Comparison of samplers collecting airborne influenza viruses: 1. Primarily impingers and cyclones

Researchers must be able to measure concentrations, sizes, and infectivity of virus-containing particles in animal agriculture facilities to know how far infectious virus-containing particles may travel through air, where they may deposit in the human or animal respiratory tract, and the most effective ways to limit exposures to them. The objective of this study was to evaluate a variety of impinger and cyclone aerosol or bioaerosol samplers to determine approaches most suitable for detecting and measuring concentrations of virus-containing particles in air. Six impinger/cyclone air samplers, a filter-based sampler, and a cascade impactor were used in separate tests to collect artificially generated aerosols of MS2 bacteriophage and swine and avian influenza viruses. Quantification of infectious MS2 coliphage was carried out using a double agar layer procedure. The influenza viruses were titrated in cell cultures to determine quantities of infectious virus. Viral RNA was extracted and used for quantitative real time RT-PCR, to provide total virus concentrations for all three viruses. The amounts of virus recovered and the measured airborne virus concentrations were calculated and compared among the samplers. Not surprisingly, high flow rate samplers generally collected greater quantities of virus than low flow samplers. However, low flow rate samplers generally measured higher, and likely more accurate, airborne concentrations of Infectious virus and viral RNA than high flow samplers. To assess airborne viruses in the field, a two-sampler approach may work well. A suitable high flow sampler may provide low limits of detection to determine if any virus is present in the air. If virus is detected, a suitable lower flow sampler may measure airborne virus concentrations accurately.

Survival assessment of Salmonella enterica in inoculated pork salami

Pork salami is an embedded, cured and ripened product commonly consumed in Brazil, and the presence of Salmonella enterica has already been reported in this product. During its preparation, the microbiological safety depends on the meat quality, addition of ingredients with antimicrobial activity, hygiene during processing, pH and water activity (Aw) reduction during maturation. In Brazil, the maturation protocol has not been determined in food regulation; therefore, the objectives of this study were (a) to identify the fermentation and drying phases during salami maturation; (b) to test the survival of S. enterica during salami processing; and (c) to compare xylose lysine deoxycholate (XLD) and thin agar layer (TAL) agar for recovering Salmonella. The salami samples were prepared with a cocktail of S. enterica strains, fermented at 30°C and dried at 20°C with controlled relative humidity (RH). Periodic sampling for S. enterica quantification and Aw and pH analyses were performed during maturation, and curves were constructed. Fermentation occurred during the first 66 hours, and the pH decreased while the population of S. enterica increased over the first 21 hours. The drying step was able to reduce the bacterial population by approximately 5 log CFU after 875 hours, reaching an Aw of less than 0.78. However, elimination of S. enterica was not achieved. For Salmonella recovery, TAL agar was more efficient than XLD agar.

Isolation of Bacteriophage from Eggs and Use of Excreta as Biocontrol Agent in Controlling Eggs Related Salmonellosis

Poultry and poultry products are the leading causes of foodborne salmonellosis worldwide. Antibiotics are used to control Salmonella spp. in poultry but its uncontrolled use results in the emergence of resistant pathogens.The use of bacteriophages as antimicrobial agents to control antibiotic resistant pathogenic bacteria could be a possible alternative. The aim of this study was to isolate, characterize and evaluate the effectiveness of bacteriophages for reducingload of Salmonella spp. on eggshells. One bacteriophage named as Sal-PE, specific to Salmonella enteritidiswas isolated from poultry excreta. For isolation, samples were subject to an enrichment protocol and then double agar layer method was performed to detect plaque. It had the capability to survive in wide range of pH between 4 to 10and found to be resistant at 60°C for 1 hour. Sal” PE showed its lytic effect on 13 of the 15 (87%) isolates including Salmonella enteritidis and Salmonella typhimurium which were recovered from 50 poultry excreta samples. After enrichment and growth on selective media, isolates were identified based on cultural characteristics, microscopic observation and biochemical tests. Amplification of three different genes (invA, sdfI, fliC) were carried out tocharacterize those isolates in molecular level. All isolates were found to be resistant to penicillin G, ampicillin, oxacillin and clindamycin but sensitive to ciprofloxacin, streptomycin, cefixime and chloramphenicol. Lytic efficiency of Sal-PE was determined by observing the reduction in optical density due to destruction of pathogens. Though more studies are needed in order to evaluate phage effectiveness, our findingsare expected to help us in initiating the development of a better preventive approach to control the occurrence of Salmonella spp. on eggshells. Bangladesh J Microbiol, Volume 35 Number 1 June 2018, pp 37-44

Effect of Different Cell Suspension Culture Densities of Physalis angulata L. by Using Plating or Embedding Methods on the Callus Formation

