Evaluating the efficacy of a phage cocktail against Pseudomonas fluorescens group strains in raw milk: microbiological, physical, and chemical analyses.

The objective of this study was to investigate the effectiveness of a phage cocktail against Pseudomonas fluorescens group and its effect on the microbial, physical and chemical properties of raw milk during different storage conditions. A phage cocktail consisting of Pseudomonas fluorescens, Pseudomonas tolaasii, and Pseudomonas libanensis phages was prepared. As a result, reductions in fluorescent Pseudomonas counts of up to 3.44 log units for the storage at 4 °C and 2.38 log units for the storage at 25 °C were achieved. Following the phage application, it is found that there was no significant difference in the total mesophilic aerobic bacteria and Enterobacteriaceae counts. However, it was observed that the number of lactic acid bacteria was higher in phage-treated groups. The results also showed that pH values in the phage added groups were lower than the others and the highest titratable acidity was obtained only in the bacteria-inoculated group. As a future perspective, this study suggests that, while keeping the number of target microorganisms under control in the milk with the use of phages during storage, the microbiota and accordingly the quality parameters of the milk can be affected. This work contributes to the development of effective strategies for maintaining the quality and extending the shelf life of milk and dairy products.

Evaluation of antibacterial efficacy of Lawsonia inermis Linn (henna) on periodontal pathogens using agar well diffusion and broth microdilution methods: An in-vitro study.

Background: Although widely explored in medicine, limited evidence exists in the literature regarding the efficacy of Lawsonia inermis Linn (henna) in the dental field. Aim: This study aimed to investigate the antibacterial effect of henna on Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in vitro. Methods: The agar well diffusion and broth microdilution methods were used to evaluate the antibacterial effect of henna extracts. Dimethyl sulfoxide was used to prepare the ethanol extract of henna, and distilled water was used to prepare the water extract. For both ethanol and water extracts, 4 different concentrations were prepared as 15, 30, 60, and 120 mg/mL. Results: It was determined that the water and ethanol extracts of the henna samples did not show an inhibition zone on P.gingivalis and A.actinomycetemcomitans. As a result of the evaluations made with the broth microdilution method, it was found that the ethanol extract had a higher inhibitory effect on both bacteria, and both extracts had more inhibitory effects against A.actinomycetemcomitans. Conclusion: To understand the effect of henna on periodontal pathogens, more comprehensive in vitro studies should be performed on henna samples at different concentrations and with different bases.

Isolation, characterization, and application of bacteriophage cocktails for the biocontrol of Pseudomonas fluorescens group strains in whole and skimmed milk.

Pseudomonas fluorescens group strains can lead to spoilage of milk as well as loss of quality in dairy products through their heat-resistant enzymes. Phages are important alternatives for combating spoilage bacteria in food industry and used successfully in many applications. The aim of this study was the isolation and characterization of phages and to assess the efficiency of a phage cocktail in whole and skimmed milk. For this purpose, phages effective against Pseudomonas fluorescens (L23.2), Pseudomonas tolaasii (P22.1), and Pseudomonas rhodesiae (A11.1) were isolated. Their host range was found to be highly specific, and the transmission electron micrographs indicates that they belonged to Tectiviridae family. Their genome sizes were found to be vary between 38.3 and 53.5 kb. The latent periods and burst sizes were determined as 15, 10, 15 min and 91, 20, 80 PFU/infected cell for L23.2, P22.1, and A11.1, respectively. All three phages were found to be sensitive to low pH and high temperature. The effect of the phage cocktail was monitored in milk with different fat contents during storage at 4 °C for 5 days. As a result, bacterial reductions up to 4.09 and 5.29 log-units were observed for the whole and skimmed milk, respectively. Thus, the efficacy of a phage cocktail against a bacterial mixture of different P. fluorescens strains was tested in milk samples with different fat contents in accordance with real-life scenarios for the first time.

Applications of Bacteriophage Cocktails to Reduce Salmonella Contamination in Poultry Farms.

