Viable Plate Counts Research Articles

Corynebacteria from the respiratory microbiota modulate inflammatory responses and associate with a reduced pneumococcal burden in the lungs

BackgroundCertain species from the normal respiratory tract microbiota have recently been proposed to positively influence human health. Corynebacterium propinquum and C. pseudodiphtheriticum (Corynebacteria) are two Gram-positive species that frequently colonize the upper respiratory tract and strongly associate with a reduced incidence of respiratory tract infections. The specific role of Corynebacteria during respiratory health and disease is, however, largely uncharacterized.MethodRespiratory tract epithelial cells NCI-H292 and BALB/cByJ mice were inoculated with Corynebacteria (C. propinquum 2018M3 and 2019M4, and C. pseudodiphtheriticum 2019M8 and 2020M12) alone or with subsequent challenge with Streptococcus pneumoniae (pneumococci). The inflammatory response and the bacterial burden of both species over time were determined by Western blot, luciferase assay, cytokine bead array, flow cytometry and viable plate counts on blood agar plates.ResultsClinical isolates of Corynebacteria were well tolerated by human cells and mice. Corynebacteria induced a transient inflammatory response during healthy conditions in the absence of known pathogens. Pre-exposure or nasal priming with Corynebacteria did not affect subsequent acquisition of pneumococci but were associated with a modulated inflammatory response in vitro and in vivo as well as with a reduced pneumococcal burden in the respiratory tract of mice. This indicates that the presence of C. propinquum or C. pseudodiphtheriticum may protect against severe pneumococcal infections.ConclusionsIn this study, we delineate the role of Corynebacteria from the normal microbiota that epidemiologically associate with respiratory health. We show that the presence of Corynebacteria modulates the inflammatory response to pneumococci and associate with faster decrease in pneumococcal burden, primarily in the lower respiratory tract. Our data indicate that Corynebacteria has potential to protect against severe pneumococcal infections.

Resveratrol alters oral biofilm in vitro and in vivo.

To evaluate the ability of resveratrol to reduce dental caries in vitro and in vivo. In part 1, a microcosm biofilm protocol was employed. One hundred twenty-six bovine enamel specimens were treated with: Resveratrol (50, 100, 200, 400 µg/mL), Phosphate buffered saline (negative control), Dimethyl sulfoxide (negative control) and 0.12% Chlorhexidine (positive control). The biofilm was produced from the saliva of 10 volunteers, under 0.2% sucrose exposure for 5 days, and daily treated with the solutions (1 min). At the end of the experimental period, resazurin and viable plate count assays were performed. Enamel demineralization was evaluated by transverse microrradiography (TMR). In part 2, 12 volunteers participated in a triple-blind crossover protocol for 7 weeks, according to the following treatments: 1) 100 mg/L resveratrol; 2) 0.05% NaF (226 mg/L F); 3) 100 mg/L resveratrol + 0.05% NaF; 4) Deionized water (negative control). Biofilm samples were collected from both sides of the mouth 12 h after the use of the solutions. Resveratrol at 50 and 200 µg/mL significantly reduced biofilm metabolic activity and mutans streptococci, respectively. Chlorhexidine was an effective treatment to significantly reduce all parameters, being an important antimicrobial and anticaries agent in vitro. Resveratrol alone or associated with NaF modulated several caries-associated bacteria in vivo. The present study represents the first step regarding the use of resveratrol within the concept of acquired enamel pellicle and biofilm engineering to prevent dental caries.

Synergistic effects of chitosan and gamma irradiation on enhanced fungal growth inhibition and biodegradability of natural rubber latex film

