High Multiplicity Of Infection Research Articles

Impact of swine influenza A virus on porcine reproductive and respiratory syndrome virus infection in alveolar macrophages.

Porcine respiratory disease complex represents a major challenge for the swine industry, with swine influenza A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV) being major contributors. Epidemiological studies have confirmed the co-circulation of these viruses in pig herds, making swIAV-PRRSV co-infections expected. A couple of in vivo co-infection studies have reported replication interferences between these two viruses. Herein, using a reductionist in vitro model, we investigated the potential mechanisms of these in vivo interferences. We first examined the impact of swIAV on porcine alveolar macrophages (AMs) and its effects on AMs co-infection by PRRSV. This was done either in monoculture or in co-culture with respiratory tracheal epithelial cells to represent the complexity of the interactions between the viruses and their respective target cells (epithelial cells for swIAV and AMs for PRRSV). AMs were obtained either from conventional or specific pathogen-free (SPF) pigs. SwIAV replication was abortive in AMs, inducing cell death at high multiplicity of infections. In AMs from three out of four conventional animals, swIAV showed no impact on PRRSV replication. However, inhibition of PRRSV multiplication was observed in AMs from one animal, accompanied by an early increase in the expression of interferon (IFN)-I and IFN-stimulated genes. In AMs from six SPF pigs, swIAV inhibited PRRSV replication in all animals, with an early induction of antiviral genes. Co-culture experiments involving tracheal epithelial cells and AMs from either SPF or conventional pigs all showed swIAV-induced inhibition of PRRSV replication, together with early induction of antiviral genes. These findings highlight the complex interactions between swIAV and PRRSV in porcine AMs, and would suggest a role of host factors, such as sanitary status, in modulating viral propagation. Our co-culture experiments demonstrated that swIAV inhibits PRRSV replication more effectively in the presence of respiratory tracheal epithelial cells, suggesting a synergistic antiviral response between AMs and epithelial cells, consistent with in vivo experiments.

Bacteriophages as potential antibiotic potentiators in cystic fibrosis: A new model to study the combination of antibiotics with a bacteriophage cocktail targeting dual species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa

Staphylococcus aureus and Pseudomonas aeruginosa co-infections in patients with cystic fibrosis (CF) are associated with disease severity. Their treatment is complicated by biofilm formation in the sticky mucus obstructing the airways. We investigated the activity of phages-antibiotics combinations using a dual species biofilm (P. aeruginosa/S. aureus) formed in artificial sputum medium. Biofilmswere incubated with broad-spectrum antibiotics (meropenem, ceftazidime, ciprofloxacin, tobramycin) combined with a cocktail of two (bacterio)phages (PSP3 and ISP) proven active via spot tests and double agar on P. aeruginosa PAO1 and S. aureus ATCC 25923. At the highest tested concentrations (100 x MIC), antibiotics alone caused a 20-50% reduction in biomass and reduced S. aureus and P. aeruginosa CFU of 2.3 to 2.8 and 2.1 to 3.6 log10, respectively. Phages alone reduced biofilm biomass by 23% and reduced P. aeruginosa CFU of 2.1 log10, but did not affect S. aureus viability. Phages enhanced antibiotic effects on biomass and exhibited additive effects with antibiotics against P. aeruginosa, but not against S. aureus. Following inhibition of bacterial respiration by phages in planktonic cultures rationalised these observations by demonstrating that PSP3 was effective at multiplicities of infection (MOI) as low as 10-4 plaque forming units (PFU)/CFU on P. aeruginosa, but ISP, at higher MOI (> 0.1) against S. aureus. Pre-screening inhibition of bacterial respiration by phages may assist in selecting those showing activity at sufficiently low titers to showcase anti-biofilm activity in this complex but clinically-relevant in vitro model of biofilm.

Evolution of RNA Viruses: Reasons for the Existence of Separate Plus, Minus, and Double-Strand Replication Strategies.