Salmonella contamination is a critical problem in poultry farms, with serious consequences for both animals and food products. The aim of this study is to investigate the use of phage cocktails to reduce Salmonella contamination in poultry farms. Within the scope of the study, Salmonella phages were isolated from chicken stool. After the host range of phages was determined, morphological characterization was performed through transmission electron microscopy analysis. Then, replication parameters and adsorption rates were determined by one-step growth curves. After that, phage cocktail was prepared, and its effectiveness was tested in three environments, which were drinking water, shavings, and plastic surfaces. The results obtained have demonstrated that the phage cocktail can reduce Salmonella count up to 2.80 log10 units in drinking water, up to 2.30 log10 units on shavings, and 2.31 log10 units on plastic surfaces. It has been determined that phage cocktails could be a successful alternative in reducing Salmonella contamination in poultry environment. This work is the first study to investigate the use of phage cocktails for reducing Salmonella contamination in poultry water and on shavings, and it is presumed that the results obtained will contribute to the fight against pathogens by making them applicable to poultry farms.

Investigation of phage and molasses interactions for the biocontrol of E. coli O157:H7.

Resistance to antibiotics is one of the most critical health problems in the world. Therefore, finding new treatment methods to be used as alternatives to antibiotics has become a priority for researchers. Similar to phages, certain products containing antimicrobial components, such as molasses, are widely used to eliminate resistant bacteria. Molasses has a strong antimicrobial effect on bacterial cells, and this effect is thought to be due to the breakdown of the cytoplasmic cell membrane and cell proteins of the polyphenols in molasses. In the present study, phage-molasses interactions were investigated to examine the effects of concomitant use. It was found that molasses samples increased the size of phage plaques by up to 3-fold, and MIC and 1/2 × MIC concentrations of molasses increased the burst size of phages. Although no synergistic effect was found between the phage and molasses, the antimicrobial activities of the components and the effect of molasses on phage activity were demonstrated.

Replacement of antibodies with bacteriophages in lateral flow assay of Salmonella Enteritidis.

In this study, the analytical performance of bacteriophages for Salmonella Enteritidis was investigated using lateral flow assay (LFA) technique. The analytical performance characteristics of bacteriophages were compared with antibodies which are regularly used as analyte-specific agents in the lateral flow immunoassay test strip. Bacteriophages could be an alternative analyte-specific agents to antibodies in lateral flow assay testing of bacteria since they offer comparable sensitivity, specificity, and accuracy. In the present study, Surface Enhanced Raman Spectroscopy (SERS) and colorimetric measurements were combined in one platform and sensitive quantitation of target bacteria was accomplished with a total quantitative analysis time of less than 30 min. The developed Salmonella Enteritidis F5-4 phage-based LFA specifically responds to Salmonella Enteritidis, while lower SERS responses to different bacteria types including Bacillus subtilis, Micrococcus luteus, Escherichia coli, Salmonella Typhimurium were observed. The developed test strips were also applied for the determination of Salmonella Enteritidis in spiked chicken and egg samples.

A disposable gold-cellulose nanofibril platform for SERS mapping.

In this study, we present a disposable and inexpensive paper-like gold nanoparticle-embedded cellulose nanofibril substrate for the rapid enumeration of Escherichia coli (E. coli) using surface-enhanced Raman scattering (SERS) mapping. A disposable SERS substrate was simply constructed by mixing CNF and gold chloride solution at 120 °C in a water bath. The application of the resulting substrate was carried out by enrichment and SERS detection of E. coli. To this end, the spherical gold nanoparticle-embedded cellulose nanofibril substrate was used as a scavenger for E. coli. After the target bacteria E. coli were separated from the matrix via oriented antibodies, the sandwich assay procedure was carried out using 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)-coated Au nanorod particles that acted as SERS mapping probes. The distribution density of DTNB was demonstrated visually using SERS mapping, and the assay was completed in one hour. The correlation between the E. coli and SERS mapping signals was found to be linear within the range of 15 cfu mL-1 to 1.5 × 105 cfu mL-1. The limit of detection for the SERS mapping assay was determined to be 2 cfu mL-1. The selectivity of the developed method was examined with Micrococcus luteus (M. luteus), Bacillus subtilis (B. subtilis), and Enterobacter aerogenes (E. aerogenes), which did not produce any significant response. Furthermore, the developed method was evaluated for detecting E. coli in artificially contaminated samples, and the results were compared with those of the plate-counting method.

