Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger

Prophage induction reduces Shiga toxin producing \u3ci\u3eEscherichia coli\u3c/i\u3e (STEC) and Salmonella enterica on tomatoes and spinach: A model study

Fresh produce is increasingly implicated in foodborne outbreaks and most fresh produce is consumed raw, emphasizing the need to develop non-thermal methods to control foodborne pathogens. This study investigates bacterial cell lysis through induction of prophages as a novel approach to control foodborne bacterial pathogens on fresh produce. Shiga toxin producing Escherichia coli (STEC) and Salmonella enterica isolates were exposed to different prophage inducers (i.e. mitomycin C or streptonigrin) and growth of the cells was monitored by measuring the optical density (OD600) during incubation at 37C. Beginning at three hours after addition of the inducer, all concentrations (0.5, 1, 2 mg/mL) of mitomycin C, or 2 mg/mL streptonigrin significantly reduced the OD600 in broth cultures, in a concentration dependent manner, relative to cultures where no inducer was added. PCR confirmed bacterial release of induced bacteriophages and demonstrated that a single compound could successfully induce multiple types of prophages. The ability of mitomycin C to induce prophages in STEC O157:H7 and in S. enterica (serovars Typhimurium and Newport) on fresh produce was evaluated by inoculating red greenhouse tomatoes or spinach leaves with 5 x 107 and 5 x 108 colony forming units, respectively. After allowing time for the inoculum to dry on the fresh produce samples, 6 mg/mL mitomycin C was sprayed onto each sample, while control samples were sprayed with water. Following overnight incubation at 4C, the bacterial cells were recovered and plate counts were performed. A 3 log reduction in STEC O157:H7 cells was observed on tomatoes sprayed with mitomycin C compared to those sprayed with water, while a 1 log reduction was obtained on spinach. Similarly, spraying mitomycin C on tomatoes and spinach inoculated with S. enterica isolates resulted in a 1-1.5 log and 2 log reduction, respectively. These findings serve as a proof of concept that prophage induction can effectively control bacterial foodborne pathogens on fresh produce

Brugia malayi Antigen (BmA) inhibits HIV-1 trans-infection but neither BmA nor ES-62 alter HIV-1 infectivity of DC induced CD4+ Th-cells

One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs

Stage- and Gender-Specific Proteomic Analysis of Brugia malayi Excretory-Secretory Products

To succeed in infection, parasites must have ways to reach the host, penetrate its tissues and escape its defense systems. As they are not necessarily fatal, most helminth parasites remain viable within their host for many years, exerting a strong influence over the host immune function. Many of these functions are performed by products that are released from the parasite. We exploited the remarkable sensitivity of modern proteomics tools together with the availability of a sequenced genome to identify and compare the proteins released in vitro by adult males, adult females and the microfilariae of the filarial nematode Brugia malayi. This parasite is one of the etiological agents of lymphatic filariasis, a disease that poses continuing and significant threats to human health. The different forms of the parasite inhabit different compartments in the mammalian host. We found that the set of proteins released by each form is unique; they must reflect particular developmental processes and different strategies for evasion of host responses. The identification of these proteins will allow us to illuminate the biology of secretory processes in this organism and to establish a path for developing an understanding of how these parasite proteins function in immune evasion events

Bacteriophages Contribute to the Spread of Antibiotic Resistance Genes among Foodborne Pathogens of the Enterobacteriaceae Family – A Review

Foodborne illnesses continue to have an economic impact on global health care systems. There is a growing concern regarding the increasing frequency of antibiotic resistance in foodborne bacterial pathogens and how such resistance may affect treatment outcomes. In an effort to better understand how to reduce the spread of resistance, many research studies have been conducted regarding the methods by which antibiotic resistance genes are mobilized and spread between bacteria. Transduction by bacteriophages (phages) is one of many horizontal gene transfer mechanisms, and recent findings have shown phage-mediated transduction to be a significant contributor to dissemination of antibiotic resistance genes. Here, we review the viability of transduction as a contributing factor to the dissemination of antibiotic resistance genes in foodborne pathogens of the Enterobacteriaceae family, including non-typhoidal Salmonella and Shiga toxin-producing Escherichia coli, as well as environmental factors that increase transduction of antibiotic resistance genes

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger

Bacteriophage-Insensitive Mutants of Antimicrobial-Resistant Salmonella Enterica are Altered in their Tetracycline Resistance and Virulence in Caco-2 Intestinal Cells