Plus, minus, and double-strand RNA viruses are all found in nature. We use computational models to study the relative success of these strategies. We consider translation, replication, and virion assembly inside one cell, and transmission of virions between cells. For viruses which do not incorporate a polymerase in the capsid, transmission of only plus strands is the default strategy because virions containing minus strands are not infectious. Packaging only plus strands has a significant advantage if the number of RNA strands produced per cell is larger than the number of capsids. In this case, by not packaging minus strands, the virus produces more plus-strand virions. Therefore, plus-strand viruses are selected at low multiplicity of infection. However, at high multiplicity of infection, it is preferable to package both strands because the additional minus virions produced are helpful when there are multiple infections per cell. The fact that plus-strand viruses are widespread while viruses that package both strands are not seen in nature suggests that RNA strands are indeed produced in excess over capsids, and that the multiplicity of infection is not sufficiently high to favor the production of both kinds of virions. For double-strand viruses, we show that it is advantageous to produce only plus strands from the double strand within the cell, as is observed in real viruses. The reason for the success of minus-strand viruses is more puzzling initially. For viruses that incorporate a polymerase in the virion, minus virions are infectious. However, this is not sufficient to explain the success of minus-strand viruses, because in this case, viruses that package both strands outcompete those that package only minus or only plus. Real minus-strand viruses make use of replicable strands that are coated by a nucleoprotein, and separate translatable plus strands that are uncoated. Here we show that when there are distinct replicable and translatable strands, minus-strand viruses are selected.

A model for accurate quantification of CRISPR effects in pooled FACS screens.

CRISPR screens are powerful tools to identify key genes that underlie biological processes. One important type of screen uses fluorescence activated cell sorting (FACS) to sort perturbed cells into bins based on the expression level of marker genes, followed by guide RNA (gRNA) sequencing. Analysis of these data presents several statistical challenges due to multiple factors including the discrete nature of the bins and typically small numbers of replicate experiments. To address these challenges, we developed a robust and powerful Bayesian random effects model and software package called Waterbear. Furthermore, we used Waterbear to explore how various experimental design parameters affect statistical power to establish principled guidelines for future screens. Finally, we experimentally validated our experimental design model findings that, when using Waterbear for analysis, high power is maintained even at low cell coverage and a high multiplicity of infection. We anticipate that Waterbear will be of broad utility for analyzing FACS-based CRISPR screens.

From multiplicity of infection to force of infection for sparsely sampled Plasmodium falciparum populations at high transmission.

High multiplicity of infection or MOI, the number of genetically distinct parasite strains co-infecting a single human host, characterizes infectious diseases including falciparum malaria at high transmission. It accompanies high asymptomatic Plasmodium falciparum prevalence despite high exposure, creating a large transmission reservoir challenging intervention. High MOI and asymptomatic prevalence are enabled by immune evasion of the parasite achieved via vast antigenic diversity. Force of infection or FOI, the number of new infections acquired by an individual host over a given time interval, is the dynamic sister quantity of MOI, and a key epidemiological parameter for monitoring the impact of antimalarial interventions and assessing vaccine or drug efficacy in clinical trials. FOI remains difficult, expensive, and labor-intensive to accurately measure, especially in high-transmission regions, whether directly via cohort studies or indirectly via the fitting of epidemiological models to repeated cross-sectional surveys. We propose here the application of queuing theory to obtain FOI on the basis of MOI, in the form of either a two-moment approximation method or Little's law. We illustrate these methods with MOI estimates obtained under sparse sampling schemes with the recently proposed " var coding" method, based on sequences of the var multigene family encoding for the major variant surface antigen of the blood stage of malaria infection. The methods are evaluated with simulation output from a stochastic agent-based model, and are applied to an interrupted time-series study from Bongo District in northern Ghana before and immediately after a three-round transient indoor residual spraying (IRS) intervention. We incorporate into the sampling of the simulation output, limitations representative of those encountered in the collection of field data, including under-sampling of var genes, missing data, and usage of antimalarial drug treatment. We address these limitations in MOI estimates with a Bayesian framework and an imputation bootstrap approach. We demonstrate that both proposed methods give good and consistent FOI estimates across various simulated scenarios. Their application to the field surveys shows a pronounced reduction in annual FOI during intervention, of more than 70%. The proposed approach should be applicable to the many geographical locations where cohort or cross-sectional studies with regular and frequent sampling are lacking but single-time-point surveys under sparse sampling schemes are available, and for MOI estimates obtained in different ways. They should also be relevant to other pathogens of humans, wildlife and livestock whose immune evasion strategies are based on large antigenic variation resulting in high multiplicity of infection.