Microencapsulation of phages to analyze their demeanor in physiological conditions.

Nowadays, phage therapy emerges as one of the alternative solutions to the problems arising from antibiotic resistance in pathogenic bacteria. Although phage therapy has been successfully applied both in vitro and in vivo, one of the biggest concerns in this regard is the stability of phages in body environment. Within the scope of this study, microencapsulation technology was used to increase the resistance of phages to physiological conditions, and the resulting microcapsules were tested in environments simulating body conditions. For this purpose, Bacillus subtilis, Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis), and Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella Typhimurium) phages were isolated from different sources and then microencapsulated with 1.33% (w/v) sodium alginate using a spray dryer to minimize the damage of physiological environment. Stability of microcapsules in simulated gastric fluid and bile salt presence was tested. As a consequence, the maximum titer decrease of microencapsulated phages after 2-h incubation was found to be 2.29 log unit for B. subtilis phages, 1.71 log unit for S. Enteritidis phages, and 0.60 log unit for S. Typhimurium phages, while free phages lost their viability even after a 15-min incubation. Similarly, microencapsulation was found to increase the stability of phages in the bile salt medium and it was seen that after 3 h of incubation, the difference between the titers of microencapsulated phages and free phages could reach up to 3 log unit.

A Raman-spectroscopy-based approach for detection and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages at low titer in raw milk.

In this study, a method combining Raman spectroscopy with chemometric analysis was developed for detection of phage presence in raw milk and discrimination of Streptococcus thermophilus and Lactobacillus bulgaricus phages which are among the main phages causing problems in dairy industry. For this purpose, S. thermophilus and L. bulgaricus phages were added into raw milk separately, and then some pretreatments such as fat separation, removal of casein, and filtration were applied to the raw milk samples. Raman spectra of the samples were collected and then analyzed using principal component analysis in order to discriminate these phages in raw milk. In the next step, dilutions of S. thermophilus phages in pretreated raw milk were prepared, and Raman spectra were collected. These spectra were analyzed by using partial least squares method to quantify phages in low titer. Consequently, it has been demonstrated that S. thermophilus and L. bulgaricus phages, which have titers sufficient to fail the fermentation (~ 107 pfu/mL) and have lower titers (102-103 pfu/mL), could be discriminated from antibiotic and each other. Additionally, low concentrations of S. thermophilus phages (102 pfu/mL) could be detected through Raman spectroscopy with a short analysis time (60 min) and high coefficient of determination (R2) values for both calibration (0.985) and validation (0.906) with a root mean square error of calibration of 70.54 and root mean square error of prediction of 165.47. However, a lower success was achieved with L. bulgaricus phages and the obtained coefficient of determination values were not sufficiently high (0.649).

A simple and fast method for discrimination of phage and antibiotic contaminants in raw milk by using Raman spectroscopy.

Phage and antibiotic in raw milk poses significant risks for starter culture activity in fermented products. Therefore, rapid detection of phage and antibiotic contaminations in raw milk is a crucial process in dairy science. For this purpose, a preliminary novel method for detection of phage and antibiotic was developed by using Raman spectroscopy. Streptococcus thermophilus phages and ampicillin which are quite important elements in dairy industry were used as model. The phage and antibiotic samples were added to raw milk separately, and Raman measurements were carried out. The obtained spectra were processed with a chemometric method. In this study, it has been demonstrated that the presence of phage has a titer sufficient to stop the fermentation (107 pfu/ml), and antibiotic in a concentration which inhibits the growth of starter cultures (0.5 µg/ml) in raw milk could be discriminated through Raman spectroscopy with a short analysis time (30 min).