Developing novel formulations and processes to improve the fungal growth inhibition and biodegradability of NR products has become a major challenge for the scientific community, given the health risks associated with fungi in natural rubber (NR) products and the environmental concerns regarding NR waste. Consequently, this study comprehensively investigated the synergistic effects of chitosan and gamma irradiation on enhancing fungal growth inhibition and biodegradability in natural rubber latex (NRL) films. The research involved incorporating varying chitosan contents (0, 3, 6, or 9 phr) into NRL composites and exposing them to different gamma doses (0, 5, 10, or 15 kGy). The results showed that increasing the chitosan content and the gamma dose improved the ability of the NRL samples to inhibit fungal growth on their surfaces. This was evidenced by the absence of fungal colonies after 7 days of incubation on potato dextrose agar (PDA) plates for NRL samples irradiated at 5–15 kGy with 9 phr of chitosan. This determination was based on isolating fungi from the film surfaces, followed by serial dilution and a viable plate count. The biodegradability tests also revealed that the NRL films irradiated at 15 kGy with 9 phr of chitosan had the highest weight loss, reaching as high as 10.42 ± 0.62 % after soil burial for 8 weeks. However, the results indicated that gamma irradiation on the pristine NRL and chitosan/NRL films did not substantially alter the thermal stabilities, density, morphology, and functional groups of the samples. Lastly, by comparing the tensile properties of all the NRL films to the ASTM D3578-01a standard for examination gloves, the optimum conditions for the NRL films were 5 kGy of gamma irradiation with 9 phr of chitosan. This combination resulted in gloves with sufficient tensile strength and complete fungal growth inhibition.

Impact of Airborne Exposure to PM10 Increases Susceptibility to P. aeruginosa Infection.

The effects of exposure to airborne particulate matter with a size of 10 μm or less (PM10) on C57BL/6 mouse corneas, their response to Pseudomonas aeruginosa (PA) infection, and the protective effects of SKQ1 were determined. C57BL/6 mouse corneas receiving PBS or SKQ1 were exposed to control (air) or PM10 for 2 weeks, infected, and the disease was documented by clinical score, PMN quantitation, bacterial plate count, RT-PCR and Western blot. PBS-treated, PM10-exposed corneas did not differ at 1 day postinfection (dpi), but exhibited earlier (3 dpi) corneal thinning compared to controls. By 3 dpi, PM10 significantly increased corneal mRNA levels of several pro-inflammatory cytokines, but decreased IL-10, NQO1, GR1, GPX4, and Nrf2 over control. SKQ1 reversed these effects and Western blot selectively confirmed the RT-PCR results. PM10 resulted in higher viable bacterial plate counts at 1 and 3 dpi, but SKQ1 reduced them at 3 dpi. PM10 significantly increased MPO in the cornea at 3 dpi and was reduced by SKQ1. SKQ1, used as an adjunctive treatment to moxifloxacin, was not significantly different from moxifloxacin alone. Exposure to PM10 increased the susceptibility of C57BL/6 to PA infection; SKQ1 significantly reversed these effects, but was not effective as an adjunctive treatment.

Study on the inhibition activity and mechanism of Tanreqing against Klebsiella pneumoniae biofilm formation in vitro and in vivo.

To evaluate the antibacterial effect of Tanreqing (TRQ) against K. pneumoniae and its inhibition activity on bacterial biofilm formation in vitro and in vivo, and to explore the mechanism of the inhibitory effects of TRQ on K. pneumoniae biofilm formation. An in vitro biofilm model of K. pneumoniae was established, and the impact of TRQ on biofilm formation was evaluated using crystal violet staining and scanning electron microscopy (SEM). Furthermore, the clearance effect of TRQ against K. pneumoniae in the biofilm was assessed using the viable plate counting method; q-RT PCR was used to evaluate the inhibitory effect of different concentrations of TRQ on the expression of biofilm-related genes in Klebsiella pneumoniae; The activity of quorum sensing signal molecule AI-2 was detected by Vibrio harveyi bioluminescence assay; Meanwhile, a guinea pig lung infection model of Klebsiella pneumoniae was constructed, and after treated with drugs, pathological analysis of lung tissue and determination of bacterial load in lung tissue were performed. The treatment groups included TRQ group, imipenem(IPM) group, TRQ+IPM group, and sterile saline group as the control. The formation of K. pneumoniae biofilm was significantly inhibited by TRQ in vitro experiments. Furthermore, when combined with IPM, the clearance of K. pneumoniae in the biofilm was notably increased compared to the TRQ group and IPM group alone. q-RT PCR analysis revealed that TRQ down-regulated the expression of genes related to biofilm formation in K. pneumoniae, specifically luxS, wbbm, wzm, and lsrK, and also inhibited the activity of AI-2 molecules in the bacterium. In vivo experiments demonstrated that TRQ effectively treated guinea pig lung infections, resulting in reduced lung inflammation. Additionally, when combined with IPM, there was a significant reduction in the bacterial load in lung tissue. TRQ as a potential therapeutic agent plays a great role in the treatment of K. pneumoniae infections, particularly in combination with conventional antibiotics. And TRQ can enhanced the clearance effect on the bacterium by inhibiting the K. pneumoniae biofilm formation, which provided experimental evidence in support of clinical treatment of TRQ against K. pneumoniae infections.