Accumulation Dynamics of Defective Genomes during Experimental Evolution of Two Betacoronaviruses.

Virus-encoded replicases often generate aberrant RNA genomes, known as defective viral genomes (DVGs). When co-infected with a helper virus providing necessary proteins, DVGs can multiply and spread. While DVGs depend on the helper virus for propagation, they can in some cases disrupt infectious virus replication, impact immune responses, and affect viral persistence or evolution. Understanding the dynamics of DVGs alongside standard viral genomes during infection remains unclear. To address this, we conducted a long-term experimental evolution of two betacoronaviruses, the human coronavirus OC43 (HCoV-OC43) and the murine hepatitis virus (MHV), in cell culture at both high and low multiplicities of infection (MOI). We then performed RNA-seq at regular time intervals, reconstructed DVGs, and analyzed their accumulation dynamics. Our findings indicate that DVGs evolved to exhibit greater diversity and abundance, with deletions and insertions being the most common types. Notably, some high MOI deletions showed very limited temporary existence, while others became prevalent over time. We observed differences in DVG abundance between high and low MOI conditions in HCoV-OC43 samples. The size distribution of HCoV-OC43 genomes with deletions differed between high and low MOI passages. In low MOI lineages, short and long DVGs were the most common, with an additional cluster in high MOI lineages which became more prevalent along evolutionary time. MHV also showed variations in DVG size distribution at different MOI conditions, though they were less pronounced compared to HCoV-OC43, suggesting a more random distribution of DVG sizes. We identified hotspot regions for deletions that evolved at a high MOI, primarily within cistrons encoding structural and accessory proteins. In conclusion, our study illustrates the widespread formation of DVGs during betacoronavirus evolution, influenced by MOI and cell- and virus-specific factors.

Epistatic pathways can drive HIV-1 escape from integrase strand transfer inhibitors.

People living with human immunodeficiency virus (HIV) receiving integrase strand transfer inhibitors (INSTIs) have been reported to experience virological failure in the absence of resistance mutations in integrase. To elucidate INSTI resistance mechanisms, we propagated HIV-1 in the presence of escalating concentrations of the INSTI dolutegravir. HIV-1 became resistant to dolutegravir by sequentially acquiring mutations in the envelope glycoprotein (Env) and the nucleocapsid protein. The selected Env mutations enhance the ability of the virus to spread via cell-cell transfer, thereby increasing the multiplicity of infection (MOI). While the selected Env mutations confer broad resistance to multiple classes of antiretrovirals, the fold resistance is ~2 logs higher for INSTIs than for other classes of drugs. We demonstrate that INSTIs are more readily overwhelmed by high MOI than other classes of antiretrovirals. Our findings advance the understanding of how HIV-1 can evolve resistance to antiretrovirals, including the potent INSTIs, in the absence of drug-target gene mutations.

Discovery of a Novel Antiviral Effect of the Restriction Factor SPOC1 against Human Cytomegalovirus.

The chromatin-remodeler SPOC1 (PHF13) is a transcriptional co-regulator and has been identified as a restriction factor against various viruses, including human cytomegalovirus (HCMV). For HCMV, SPOC1 was shown to block the onset of immediate-early (IE) gene expression under low multiplicities of infection (MOI). Here, we demonstrate that SPOC1-mediated restriction of IE expression is neutralized by increasing viral titers. Interestingly, our study reveals that SPOC1 exerts an additional antiviral function beyond the IE phase of HCMV replication. Expression of SPOC1 under conditions of high MOI resulted in severely impaired viral DNA replication and viral particle release, which may be attributed to inefficient viral transcription. With the use of click chemistry, the localization of viral DNA was investigated at late time points after infection. Intriguingly, we detected a co-localization of SPOC1, RNA polymerase II S5P and polycomb repressor complex 2 (PRC2) components in close proximity to viral DNA in areas that are hypothesized to harbor viral transcription sites. We further identified the N-terminal domain of SPOC1 to be responsible for interaction with EZH2, a subunit of the PRC2 complex. With this study, we report a novel and potent antiviral function of SPOC1 against HCMV that is efficient even with unrestricted IE gene expression.

