Future research priorities for soft-tissue knee injuries.

Aims: To identify unanswered questions about the prevention, diagnosis, treatment, and rehabilitation and delivery of care of first-time soft-tissue knee injuries (ligament injuries, patella dislocations, meniscal injuries, and articular cartilage) in children (aged 12 years and older) and adults. Methods: The James Lind Alliance (JLA) methodology for Priority Setting Partnerships was followed. An initial survey invited patients and healthcare professionals from the UK to submit any uncertainties regarding soft-tissue knee injury prevention, diagnosis, treatment, and rehabilitation and delivery of care. Over 1,000 questions were received. From these, 74 questions (identifying common concerns) were formulated and checked against the best available evidence. An interim survey was then conducted and 27 questions were taken forward to the final workshop, held in January 2023, where they were discussed, ranked, and scored in multiple rounds of prioritization. This was conducted by healthcare professionals, patients, and carers. Results: The top ten included questions regarding prevention, diagnosis, treatment, and rehabilitation. The number one question was, 'How urgently do soft-tissue knee injuries need to be treated for the best outcome?'. This reflects the concerns of patients, carers, and the wider multidisciplinary team. Conclusion: This validated process has generated ten important priorities for future soft-tissue knee injury research. These have been submitted to the National Institute for Health and Care Research. All 27 questions in the final workshop have been published on the JLA website.

Environmental persistence of Listeria monocytogenes and its implications in dairy processing plants.

Listeriosis, an invasive illness with a fatality rate between 20% and 30%, is caused by the ubiquitous bacterium Listeria monocytogenes. Human listeriosis has long been associated with foods. This is because the ubiquitous nature of the bacteria renders it a common food contaminant, posing a significant risk to the food processing sector. Although several sophisticated stress coping mechanisms have been identified as significant contributing factors toward the pathogen's persistence, a complete understanding of the mechanisms underlying persistence across various strains remains limited. Moreover, aside from genetic aspects that promote the ability to cope with stress, various environmental factors that exist in food manufacturing plants could also contribute to the persistence of the pathogen. The objective of this review is to provide insight into the challenges faced by the dairy industry because of the pathogens' environmental persistence. Additionally, it also aims to emphasize the diverse adaptation and response mechanisms utilized by L. monocytogenes in food manufacturing plants to evade environmental stressors. The persistence of L. monocytogenes in the food processing environment poses a serious threat to food safety and public health. The emergence of areas with high levels of L. monocytogenes contamination could facilitate Listeria transmission through aerosols, potentially leading to the recontamination of food, particularly from floors and drains, when sanitation is implemented alongside product manufacturing. Hence, to produce safe dairy products and reduce the frequency of outbreaks of listeriosis, it is crucial to understand the factors that contribute to the persistence of this pathogen and to implement efficient control strategies.

Development of Value-Added Butter by Incorporating Whey Protein Hydrolysate-Encapsulated Probiotics.

The probiotic foods market is growing exponentially; however, probiotics' survivability and interaction with product attributes pose major challenges. A previous study of our lab developed a spray-dried encapsulant utilizing whey protein hydrolysate-maltodextrin and probiotics with high viable counts and enhanced bioactive properties. Viscous products such as butter could be suitable carriers for such encapsulated probiotics. The objective of the current study was to standardize this encapsulant in salted and unsalted butter, followed by storage stability studies at 4 °C. Butter was prepared at a lab-scale level, and the encapsulant was added at 0.1% and 1%, followed by physiochemical and microbiological characterization. Analyses were conducted in triplicates, and means were differentiated (p < 0.05). The viability of probiotic bacteria and the physicochemical characteristics of the butter samples with 1% encapsulant were significantly higher as compared to 0.1%. Furthermore, the 1% encapsulated probiotics butter variant showed a relatively higher stability of probiotics ratio (LA5 and BB12) than the control with unencapsulated probiotics during storage conditions. Although the acid values increased along with a mixed trend of hardness, the difference was insignificant. This study thus provided a proof of concept for incorporating encapsulated probiotics in salted and unsalted butter samples.

Development and Control of Biofilms: Novel Strategies Using Natural Antimicrobials.