Impact of different sterilisation techniques on sorption and NER formation of test chemicals in soil

Standard OECD tests are used to generate data on biodegradation (OECD 307) and sorption (OECD 106) of test chemicals in soil. In such tests, data on abiotic degradation using sterile samples are utilised to investigate any losses due to abiotic processes. The data from sterile samples are also used to interpret results and findings of non-sterile samples, especially in the context of sorption and non-extractable residue (NER) formation. However, to ensure the comparability of the data obtained from sterile and non-sterile experiments, effects of sterilisation on the soil matrix should be minimal. The objective of this study was to investigate the efficiencies of different sterilisation techniques and the impact of the sterilisation on sorption and NER formation in soil. In this study, experiments in accordance with OECD 307 and OECD 106 guidelines were performed with two soils covering wide range of soil characteristics and treated with the three sterilisation techniques autoclaving, gamma(γ)-radiation and adding 1% (w/w) sodium azide. As a test item, 14C-labelled phenanthrene and bromoxynil was used for OECD 307 test, whereas non-labelled phenanthrene and atrazine was used for OECD 106. The sterilisation efficiencies were investigated using traditional viable plate count and molecular approaches (RNA extraction method). The results suggest that none of the tested techniques resulted in completely sterilised soil with autoclaving being the most efficient technique. Adding sodium azide led to most inefficient sterilisation and a significant increase (0.56 units) in soil pH. OECD 307 results showed differences in NER formation of the test chemicals, especially for soil poisoning and γ-radiation, which could be due to inefficient sterilisation and/or change in soil physico-chemical properties. OECD 106 results suggest that none of the sterilisation techniques considerably affected sorption behaviour of the test chemicals. Based on our results, we recommend autoclaving as most suitable sterilisation technique.

The impact of processing technology on microbial community composition and functional properties of Beninese maize ogi

Traditionally fermented maize starch, called ogi, is produced to prepare akpan, a yoghurt-like street food widely consumed in Benin. Current maize ogi production practices were compared to assess the impact of different processing technologies on the characteristics of the fermented product as a basis to determine best practices. Maize starch slurry samples were collected from processors in five municipalities in southern Benin and analysed before fermentation (starch samples) and after spontaneous fermentation (ogi samples). Four technological pathways for maize starch production were distinguished based on variations in the duration of steeping the grains, which ranged from 6 to 72 h, and whether or not kneading of the wet flour before filtration was practised. Six categories of maize ogi were derived from the four technology groups based on the duration of the fermentation, which lasted from 6 to 24 h. The average pH of maize starch varied from 3.2 to 5.3, with the lowest values for the two technology groups that also had the highest lactate concentrations (9–11.8 g/L). The six maize ogi categories had a pH ranging from 3.1 to 4.0. Viable plate counts of lactic acid bacteria were similar for maize starch samples and for ogi samples, whereas yeast counts showed clear differences. Members of the genera Limosilactobacillus, Lactobacillus, Weissella, Streptococcus and Ligilactobacillus, dominated the bacterial community in maize starch, and were also dominant in maize ogi. The members of the genera dominating the fungal community in maize starch were also dominant in maize ogi, except for Aspergillus and Stenocarpella spp., which decreased in relative abundance by fermentation. The highest total free essential amino acid concentration was 61.6 mg/L in maize starch and 98.7 mg/L in ogi. The main volatile organic compounds in maize starch samples were alcohols, esters, and carboxylic acids, which also prevailed in maize ogi samples. The results indicate that the characteristics of traditional maize ogi depend on the processing technologies used to produce the maize starch before the intentional fermentation into ogi, with no clear-cut connection with the production practices due to high variations between samples from the same technology groups. This revealed the importance of a standardized maize starch production process, which would benefit controlling the starch fermentation and the characteristics of maize ogi. Further research is needed to understand the hidden fermentation during maize starch production for determination of the best practices that support the production of quality maize ogi.