A cytoprotective role for optineurin during mycobacterial infection of macrophages

Autophagy has emerged as a critical innate immune mechanism for host elimination of intracellular pathogens, however, the role of the autophagy receptor Optineurin during mycobacterial infection is not fully understood. To address this lacuna, we infected bone marrow-derived macrophages (BMDMs) derived from Optn+/+ and Optn−/− mice with Mycobacterium smegmatis, and observed the infection outcome at sequential time points. While low multiplicity of infection (MOI) did not show any significant difference between BMDMs from the two groups, at high MOI Optn−/− mice-derived BMDMs showed significantly lower colony forming unit counts, as well as lower cell counts at 12 h and 24 h post-infection. Quantification of cell numbers and nuclear morphologies at various time points post-infection indicated a markedly higher cell death in the Optineurin-deficient macrophages. Optineurin-deficient BMDMs showed significantly lower levels of the autophagosomal protein LC3-II upon infection, indicating a potential role for Optineurin in regulating autophagy during mycobacterial infection. Moreover, when stimulated by bacterial LPS, Optineurin deficient macrophages, showed altered levels of the inflammatory cytokine pro-IL-1β. These observations taken together suggest a novel regulatory role for Optineurin during mycobacterial infection. Its deficiency leads to an impairment in macrophage responses, directly impacting the pathophysiology of infection.

Expression activation of over 70% of Chlamydia trachomatis genes during the first hour of infection.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Vaccinia Virus Defective Particles Lacking the F17 Protein Do Not Inhibit Protein Synthesis: F17, a Double-Edged Sword for Protein Synthesis?

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

Integrator Complex Subunit 3 Knockdown Has Minimal Effect on Lytic Herpes Simplex Virus Type-1 Infection in Fibroblast Cells.

Proteomic analysis of viral and cellular proteins that copurify with the herpes simplex virus type-1 (HSV-1) genome revealed that the cellular Integrator complex associates with viral DNA throughout infection. The Integrator complex plays a key role in the regulation of transcription of cellular coding and non-coding RNAs. We therefore predicted that it may regulate transcription of viral genes. Here, we demonstrate that knockdown of the Integrator complex subunit, Ints3, has minimal effect on HSV-1 infection. Despite reducing viral yield during low multiplicity infection, Ints3 knockdown had no effect on viral DNA replication, mRNA expression, or yield during high multiplicity infection.

Control of Escherichia coli in Fresh-Cut Mixed Vegetables Using a Combination of Bacteriophage and Carvacrol.