Separation membranes have a wide application in the food industry, for instance, in the clarification/fractionation of milk, the concentration/separation of selected components, and wastewater treatment. They provide a large area for bacteria to attach and colonize. When a product comes into contact with a membrane, it initiates bacterial attachment/colonization and eventually forms biofilms. Several cleaning and sanitation protocols are currently utilized in the industry; however, the heavy fouling of the membrane over a prolonged duration affects the overall cleaning efficiency. In view of this, alternative approaches are being developed. Therefore, the objective of this review is to describe the novel strategies for controlling membrane biofilms such as enzyme-based cleaner, naturally produced antimicrobials of microbial origin, and preventing biofilm development using quorum interruption. Additionally, it aims to report the constitutive microflora of the membrane and the development of the predominance of resistant strains over prolonged usage. The emergence of predominance could be associated with several factors, of which, the release of antimicrobial peptides by selective strains is a prominent factor. Therefore, naturally produced antimicrobials of microbial origin could thus provide a promising approach to control biofilms. Such an intervention strategy could be implemented by developing a bio-sanitizer exhibiting antimicrobial activity against resistant biofilms.

A review of oleogels applications in dairy foods.

The characteristics of dairy products, such as texture, color, flavor, and nutritional profile, are significantly influenced by the presence of milk fat. However, saturated fatty acids account for 65% of total milk fat. With increased health awareness and regulatory recommendations, consumer preferences have evolved toward low/no saturated fat food products. Reducing the saturated fat content of dairy products to meet market demands is an urgent yet challenging task, as it may compromise product quality and increase production costs. In this regard, oleogels have emerged as a viable milk fat replacement in dairy foods. This review focuses on recent advances in oleogel systems and explores their potential for incorporation into dairy products as a milk fat substitute. Overall, it can be concluded that oleogel can be a potential alternative to replace milk fat fully or partially in the product matrix to improve nutritional profile by mimicking similar rheological and textural product characteristics as milk fat. Furthermore, the impact of consuming oleogel-based dairy foods on digestibility and gut health is also discussed. A thorough comprehension of the application of oleogels in dairy products will provide an opportunity for the dairy sector to develop applications that will appeal to the changing consumer needs.

A fluorescence spectroscopic method for rapid detection of bacterial endospores: Proof of concept.

Current spore detection methods rely on culture techniques, with limitations of time, efficiency, and sensitivity. The bacterial spore coat contains calcium dipicolinic acid (CaDPA) as a major constituent, which could serve as a biomarker for bacterial endospores. We report proof of concept for a rapid and sensitive technique for the detection of bacterial endospores by using ratiometric fluorescence-based sensors. This method is based on the detection of CaDPA, which enhances the luminescence of lanthanide ions when complexed with a semiconducting polymer. A CaDPA standard curve was generated at an excitation-emission wavelength (λ) of λ275-λ544 by using a spectrophotometer. The intensity was recorded after chelating semiconducting fluorescent polyfluorene (PFO) dots with terbium (lanthanide) ions, sensitized by different volumes of CaDPA (0.1 µM). The resultant standard curve showed a linear relationship (R2 = 0.98) in the experimental concentration range of 2.5 to 25 nM CaDPA, with corresponding intensity (arbitrary units) of 545 to 2,130. Endospores of the aerobic sporeformer Bacillus licheniformis ATCC 14580 were produced at 37°C for 15 d on brain heart infusion agar plates. The efficiency of sporulation was evaluated by spore staining and plating techniques. Total CaDPA content on spores was estimated after suspending decreasing concentrations of spores (logs 9.0 through 1.0 cfu/mL, at 1-log intervals) in HPLC-grade water (to serve as control) and skim milk samples. In HPLC-grade water, for higher spiking levels such as (mean ± SD) 9.2 ± 0.03, 8.4 ± 0.05, 7.1 ± 0.13, and 6.3 ± 0.02 logs, the corresponding mean CaDPA from the standard curve were 9.4, 7.2, 6.2, and 5.3 nM, respectively. For lower spiking levels of 4.2 ± 0.05, 3.1 ± 0.04, 2.0 ± 0.11, and 1.36 ± 0.09 logs, we observed mean CaDPA contents of 3.8, 3.3, 2.2, and 1.3 nM, respectively. For raw skim milk spiked with B. licheniformis ATCC 14580 spores, the mean CaDPA content on spores was approximately 2.5, 3.8, and 5.0 nM for spiking levels of 5.21, 6.39, and 9.47 log cfu/mL, respectively. Trials were conducted in replicates of 3 and means were compared. Trials conducted using HPLC-grade water showed a linear relationship for the CaDPA content of endospores and for endospore counts with the standard CaDPA concentration curve. For skim milk-spiked samples, we observed reduced fluorescence detection, which was 5 times lower than that of spiked samples in HPLC-grade water. The reduced fluorescence in skim milk could be due to the turbidity of the solution or to interference from proteins, amino acids, and other ions in milk. This study thus provides proof of concept for a potential application of this technique to rapidly detect bacterial endospores in the dairy and food industry. Further work is required to remove the interference of ionic components in milk to improve detection limits in milk and other dairy product matrices such as cheese, whey proteins, and reconstituted powders.