Efficiency and novelty of using environmental swabs for dry-surface biofilm recovery.

Studies on the epidemiology of dry-surface biofilms (DSBs) within healthcare settings have shown an almost universal distribution across frequently touched items. Despite a growing body of evidence for DSBs in hospitals, little attention has been paid to the recovery capacity of techniques used to detect these microbial communities. Biofilms are inherently difficult to remove from surfaces due to adhesive substances within their matrix and may act as sources of infection, but to what extent is largely unknown. In this study, we evaluate the recovery efficiencies of commonly used environmental swabs against DSBs containing 7.24 log10 Acinetobacter baumannii cm-2, using a drip flow reactor and desiccation cycle. Biofilm presence was visually confirmed using episcopic differential interference contrast microscopy combined with epifluorescence and quantified using sonicated viable plate counts. The swab materials used comprised foam, viscose and cotton, all of which were pre-moistened using a buffer solution. The surfaces were vigorously swabbed by each material type and the resultant microbe populations for both swabs and remaining DSBs were quantified. Our results found foam-tipped swabs to be superior, detecting on average 30 % of the original DSB contamination; followed by viscose (6 %) and cotton (3 %). However, no distinct difference was revealed in the concentration of microbes remaining on the surface after swabbing for each swab type, suggesting there is variation in the capacity for each swab to release biofilm-associated micro-organisms. We conclude whilst environmental swabs do possess the ability to detect biofilms on dry surfaces, the reduced efficiencies are likely to cause an underestimation of the microbes present and should be considered during clinical application.

Tracking the microbial communities from the farm to the processing facility of a washed-rind cheese operation.

Milk residue and the accompanying biofilm accumulation in milking systems can compromise the microbial quality of milk and the downstream processes of cheese production. Over a six-month study, the microbial ecosystems of milk (n = 24), tap water (n = 24) and environmental swabs (n = 384) were cultured by plating decimal dilutions to obtain viable counts of total aerobic mesophilic lactose-utilizing bacteria (lactose-M17), lactic acid bacteria (MRS), yeasts and molds (Yeast, Glucose, Chloramphenicol (YGC) medium). Viable aerobic lactose-M17 plate counts of milk remained well below 4.7 log CFU/ml over five of the months, except for 1 week in November where milk at the facility exceeded 5 log CFU/ml. Swab samples of the farm milking equipment showed consistent viable counts after sanitation, while the bulk tank swabs contained the lowest counts. Viable counts from swabs of the facility were generally below the detection limit in the majority of samples with occasional residual contamination on some food contact surfaces. Extracted DNA was amplified using primers targeting the V3-V4 region of the 16S rRNA gene, and the amplicons were sequenced by MiSeq to determine the shared microbiota between the farm and the processing facility (8 genera). Culture independent analysis of bacterial taxa in milk, water and residual contamination after sanitation with swab samples revealed the shared and distinct microbiota between the sample types of both facilities. Amplicon sequence variants (ASVs) of the V3-V4 region of the 16S rRNA gene revealed that the microbiota of milk samples had lower diversity than water or environmental swabs (279 ASVs compared to 3,444 in water and 8,747 in environmental swabs). Brevibacterium and Yaniella (both Actinomycetota) were observed in all sampling types. Further studies will include whole genome sequencing of Brevibacterium spp. isolates to determine their functionality and diversity within the system.

Sensitive detection of Mycobacterium bovis in spiked milk using polymerase chain reaction assay

Bovine tuberculosis is a chronic zoonotic disease that affects both animal and human health and imposes serious public health concerns in the world. Intake of non-pasteurized milk is considered the most probable vehicle for the transmission of pathogenic bacteria. In this study, the detection of Mycobacterium bovis BCG in spiked milk using a polymerase chain reaction was performed. The performance of two DNA extraction methods, CTAB/phenol:chloroform:isoamyl alcohol and EXTRAGENMB were also evaluated. In addition, Mycobacterial concentration was tried to determine using the Standard/ Viable Plate Count Method and Spectrophotometric (Turbidimetric) Method. PCR successfully detected M. bovis BCG in spiked milk, detecting approximately up to two bacilli per reaction. The two DNA extraction methods were effective in the isolation of amplifiable DNA, having the advantage of EXTRAGENMB in terms of (1) shorter duration of DNA extraction, (2) less sample manipulation, and (3) ease of execution of the procedure. Quantitative determination of the Mycobacterial population however failed to quantify the bacterial concentration per dilution, suggesting that CFU concentration should be considered an approximation. It is expected that this method can be used for the detection of M. bovis in raw milk samples.

