is in a unique organization not found in previously analyzed phages. The JC1 genome also harbours 3 interesting moron genes, that encode a carbon storage regulator (CsrA), an N-acetyltransferase, and a phosphoadenosine phosphosulfate (PAPS) reductase. JC1 can stably lysogenize its host Van1 and integrates into the 5' end of the gene rimO. This is the first account of stable integration identified for Bcep22-like phages. JC1 has a higher global virulence index at 37 °C than at 30 °C (0.8 and 0.21, respectively); however, infection efficiency and lysogen stability are not affected by a change in temperature, and no observable temperature-sensitive switch between lytic and lysogenic lifestyle appears to exist. Although JC1 can stably lysogenize its host, it possesses some desirable characteristics for use in phage therapy. Phage JC1 has a broad host range and requires the inner core of the bacterial LPS for infection. Bacteria that mutate to evade infection by JC1 may develop a fitness disadvantage as seen in previously characterized LPS mutants lacking inner core.

Aztreonam Lysine Increases the Activity of Phages E79 and phiKZ against Pseudomonas aeruginosa PA01.

Pseudomonas aeruginosa is a pernicious bacterial pathogen that is difficult to treat because of high levels of antibiotic resistance. A promising alternative treatment option for such bacteria is the application of bacteriophages; the correct combination of phages plus antibiotics can produce synergistic inhibitory effects. In this study, we describe morphological changes induced by sub-MIC levels of the antibiotic aztreonam lysine (AzLys) on P. aeruginosa PA01, which may in part explain the observed phage-antibiotic synergy (PAS). One-step growth curves for phage E79 showed increased adsorption rates, decreased infection latency, accelerated time to lysis and a minor reduction in burst size. Phage E79 plus AzLys PAS was also able to significantly reduce P. aeruginosa biofilm growth over 3-fold as compared to phage treatment alone. Sub-inhibitory AzLys-induced filamentation of P. aeruginosa cells resulted in loss of twitching motility and a reduction in swimming motility, likely due to a reduction in the number of polar Type IV pili and flagella, respectively, on the filamented cell surfaces. Phage phiKZ, which uses Type IV pili as a receptor, did not exhibit increased activity with AzLys at lower sub-inhibitory levels, but still produced phage-antibiotic synergistic killing with sub-inhibitory AzLys. A one-step growth curve indicates that phiKZ in the presence of AzLys also exhibits a decreased infection latency and moderately undergoes accelerated time to lysis. In contrast to prior PAS studies demonstrating that phages undergo delayed time to lysis with cell filamentation, these PAS results show that phages undergo accelerated time to lysis, which therefore suggests that PAS is dependent upon multiple factors, including the type of phages and antibiotics used, and the bacterial host being tested.

Duckweed (Lemna minor) and Alfalfa (Medicago sativa) as Bacterial Infection Model Systems.

Alternative animal host models of bacterial infection have been developed which reproduce some of the disease conditions observed in higher animals. Analogously, plants are useful for modeling bacterial pathogenesis, in some cases revealing broadly conserved infection mechanisms. Similar to animals, plants have been shown to possess innate immune systems that respond to invading viruses, bacteria, and fungi. Plant infection models often yield results faster, are more convenient, and less expensive than many animal infection models. Here, we describe the use of two different plant-based infection models for the discovery of virulence genes and factors involved in bacterial pathogenesis.