Overall Adverse Event Profile of Vadadustat versus Darbepoetin Alfa for the Treatment of Anemia Associated with Chronic Kidney Disease in Phase 3 Trials.

INTRODUCTION: Anemia frequently occurs in chronic kidney disease (CKD), is associated with poor quality of life and cardiovascular outcomes, and its treatment represents a considerable economic burden to the healthcare system. Although effective, the current standard of care for the treatment of anemia in chronic kidney disease patients with erythropoiesis-stimulating agents requires chronic/ongoing injections, making the treatment less accessible or desirable to patients not treated by in-center maintenance hemodialysis. Furthermore, safety concerns, including an increased risk of cardiovascular events and mortality, have emerged from their use in studies targeting hemoglobin concentrations in the normal or near-normal range. The orally active hypoxia-inducible factor prolyl hydroxylase inhibitor vadadustat may offer advantages over erythropoiesis-stimulating agents by correcting anemia via pathways activating endogenous erythropoietin production. METHODS: To comprehensively analyze the safety profile of vadadustat in patients with dialysis-dependent and non-dialysis-dependent CKD-related anemia, we pooled the safety populations from each of the four trials in the phase 3 clinical program (n = 7,373) and compared the risk of treatment-emergent adverse events (TEAEs) for each treatment arm. RESULTS: In patients randomized to vadadustat versus darbepoetin alfa, rates of TEAEs (88.9% vs. 89.3%), treatment-emergent serious adverse events (58.0% vs. 59.3%), and TEAEs leading to death (16.1% vs. 16.2%) were similar, as were rates of adverse events of special interest, including cardiovascular-, hepatic-, and neoplasm-related adverse events. DISCUSSION/CONCLUSION: Among patients with CKD-related anemia treated with vadadustat, we observed similar rates of adverse events relative to those treated with darbepoetin alfa.

Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms.

This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.

A Pan-respiratory Antiviral Chemotype Targeting a Transient Host Multiprotein Complex.

We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. One Sentence Summary: A host-targeted drug to treat all respiratory viruses without viral resistance development.

Effect of midday pasteurizer washing on thermoduric organisms and their progression through Cheddar cheese manufacturing and ripening.

Thermoduric bacteria are known to affect the quality of Cheddar cheese, with manifested defects including slits, weak body, and blowing. Thermoduric bacteria are likely to increase in numbers during cheese-making, as in-process conditions are conducive to proliferation. The present study was conducted to track thermoduric bacterial progression during an 18- to 20-h Cheddar cheese production run and during ripening when the pasteurizer was washed at midway through the production day. This study also correlated a broad range of chemical changes to the growth of thermoduric bacteria during ripening. Three independent cheese trials were performed at 3.5- ± 0.5-mo intervals. Samples were drawn in duplicates at 4 different times of the day: at the start of the run (vat 1), prior to a midday wash of the pasteurizer (vat 20), after the midday wash of the pasteurizer (vat 21), and at the end of the run (vat 42) for raw milk, pasteurized milk, and cheese. Cheeses were also tested during ripening for 6 mo. Results showed that raw milk total bacterial counts comprised 0.24% thermoduric mesophiles (TM) and 0.12% thermoduric thermophiles (TT). The thermoduric thermophilic bacterial counts increased by log10 1.23 during the pasteurizer run of 9 to 10 h, indicating a buildup of thermoduric thermophilic bacteria during the pasteurization process itself. Midday washing reduced thermophilic counts by log10 1.36, as evident by pre- and post-midday wash counts. However, a thermophilic buildup during post-midday wash was again noticed near the end of the 20-h run. We found that TT bacteria decreased in the first 60 d of ripening, whereas TM bacteria increased during the same period. However, TT bacteria increased later during 60 to 180 d of ripening. Bacillus licheniformis was the most frequently isolated bacteria in this study and was recovered at all production stages sampled during the cheese-making and ripening. We observed a significant increase in the level of orotic and uric acids in the vat made at the end of the day. No significant difference in the overall chemical composition, proteolysis, sugar, or other organic acids was observed in cheese made at the start versus the end of the production run.