The continual emergence of antibiotic-resistant bacteria and the slow development of new antibiotics has driven the resurgent interest in the potential application of bacteriophages as antimicrobial agents in different medical and industrial sectors. In the present study, the potential of combining phage biocontrol and a natural plant compound (carvacrol) in controlling Escherichia coli on fresh-cut mixed vegetable was evaluated. Four coliphages, designated Escherichia phage SUT_E420, Escherichia phage SUT_E520, Escherichia phage SUT_E1520 and Escherichia phage SUT_E1620, were isolated from raw sewage. Biological characterization revealed that all four phages had a latent period of 20-30 min and a burst size ranging from 116 plaque-forming units (PFU)/colony forming units (CFU) to 441 PFU/CFU. The phages effectively inhibited the growth of respective host bacteria in vitro, especially when used at a high multiplicity of infection (MOI). Based on transmission electron microscopy analysis, all phages were classified as tailed phages in the class of Caudoviricetes. Additionally, next generation sequencing indicated that none of the selected coliphages contained genes encoding virulence or antimicrobial resistance factors, highlighting the suitability of isolated phages as biocontrol agents. When a phage cocktail (~109 PFU/mL) was applied alone onto fresh-cut mixed vegetables artificially contaminated with E. coli, no bacteria were recovered from treated samples on Day 0, followed by a gradual increase in the E. coli population after 24 h of incubation at 8 °C. On the other hand, no significant differences (p < 0.05) were observed between treated and non-treated samples in terms of E. coli viable counts when carvacrol at the minimum inhibitory concentration (MIC) of 6.25 μL/mL was applied alone. When a phage cocktail at an MOI of ~1000 and MIC carvacrol were applied in combination, no E. coli were recovered from treated samples on Day 0 and 1, followed by a slight increase in the E. coli population to approximately 1.2-1.3 log CFU/mL after 48 h of incubation at 8 °C. However, total elimination of E. coli was observed in samples treated with a phage cocktail at a higher MOI of ~2000 and carvacrol at MIC, with a reduction of approximately 4 log CFU/mL observed at the end of Day 3. The results obtained in this study highlight the potential of combined treatment involving phage biocontrol and carvacrol as a new alternative method to reduce E. coli contamination in minimally processed ready-to-eat foods.

Impedance In Vitro Assessment for the Detection of Salmonella typhimurium Infection in Intestinal Human Cancer Cells

A significant number of research papers regarding biosensor-related assays for key food safety pathogens based on the use of mammalian cells has been reported. In this study, the Salmonella typhimurium infection progression was monitored in the human colon cancer cell line Caco-2 and the mucus-secreting HT29-MTX-E12, after treatment with five different bacterial MOI for 30 min by comparing the alterations of frequencies recordings with impedance spectroscopy measurements. For this purpose, bacterial adhesion and invasion assays were initially performed. Then, the data obtained from impedance spectroscopy recordings were compared to cell viability data derived from the MTT uptake cell proliferation assay as well as from live cell analysis assays of mitochondrial membrane potential alterations. From our findings a concentration-dependent increase in bacterial colonies occurring from invaded cells was observed upon a higher multiplicity of infection (MOI) bacterial infection at both cell lines. On the contrary, the bacteria infection did not have any impact on the viability of the cells after 1 h of treatment. Differential results were obtained from the measurement of mitochondrial potential at both cell lines. Finally, the impedance values recorded from the 2D, and 3D cultures were concentration-dependent for both cell lines whereas a characteristic pattern specific to each cell line was revealed. Our results indicate that human cell-based bio-electric assays can be a valuable tool for obtaining a unique fingerprint for each bacterial infection in the near future.

A novel coli myophage and antibiotics synergistically inhibit the growth of the uropathogenic E. coli strain CFT073 in stoichiometric niches.

Urinary tract infections are widespread bacterial infections affecting millions of people annually, with Escherichia coli being the most prevalent. Although phage therapy has recently gained interest as a promising alternative therapy for antibiotic-resistant bacteria, several studies have raised concerns regarding the evolution of phage resistance, making the therapy ineffective. In this study, we discover a novel coli myophage designated as Killian that targets E. coli strains, including the uropathogenic E. coli (UPEC) strain CFT073. It requires at least 20 minutes for 90% of its particles to adsorb to the host cells, undergoes subcellular activities for replication for 30 minutes, and eventually lyses the cells with a burst size of about 139 particles per cell. Additionally, Killian can withstand a wide variety of temperatures (4-50°C) and pHs (4-10). Genome analysis reveals that Killian's genome consists of 169,905 base pairs with 35.5% GC content, encoding 276 open reading frames; of these, 209 are functionally annotated with no undesirable genes detected, highlighting its potential as an antibiotic alternative against UPEC. However, after an 8-hour phage treatment at high multiplicities of infection, bacterial density continuously increases, indicating an onset of bacterial growth revival. Thus, the combination study between the phage and three different antibiotics, including amikacin, ciprofloxacin, and piperacillin, was performed and showed that certain pairs of phage and antibiotics exhibited synergistic interactions in suppressing the bacterial growth revival. These findings suggest that Killian-antibiotic combinations are effective in inhibiting the growth of UPEC. IMPORTANCE Phage therapy has recently been in the spotlight as a viable alternative therapy for bacterial infections. However, several studies have raised concerns about the emergence of phage resistance that occurs during treatment, making the therapy not much effective. Here, we present the discovery of a novel E. coli myophage that, by itself, can effectively kill the uropathogenic E. coli, but the emergence of bacterial growth revival was detected during the treatment. Phage and antibiotics are then combined to improve the efficiency of the phage in suppressing the bacterial re-growth. This research would pave the way for the future development of phage-antibiotic cocktails for the sustainable use of phages for therapeutic purposes.

