Jinyu Shan

Diverse Temperate Bacteriophage Carriage in Clostridium difficile 027 Strains

Background The hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027. Methodology/Principal Findings We induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species. Conclusion/Significance A diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes.

Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile

ABSTRACT Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation.

Infection by the ‘photosynthetic’ phage S‐PM2 induces increased synthesis of phycoerythrin in Synechococcus sp. WH7803

Phycoerythrin-containing Synechococcus strains are unicellular cyanobacteria that are of great ecological importance in the marine environment. These organisms are known to be susceptible to infection by cyanophages (viruses that infect cyanobacteria). The infection cycle takes several hours and during this time the cyanophages may potentially modify the cyanobacterial light-harvesting apparatus. This study based on a model system consisting of Synechococcus sp. WH7803 and cyanophage S-PM2 revealed a progressive increase in the content of phycoerythrin per cell and per phycobilisome postinfection using absorption and emission spectrophotometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. An increased cellular content of chlorophyll a was also revealed using absorption spectrophotometry. The transcript levels of the phycoerythrin-coding operons, mpeBA and cpeBA, were found to increase after phage infection using quantitative real-time PCR. This phage-induced increase in light-harvesting capacity could potentially increase the photosynthetic activity of the host to satisfy the phages energy demand for reproduction.

Isolation and characterization of a novel podovirus which infects burkholderia pseudomallei

Burkholderia pseudomallei is a saprophytic soil bacterium and the etiological agent that causes melioidosis. It is naturally resistant to many antibiotics and therefore is difficult to treat. Bacteriophages may provide an alternative source of treatment. We have isolated and characterised the bacteriophage ΦBp-AMP1. The phage is a member of the Podoviridae family and has a genome size of ~ 45 Kb. Molecular data based on the gene which encodes for the phage tail tubular protein suggests that the phage is distinct from known phages but related to phages which infect B. thailandensis and Ralstonia spp. The phage ΦBp-AMP1 is the first B. pseudomallei podovirus to be isolated from the environment rather than being induced from a bacterial culture. It has a broad host range within B. pseudomallei and can infect all 11 strains that we tested it on but not related Burkholderia species. It is heat stable for 8 h at 50°C but not stable at 60°C. It may potentially be a useful tool to treat or diagnose B. pseudomallei infections as it can lyse several strains of clinical relevance.

Temperature dependent bacteriophages of a tropical bacterial pathogen.

There is an increasing awareness of the multiple ways that bacteriophages (phages) influence bacterial evolution, population dynamics, physiology, and pathogenicity. By studying a novel group of phages infecting a soil borne pathogen, we revealed a paradigm shifting observation that the phages switch their lifestyle according to temperature. We sampled soil from an endemic area of the serious tropical pathogen Burkholderia pseudomallei, and established that podoviruses infecting the pathogen are frequently present in soil, and many of them are naturally occurring variants of a common virus type. Experiments on one phage in the related model B. thailandensis demonstrated that temperature defines the outcome of phage-bacteria interactions. At higher temperatures (37°C), the phage predominantly goes through a lytic cycle, but at lower temperatures (25°C), the phage remains temperate. This is the first report of a naturally occurring phage that follows a lytic or temperate lifestyle according to temperature. These observations fundamentally alter the accepted views on the abundance, population biology and virulence of B. pseudomallei. Furthermore, when taken together with previous studies, our findings suggest that the phenomenon of temperature dependency in phages is widespread. Such phages are likely to have a profound effect on bacterial biology, and on our ability to culture and correctly enumerate viable bacteria.