Development of a spray-dried conjugated whey protein hydrolysate powder with entrapped probiotics.

Bifidobacterium animalis ssp. lactis ATCC27536 and Lactobacillus acidophilus ATCC4356 were encapsulated in a conjugated whey protein hydrolysate (WPH10) through spray drying. Probiotic cultures were added at the ratio of 1:1 into the conjugated WPH10 solution at a spiking level of about 10 log10 cfu/mL. The mixture was spray dried in a Niro drier with inlet and outlet temperatures of 200°C and 90°C, respectively. The final dried product was determined for cell viability and further stored for 16 wk at 25°, 4°, and -18°C to monitor viability and functionality. Micro images showed the presence of link bridges in non-conjugated WPH10, whereas, in the case of conjugated WPH10, round particles with pores were observed. The mean probiotic counts before and after spray drying were 10.59 log10 cfu/mL and 8.98 log10 cfu/g, respectively, indicating good retention of viability after spray drying. The solubility and wetting time of the WPH10-maltodextrin (MD) encapsulated probiotic powder were 91.03% and 47 min, whereas for WPH10, the solubility and wetting time were 82.03% and 53 min, respectively. At the end of storage period, the counts were 7.18 log10 cfu/g at 4°C and 7.87 log10 cfu/g at -18°C, whereas at 25°C the counts were significantly reduced, to 3.97 log10 cfu/g. The solubility of WPH-MD powder was 82.36%, 83.1%, and 81.19% at -18°C, 4°C, and 25°C, respectively, and wetting times were 61 min, 60 min, and 63 min at -18°C, 4°C, and 25°C, respectively. By contrast, for WPH10 powder, the solubility significantly reduced to 69.41%, 69.97%, and 68.99% at -18°C, 4°C, and 25°C, and wetting times increased to 71 min, 70 min, and 72 min at -18°C, 4°C, and 25°C, respectively. The conjugated WPH10 is thus demonstrated as a promising carrier for probiotics and can be further used as an ingredient for developing functional foods, to harness their enhanced functionality and health benefits derived from both WPH and probiotics.

Evaluating the effect of conjugation on the bioactivities of whey protein hydrolysates.

In this study, the ability of a whey protein hydrolysate to exhibit the antimicrobial, antioxidant, and antihypertensive behavior after combining with a reducing carbohydrate was studied. Whey protein hydrolysates with varying degrees of hydrolysis (WPH10, WPH15, and WPH20) were determined for their antimicrobial, antioxidant, and antihypertensive activities. Of these, hydrolysate (WPH10) exhibited the highest antimicrobial activity (with 10-11.2 mm zone of inhibition) against tested microorganisms: Listeria innocua, Staphylococcus aureus, and Bacillus coagulans. Also, the WPH10 exhibited the highest antioxidant (866.56 TEAC µmol/L) and antihypertensive (67.52%) attributes. Hence, based on the highest bioactivity, hydrolysate WPH10 was selected for conjugation with maltodextrin, and the effect of conjugation on the bioactivities was evaluated. The conjugated WPH10 solution demonstrated higher antimicrobial (17.16 mm) and antioxidant activity (1044.37 TEAC µmol/L), whereas a slight decrease in the antihypertensive activity (65.4%) was observed, as compared to WPH10 alone. The conjugated solution was further spray dried and alternatively, freeze-dried. The dried WPH10 conjugate exhibited even higher antimicrobial (18.5 mm) and antioxidant activity (1268.89 TEAC µmol/L) while retaining the antihypertensive activity (65.6%). Overall, the results indicate the ability of the WPH10-maltodextrin to retain the bioactive behavior after combining with a reduced carbohydrate. PRACTICAL APPLICATION: Whey protein hydrolysates upon conjugation with carbohydrates retain the bioactive properties of whey protein, which provides opportunities for application as an ingredient to develop novel health formulations.

Determining the real site of peroneal nerve injury with knee dislocation: Earlierier is easier.