The study was successful in establishing cell suspensions cultures derived from the hypocotyl callus of the plant Physalis angulata L. induced in the solid MS medium supplemented with the concentration of 3.0 mg. L-1 2,4-Dichlorophenoxy acetic acid (2,4-D) plus 0.5 mg. L-1 Kinetin(Kin). The culture of the suspensions by different densities (12.36, 13.70, 14.75, 15.98)× 105 cell.cm3 using the plating and embedding methods by agar layer in the solid Murashige and Skoog medium (MS) enriched with the same callus induction concentrations, showed that the plating method exceed the embedding method in the number of cellular colonies which reached the average of 79.4 and 15.0 colony.dish-1 at the culturing density of 15.98 × 105 cell.cm3, these values were declined with the primary density to reach the average of 14.0 and 11.4 colony.dish-1 by both the plating and the embedding methods respectively, the high density significantly exceed all the rest densities and gave rate of callus primordia of 56.6 primordia.dish-1 after 28 days from the day of culturing the suspended cells by plating method, and reached 9.8 primordia.dish-1 after 33 days from the day of culturing of the suspended cells by using the embedding method, the transfer of the callus primordia to the MS medium supplemented with 3.0 mg. L-1 2,4-D plus 0.5 mg. L-1 Kin, led to the growth of callus segments and their differentiation to the somatic embryos and their development through all their stages until reaching the shoot formation.

Use of non-linear mixed-effects modelling and regression analysis to predict the number of somatic coliphages by plaque enumeration after 3 hours of incubation

The present study aimed to establish the kinetics of the appearance of coliphage plaques using the double agar layer titration technique to evaluate the feasibility of using traditional coliphage plaque forming unit (PFU) enumeration as a rapid quantification method. Repeated measurements of the appearance of plaques of coliphages titrated according to ISO 10705-2 at different times were analysed using non-linear mixed-effects regression to determine the most suitable model of their appearance kinetics. Although this model is adequate, to simplify its applicability two linear models were developed to predict the numbers of coliphages reliably, using the PFU counts as determined by the ISO after only 3 hours of incubation. One linear model, when the number of plaques detected was between 4 and 26 PFU after 3 hours, had a linear fit of: (1.48 × Counts3 h + 1.97); and the other, values >26 PFU, had a fit of (1.18 × Counts3 h + 2.95). If the number of plaques detected was <4 PFU after 3 hours, we recommend incubation for (18 ± 3) hours. The study indicates that the traditional coliphage plating technique has a reasonable potential to provide results in a single working day without the need to invest in additional laboratory equipment.

Evaluation of the Thin Agar Layer Method for the Recovery of Pressure-Injured and Heat-Injured Listeria monocytogenes

A sublethally injured bacterial cell has been defined as a cell that survives a stress such as heating, freezing, acid treatment, or other antimicrobial intervention but can repair the cellular damage exerted by the stressor and later regain its original ability to grow. Consequently, sublethally injured cells are not likely to be included in conventional enumeration procedures, which could result in unrealistically low counts unless efforts are made to encourage recovery of the injured cells before enumeration. The objective of this study was to evaluate the use of the thin agar layer (TAL) method for the recovery of pressure-injured and heat-injured Listeria monocytogenes in a tryptic soy broth with 0.6% yeast extract system. Pressure injury consisted of treatment of a culture of mixed L. monocytogenes strains with high hydrostatic pressure at 400 or 600 MPa for 1 s, 2 min, 4 min, or 6 min at a process temperature of 12 ± 2 deg;C. Heat injury consisted of treatment of a culture of mixed L. monocytogenes strains at 60 ± 1°C for 3, 6, or 9 min. Growth media were tryptic soy agar (TSA) with 0.6% yeast extract, modified Oxford medium (MOX), and TAL, which consisted of a 7-ml layer of TSA overlaid onto solidified MOX. Counts of viable L. monocytogenes on TAL were higher than those on MOX in the heat-injury experiment but not in the pressure-injury experiment. Therefore, the effectiveness of the TAL method may be specific to the type of injury applied to the microorganism and should be investigated in a variety of cellular injury scenarios.

Effects of Different Nitrite Concentrations from a Vegetable Source with and without High Hydrostatic Pressure on the Recovery of Listeria monocytogenes on Ready-to-Eat Restructured Ham

Sodium nitrite exerts an inhibitory effect on the growth of Listeria monocytogenes. The objective of this study was to investigate the effects of various nitrite concentrations from a vegetable source with and without high hydrostatic pressure (HHP) on the recovery and growth of L. monocytogenes on ready-to-eat restructured ham. A preconverted celery powder was used as the vegetable source of nitrite. Targeted concentrations of natural nitrite investigated were 0, 50, and 100 mg/kg. HHP treatments evaluated were 400 MPa for 4 min and 600 MPa for 1 or 4 min at 12 ± 2°C (initial temperature of the pressurization fluid). Viable L. monocytogenes populations were monitored on modified Oxford medium and thin agar layer medium through 98 days of storage at 4 ± 1°C. Populations on both media did not differ. The HHP treatment at 600 MPa for 4 min resulted in L. monocytogenes populations below the detection limit of our sampling protocols throughout the storage period regardless of the natural nitrite concentration. The combination of HHP at 400 MPa for 4 min or 600 MPa for 1 min with natural nitrite resulted in initial inhibition of viable L. monocytogenes. Ham formulations that did not contain natural nitrite allowed faster growth of L. monocytogenes than did those with nitrite, regardless of whether they were treated with HHP. The results indicate that nitrite from a vegetable source at the concentrations used in this study resulted in slower growth of this microorganism. HHP treatments enhanced the inhibitory effects of natural nitrite on L. monocytogenes growth. Thus, the combination of natural nitrite plus HHP appears to have a synergistic inhibitory effect on L. monocytogenes growth.