Transcriptomic Analysis of Mycobacterial Infected Macrophages Reveals a High MOI Specific Type I IFN Signaling.

Macrophage (MΦ) infection models are important tools for studying host-mycobacterial interactions. Although the multiplicity of infection (MOI) is an important experimental variable, the selection of MOI in mycobacterial infection experiments is largely empirical, without reference to solid experimental data. To provide relevant data, we used RNA-seq to analyze the gene expression profiles of MΦs 4 or 24 h after infection with Mycobacterium marinum (M. m) at MOIs ranging from 0.1 to 50. Analysis of differentially expressed genes (DEGs) showed that different MOIs are linked to distinct transcriptomic changes and only 10% of DEGs were shared by MΦ infected at all MOIs. KEGG pathway enrichment analysis revealed that type I interferon (IFN)-related pathways were inoculant dose-dependent and enriched only at high MOIs, whereas TNF pathways were inoculant dose-independent and enriched at all MOIs. Protein-protein interaction (PPI) network alignment showed that different MOIs had distinct key node genes. By fluorescence-activated cell sorting and follow-up RT-PCR analysis, we could separate infected MΦs from uninfected MΦs and found phagocytosis of mycobacteria to be the determinant factor for type I IFN production. The distinct transcriptional regulation of RAW264.7 MΦ genes at different MOIs was also seen with Mycobacterium tuberculosis (M.tb) infections and primary MΦ infection models. In summary, transcriptional profiling of mycobacterial infected MΦs revealed that different MOIs activate distinct immune pathways and the type I IFN pathway is activated only at high MOIs. This study should provide guidance for selecting the MOI most appropriate for different research questions.

Establishment of an In Vitro Model to Study Viral Infections of the Fish Intestinal Epithelium.

Viral infections are still a major concern for the aquaculture industry. For salmonid fish, even though breeding strategies and vaccine development have reduced disease outbreaks, viral diseases remain among the main challenges having a negative impact on the welfare of fish and causing massive economic losses for the industry. The main entry port for viruses into the fish is through mucosal surfaces including that of the gastrointestinal tract. The contradictory functions of this surface, both creating a barrier towards the external environment and at the same time being responsible for the uptake of nutrients and ion/water regulation make it particularly vulnerable. The connection between dietary components and viral infections in fish has been poorly investigated and until now, a fish intestinal in vitro model to investigate virus-host interactions has been lacking. Here, we established the permissiveness of the rainbow trout intestinal cell line RTgutGC towards the important salmonid viruses-infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (subtype 3, SAV3) and infectious salmon anemia virus (ISAV)-and explored the infection mechanisms of the three different viruses in these cells at different virus to cell ratios. Cytopathic effect (CPE), virus replication in the RTgutGC cells, antiviral cell responses and viral effects on the barrier permeability of polarized cells were investigated. We found that all virus species infected and replicated in RTgutGC cells, although with different replication kinetics and ability to induce CPE and host responses. The onset and progression of CPE was more rapid at high multiplicity of infection (MOI) for IPNV and SAV3 while the opposite was true of ISAV. A positive correlation between the MOI used and the induction of antiviral responses was observed for IPNV while a negative correlation was detected for SAV3. Viral infections compromised barrier integrity at early time points prior to observations of CPE microscopically. Further, the replication of IPNV and ISAV had a more pronounced effect on barrier function than SAV3. The in vitro infection model established herein can thus provide a novel tool to generate knowledge about the infection pathways and mechanisms used to surpass the intestinal epithelium in salmonid fish, and to study how a virus can potentially compromise gut epithelial barrier functions.

Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug-resistant Salmonella enterica serovar Kentucky.

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens which is isolated in high frequency from poultry meat in the recent decades and is known for its multidrug resistance. The current study was aimed to isolate and characterize a bacteriophage against S. enterica serovar Kentucky isolate, 5925, which showed resistance to at least seven antibiotics and to study its efficiency to decontaminate S. Kentucky from chicken skin. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2 representing the place, source, and host. Electron microscopy revealed that the phage possesses isometric head and contractile tail, indicative of Siphoviridae family. Molecular detection of major capsid protein E gene yielded 511bp, and NCBI blast analysis revealed that the phage belonged to the genus chivirus. The optimum temperature and pH for phage survival and multiplication were found to be - 20 to 42°C and 6-10, respectively. One-step growth curve experiment of vB_SenS_Ib_psk2 revealed a latent period of 20min and burst size of 253 phages/bacterial cell. The host susceptibility studies revealed that 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Artificial spiking studies on chicken skin revealed that high multiplicity of infection (MOI) of phages of 106 pfu/mL is required for significant reduction (p ≤ 0.01) of bacterial concentration (0.14 ± 0.04) after 24-h incubation at 8°C compared to group 1 (2.55 ± 0.89cfu/mL).

Mucin and Agitation Shape Predation of Escherichia coli by Lytic Coliphage.

The ability of bacteriophage (phage), abundant within the gastrointestinal microbiome, to regulate bacterial populations within the same micro-environment offers prophylactic and therapeutic opportunities. Bacteria and phage have both been shown to interact intimately with mucin, and these interactions invariably effect the outcomes of phage predation within the intestine. To better understand the influence of the gastrointestinal micro-environment on phage predation, we employed enclosed, in vitro systems to investigate the roles of mucin concentration and agitation as a function of phage type and number on bacterial killing. Using two lytic coliphage, T4 and PhiX174, bacterial viability was quantified following exposure to phages at different multiplicities of infection (MOI) within increasing, physiological levels of mucin (0-4%) with and without agitation. Comparison of bacterial viability outcomes demonstrated that at low MOI, agitation in combination with higher mucin concentration (>2%) inhibited phage predation by both phages. However, when MOI was increased, PhiX predation was recovered regardless of mucin concentration or agitation. In contrast, only constant agitation of samples containing a high MOI of T4 demonstrated phage predation; briefly agitated samples remained hindered. Our results demonstrate that each phage-bacteria pairing is uniquely influenced by environmental factors, and these should be considered when determining the potential efficacy of phage predation under homeostatic or therapeutic circumstances.

Application of the lytic bacteriophage Rostam to control Salmonella enteritidis in eggs

Foodborne Salmonella enteritidis infections place human health at risk, driven by regular outbreaks and individual cases by different contaminated food materials. This study was conducted to characterize and employ a single bacteriophage as a potential biocontrol agent. Phage Rostam was isolated, characterized and then applied as biocontrol agent against S. enteritidis in liquid whole eggs and eggshell. Rostam is a novel myovirus belonging to the Rosemountvirus genus and active against Escherichia coli and Salmonella spp. Rostam is stable in a pH range from 4 to 10, a salt concentration of 1–9 %, whereas UV radiation gradually reduces phage stability, and its 53 kb genome sequence indicates this phage does not contain known toxins or lysogeny-associated genes. Its latent period is short with a burst size of 151 PFU/cell, under standard growth conditions. Killing curves indicate that at higher multiplicities of infection (MOI), the reduction in S. enteritidis count is more pronounced. Phage Rostam (MOI 10,000) reduces S. enteritidis growth to below the detection limit at 4 °C in both liquid whole eggs and on the eggshell within 24 h. Due to its high lytic activity and stability in relevant conditions, Rostam has the potential to be an efficient biopreservative for egg and egg products.