Common peroneal nerve (CPN) injury is a recognised complication of traumatic knee dislocation with a direct association between the degree of ligamentous injury and the degree of CPN injury. It is essential explore and repair these injuries in good time to reduce morbidity. Often exploration only involves the portion of this nerve associated with the joint as it courses around the fibular head. However, a recent case highlighted the importance of proximal exploration to its branching point from the sciatic nerve, a known point of fragility, even if other defects have been identified.

Evaluation of immunogenicity and protection mediated by Lawsonia intracellularis subunit vaccines.

Lawsonia intracellularis is an economically important bacterium that causes ileitis in pigs. Current vaccines for L. intracellularis do not allow for differentiation between infected and vaccinated animals (DIVA), which is beneficial for disease tracking and surveillance. Previously, we identified five putative surface L. intracellularis proteins that were targeted by antibodies from pigs infected with L. intracellularis which could serve as antigens in a subunit vaccine. We conducted two trials to determine whether these antigens were immunogenic and provided protection against infectious challenge and whether truncated glycoprotein D could be used as a DIVA antigen. For Trial 1, 5 week-old piglets were administered intramuscular monovalent vaccines comprised of a recombinant (r) flagella subunit protein (rFliC,) and DIVA antigen (truncated glycoprotein D (TgD), a herpes virus antigen) both formulated with a combination adjuvant consisting of polyinosinic:polycytidylic acid(poly I:C), host defense peptide 1002 and polyphosphazene, referred to as Triple Adjuvant (TriAdj). Relative to control animals, animals vaccinated with rFliC and rTgD had significantly elevated antigen-specific humoral immunity in sera suggesting that rFliC and TgD are immunogenic. Control animals had negligible anti-TgD titres suggesting that TgD may be a suitable DIVA antigen for pigs. For Trial 2, piglets were immunized with a trivalent vaccine (FOG vaccine consisting of rFLiC, rOppA protein (a ABC Type dipeptide transport system) and rGroEL (a stress response protein)) and a divalent vaccine (CM vaccine consisting of rClpP (an ATP-dependent Clp protease proteolytic subunit) and rMetK (a S-adenosyl methionine synthase)) formulated with Emulsigen®. Relative to the control pigs, pigs immunized with the FOG vaccine produced robust and significantly higher serum IgG antibodies against rFliC and rGroEL, and significantly higher anti-FliC and anti-GroEL IgA antibodies in jejunal (GroEL only) and ileal intestinal mucosa. Pigs immunized with CM vaccine produced significantly higher serum antibodies against rClpP and rMetK and significantly higher anti-rClpP IgA antibodies in the ileum relative to the control pigs. Quantitative polymerase chain reaction (qPCR) analysis showed that 18 days after challenge with infectious L. intracellularis, challenged/control pigs and pigs that received the CM vaccine, but not the pigs vaccinated with the FOG vaccine, shed significantly more bacteria in feces than the unchallenged controls pigs. These data suggest that the FOG vaccinated pigs showed limited protection. While promising, more work is needed to enhance the efficiency of the intramuscular vaccine to show significant disease protection.

Short communication: Evaluating the recovery potential of injured cells of Listeria innocua under product temperature-abuse conditions and passage through simulated gastrointestinal fluids.

Ice cream handling and serving conditions on the consumer side may result in temperature abuse before consumption. Under some extreme conditions, even the sporadic presence of injured bacterial cells might pose a health risk due to the possibility of recovery of those cells. We conducted this investigation to evaluate the potential of injured cells of Listeria innocua to recover under ice cream temperature abuse conditions and on exposure to simulated gastrointestinal (GI) fluids. Ice cream mix samples (42% total solids), spiked with 4 log10 cfu/g of Listeria innocua, were thermally treated at 69°C for 30 min. Potential heat-injured cells were recovered in buffered Listeria broth (BLEB), followed by isolation on Listeria-specific modified Oxford agar (MOX). The ice cream mix samples, containing potentially injured cells of Listeria innocua, were followed through overnight aging (7°C), freezing (-3.3°C), and overnight hardening (-40°C) steps to obtain the final ice cream samples. To simulate temperature abuse conditions, the samples were held for 12 h at 4.4°C, followed by 30 min at room temperature (22°C); this treatment was considered the first cycle of temperature abuse. To generate a worst-case scenario, the samples were exposed to 3 such consecutive temperature abuse cycles. At the end of each cycle, direct plating was done on MOX to recover viable cells, and BLEB enrichment verified the presence of potential injured cells. In addition, the ice cream samples, containing potential injured cells, were passed through simulated GI fluids. As a first step, samples were mixed (1:1) with simulated gastric fluids (pH 1.0 and 2.0 before mixing) and held at 37°C in a shaker incubator. Samples drawn at 15, 30, and 60 min were analyzed for viable and potential injured cells. To study the effect of sequential transit through simulated intestinal fluid, a mixture of ice cream and gastric fluid (1:1) from the gastric fluid experiment above was added to simulated intestinal fluid (pH 6.8) and held at 37°C. Samples were analyzed at 30 and 360 min for viable and potential injured cells. Three trials were conducted and the samples collected in duplicates. The temperature abuse or GI fluid exposure studies did not result in the recovery of potential injured cells of Listeria innocua in the ice cream samples under the conditions tested. Exposure to gastric fluids, however, did not eliminate the potential injured cells. Further studies are necessary to understand the exact risk implications of these findings.