Bacteriophages have shown promise as therapeutic alternatives to antibiotics for the control of infectious bacteria, including the human pathogen Salmonella. However, the development of effective phage-based applications requires the elucidation of key interactions between phages and target hosts, particularly since host resistance to phage is inevitable. Little is known about the alteration of host phenotypes following the development of resistance to phage. The aim of this study is to evaluate the antibiotic susceptibility and virulence of a Salmonella isolate following the development of resistance to bacteriophage SI1. We observed enhanced susceptibility to tetracycline and decreased invasion capacity in a differentiated Caco-2 intestinal cell line. Whole genome sequence analysis revealed an array of mutations, most notably, truncations in vgrG1_2, a core gene involved in Type VI secretion and mutations in the lipopolysaccharide, thereby indicating the plausible attachment site of phage SI1. These findings shed light on understanding the underlying mechanism for phage immunity within the host. Importantly, we reveal an associated genetic cost to the bacterial host with developing resistance to phages. Taken together, these results will aid in advancing strategies to delay or eliminate the development of host resistance when designing informed phage-based antimicrobials.Land and Food Systems, Faculty ofOther UBCNon UBCReviewedFacult

Prophage induction reduces Shiga toxin producing \u3ci\u3eEscherichia coli\u3c/i\u3e (STEC) and Salmonella enterica on tomatoes and spinach: A model study

Fresh produce is increasingly implicated in foodborne outbreaks and most fresh produce is consumed raw, emphasizing the need to develop non-thermal methods to control foodborne pathogens. This study investigates bacterial cell lysis through induction of prophages as a novel approach to control foodborne bacterial pathogens on fresh produce. Shiga toxin producing Escherichia coli (STEC) and Salmonella enterica isolates were exposed to different prophage inducers (i.e. mitomycin C or streptonigrin) and growth of the cells was monitored by measuring the optical density (OD600) during incubation at 37C. Beginning at three hours after addition of the inducer, all concentrations (0.5, 1, 2 mg/mL) of mitomycin C, or 2 mg/mL streptonigrin significantly reduced the OD600 in broth cultures, in a concentration dependent manner, relative to cultures where no inducer was added. PCR confirmed bacterial release of induced bacteriophages and demonstrated that a single compound could successfully induce multiple types of prophages. The ability of mitomycin C to induce prophages in STEC O157:H7 and in S. enterica (serovars Typhimurium and Newport) on fresh produce was evaluated by inoculating red greenhouse tomatoes or spinach leaves with 5 x 107 and 5 x 108 colony forming units, respectively. After allowing time for the inoculum to dry on the fresh produce samples, 6 mg/mL mitomycin C was sprayed onto each sample, while control samples were sprayed with water. Following overnight incubation at 4C, the bacterial cells were recovered and plate counts were performed. A 3 log reduction in STEC O157:H7 cells was observed on tomatoes sprayed with mitomycin C compared to those sprayed with water, while a 1 log reduction was obtained on spinach. Similarly, spraying mitomycin C on tomatoes and spinach inoculated with S. enterica isolates resulted in a 1-1.5 log and 2 log reduction, respectively. These findings serve as a proof of concept that prophage induction can effectively control bacterial foodborne pathogens on fresh produce

Characterization of Four Novel Bacteriophages Isolated from British Columbia for Control of Non-typhoidal Salmonella in Vitro and on Sprouting Alfalfa Seeds

Alfalfa sprouts have been linked to numerous North American outbreaks of Salmonella in recent years. Conventionally, treatments involving chlorine, heat, and irradiation are used for alfalfa seed sanitation. However, such treatments may be highly variable in their efficacy for pathogen control and/or detrimental to sprout quality, therefore negatively perceived by consumers advocating for natural alternatives. The usage of bacteriophages for pathogen control in sprouts has been previously explored, although with conflicting and inconsistent results. Lytic phages, viral predators of bacteria, represent an attractive approach as they provide several advantages compared to conventional treatments, such as their high specificity for bacterial targets and their ubiquity in nature. In this study, four Salmonella phages were isolated from British Columbia, Canada and characterized with respect to host range, burst size, latent period, and environmental stability to assess their potential to control Salmonella. Phage isolate SI1 showed the greatest host range, highest burst size and shortest latent period, greatest stability across all pH and temperatures and was the most effective in control of S. Enteritidis in vitro. Therefore, SI1 was chosen for treatment of sprouting alfalfa seeds artificially contaminated with S. Enteritidis with a multiplicity of infection (MOI) of ∼110 PFU/CFU. A significant (p < 0.05) reduction of 38.3 ± 3.0% of viable Salmonella cells was observed following two h of phage treatment. On days two to six of the sprouting process, reductions of Salmonella were also observed, but were not significant compared to the control (p > 0.05). It was further demonstrated that the sprout yield was not significantly (p > 0.05) affected by phage treatment. These results highlight the potential of phages recovered from the British Columbia environment for use as biocontrol agents against Salmonella, although differing efficacies in vitro was observed. Moreover, the effectiveness of SI1 to significantly (p < 0.05) control Salmonella on sprouting alfalfa seeds on day 1 of treatment was demonstrated. Although promising, future work should aim to optimize this treatment to achieve more effective, and longer lasting, biocontrol of Salmonella in sprouting alfalfa seeds