Infection by the 'photosynthetic' phage S-PM2 induces increased synthesis of phycoerythrin in Synechococcus sp. WH7803

Phycoerythrin-containing Synechococcus strains are unicellular cyanobacteria that are of great ecological importance in the marine environment. These organisms are known to be susceptible to infection by cyanophages (viruses that infect cyanobacteria). The infection cycle takes several hours and during this time the cyanophages may potentially modify the cyanobacterial light-harvesting apparatus. This study based on a model system consisting of Synechococcus sp. WH7803 and cyanophage S-PM2 revealed a progressive increase in the content of phycoerythrin per cell and per phycobilisome postinfection using absorption and emission spectrophotometry and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. An increased cellular content of chlorophyll a was also revealed using absorption spectrophotometry. The transcript levels of the phycoerythrin-coding operons, mpeBA and cpeBA, were found to increase after phage infection using quantitative real-time PCR. This phage-induced increase in light-harvesting capacity could potentially increase the photosynthetic activity of the host to satisfy the phages energy demand for reproduction.

Bacteriophages are more virulent to bacteria with human cells than they are in bacterial culture; insights from HT-29 cells

Bacteriophage therapeutic development will clearly benefit from understanding the fundamental dynamics of in vivo phage-bacteria interactions. Such information can inform animal and human trials, and much can be ascertained from human cell-line work. We have developed a human cell-based system using Clostridium difficile, a pernicious hospital pathogen with limited treatment options, and the phage phiCDHS1 that effectively kills this bacterium in liquid culture. The human colon tumorigenic cell line HT-29 was used because it simulates the colon environment where C. difficile infection occurs. Studies on the dynamics of phage-bacteria interactions revealed novel facets of phage biology, showing that phage can reduce C. difficile numbers more effectively in the presence of HT-29 cells than in vitro. Both planktonic and adhered Clostridial cell numbers were successfully reduced. We hypothesise and demonstrate that this observation is due to strong phage adsorption to the HT-29 cells, which likely promotes phage-bacteria interactions. The data also showed that the phage phiCDHS1 was not toxic to HT-29 cells, and phage-mediated bacterial lysis did not cause toxin release and cytotoxic effects. The use of human cell lines to understand phage-bacterial dynamics offers valuable insights into phage biology in vivo, and can provide informative data for human trials.

Preparation of RNA from Bacteria Infected with Bacteriophages: A Case Study from the Marine Unicellular Synechococcus sp. WH7803 Infected by Phage S-PM2

Bacteriophages manipulate bacterial gene expression in order to express their own genes or influence bacterial metabolism. Gene expression can be studied using real-time PCR or microarrays. Either technique requires the prior isolation of high quality RNA uncontaminated by the presence of genomic DNA. We outline the considerations necessary when working with bacteriophage infected bacterial cells. We also give an example of a protocol for extraction and quantification of high quality RNA from infected bacterial cells, using the marine cyanobacterium WH7803 and the phage S-PM2 as a case study. This protocol can be modified to extract RNA from the host/bacteriophage of interest.

Temperature-dependent virus lifecycle choices may reveal and predict facets of the biology of opportunistic pathogenic bacteria.

Melioidosis, a serious illness caused by Burkholderia pseudomallei, results in up to 40% fatality in infected patients. The pathogen is found in tropical water and soil. Recent findings demonstrated that bacterial numbers can be regulated by a novel clade of phages that are abundant in soil and water. These phages differentially infect their bacterial hosts causing lysis at high temperatures and lysogeny at lower temperatures. Thus seasonal and daily temperature variations would cause switches in phage-bacteria interactions. We developed mathematical models using realistic parameters to explore the impact of phages on B. pseudomallei populations in the surface water of rice fields over time and under seasonally changing environmental conditions. Historical records were used to provide UV radiation levels and temperature for two Thailand provinces. The models predict seasonal variation of phage-free bacterial numbers correlates with the higher risk of melioidosis acquisition during the “warm and wet” season. We find that enrichment of the environment may lead to irregular large amplitude pulses of bacterial numbers that could significantly increase the probability of disease acquisition. Our results suggest that the phages may regulate B. pseudomallei populations throughout the seasons, and these data can potentially help improve the melioidosis prevention efforts in Southeast Asia.