Effect of exopolysaccharides produced by dairy starter cultures on biofilms formed on reverse osmosis membranes.

Two different cheese starter cultures producing exopolysaccharides (EPS+: Streptococcus thermophilus strain ST3534 and Lactococcus lactis ssp. cremoris strain JFR+) and their isogenic EPS-negative (EPS-: S. thermophilus strain ST5842 and L. lactis ssp. cremoris strain JFR-) variants were used to study the attachment of bacterial cells in the absence of growth (at 4°C) and the resultant biofilm formation on reverse osmosis membranes (at 30 or 35°C). We used M17 broth and a 10% solution of whey protein concentrate (with 35% protein) as growth media for biofilm development under static conditions. As expected, ST3534 (EPS+) showed significantly greater cell counts within biofilms than ST5842 (EPS-). In the absence of growth, however, cells of these 2 isogenic Streptococcus strains attached to the membrane in similar numbers. In contrast, JFR+ counts were significantly lower than those of JFR- under all conditions. These findings indicate that the EPS produced by S. thermophilus may play a greater role in building up the 3-dimensional structure of the biofilm, rather than only assisting during initial attachment of the cells to the membrane, whereas the EPS produced by L. lactis ssp. cremoris hampered both initial attachment to the membrane and biofilm formation. Although no differences were observed in the surface charge of the cells between the 2 EPS-producing cultures, surface hydrophobicity was associated with the different adhesive properties of these microorganisms. In conclusion, our results exclude the hypothesis that all EPS-producing starter cultures have an advantage in regard to their ability to form biofilm on membrane separation surfaces. In contrast, variations between different EPS, with hydrophobicity being an important influencing feature, modify adhesive behavior to reverse osmosis membranes.

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix.

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.

Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm.

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

Assessment of psychiatric co-morbidities in patient of bronchial asthma attending a tertiary medical centre (Multicentric study).

AIMS AND OBJECTIVES: Asthma is a chronic inflammatory condition, which is associated with increase in airway hyper responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing. Asthma is a very common respiratory illness, in which some of the disease related factors may increases the vulnerability to psychiatric disorders. This study was done to determine the prevalence of psychiatric co-morbidity in patients of bronchial asthma. METHODOLOGY: It is an observational study conducted in 110 follow-up patients of bronchial asthma attending respiratory medicine OPD at tertiary care centre in central India. Psychiatric co-morbidities are assessed by pre-designed short-structured questionnaire using Mini international neuropsychiatric interview. RESULT: Among 110 patients of bronchial asthma 28% had psychiatric co-morbidity mainly depressive episode (59%). A significant association is found between lower socioeconomic status (P = 0.01), duration of of active illness (more than 1 year) (P = 0.001), and age of patient above 60 years (P = 0.001) with psychiatric co-morbidity of asthma patient. CONCLUSION: Our study shows there is increased prevalence of psychiatric co-morbidities in patients of bronchial asthma, higher than the national average. The predominant psychiatric disorder seen is depressive disorder, so treatment of asthma should be a multidisciplinary approach including medical treatment of asthma and psychiatric evaluation to prevent psychiatric co-morbidity or its early management. This will greatly reduce the morbidity, visits to hospital, expenditure on treatment and thereby having better outcomes in our patients of asthma.