Quantification of Lactobacillus reuteri ProTectis in MRS Broth Using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Chemometrics.

There is an increasing global demand for probiotics because of their numerous health benefits. However, a significant percentage of commercially available probiotic products have microbial quantities that are not in accordance with their product labels. In quantifying bacteria, the viable plate count is the standard method but is considered laborious and time-consuming. We demonstrate the use of an attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy coupled with chemometrics to quantify a pure culture of Lactobacillus reuteri (L. reuteri) ProTectis grown in deMan, Rogosa, and Sharpe broth. The chemometric partial least-squares model generated was able to accurately quantify viable plate count (VPC) (root-mean-square error of cross-validation (RMSECV) = 0.115 log CFU mL-1, root-mean-square error of prediction (RMSEP) = 0.145 log CFU mL-1, R2 = 0.982). These results provide proof of concept for this quantification technique and can potentially be developed and applied for the quantification of L. reuteri ProTectis in food products.

Unlocking the microbial diversity and the chemical changes throughout the fermentation process of “hákarl”, Greenland shark

Hákarl is a unique traditional Icelandic product and is obtained by fermenting and drying Greenland shark (Somniosus microcephalus). However, little is known about the chemical and microbial changes occurring during the process. In this small-scale industrial study, fresh and frozen shark meat was fermented for eight and seven weeks, respectively, and then dried for five weeks. During the fermentation, trimethylamine N-oxide levels decreased to below the limit of detection within five weeks and pH increased from about 6 to 9. Simultaneously, trimethylamine and dimethylamine levels increased significantly. Total viable plate counts, and specific spoilage organisms increased during the first weeks of the fermentation period but decreased during drying. Culture-independent analyses (16S rRNA) revealed gradual shifts in the bacterial community structure as fermentation progressed, dividing the fermentation process into three distinct phases but stayed rather similar throughout the drying process. During the first three weeks of fermentation, Photobacterium was dominant in the fresh group, compared to Pseudoalteromonas in the frozen group. However, as the fermentation progressed, the groups became more alike with Atopostipes, Pseudomonas and Tissierella being dominant. The PCA analysis done on the chemical variables and 16S rRNA analysis variables confirmed the correlation between high concentrations of TMAO and Pseudoalteromonas, and Photobacterium at the initial fermentation phase. During the final fermentation phase, correlation was detected between high concentrations of TMA/DMA and Atopostipes, Pseudomonas and Tissierella. The results indicate the possibility to shortening the fermentation period and it is suggested that the microbial community can potentially be standardized with starter cultures to gain an optimal fermentation procedure.

The effect and mechanism of iodophors on the adhesion and virulence of Staphylococcus aureus biofilms attached to artificial joint materials

BackgroundIodophors are known to be a treatment for biofilm-related periprosthetic joint infection. However, the efficacy and mechanism of eradicating biofilms from different artificial joint materials after iodophor treatment are unknown. This study was conducted to understand the effect and mechanism of iodophors with respect to the adhesion and virulence of Staphylococcus aureus biofilms attached to artificial joint materials.MethodsBiofilms of Staphylococcus aureus strains were grown on titanium alloy, cobalt chromium molybdenum and polyethylene coupons, which are commonly used materials for artificial joints, for 24 h. Afterward, all coupons were divided into experimental and control groups: (1) exposed to a 0.5 ± 0.05% iodophor for 5 min and (2) exposed to phosphate-buffered saline for 5 min. To gauge the level of biofilm, colony forming units (CFU), live/dead staining confocal microscopy and crystal violet staining were used. Meanwhile, the expression of icaACDR and clfA, which are related to virulence and adhesion, was examined in both the experimental and control groups.ResultsA roughly three-log decrease in CFU/cm2 was seen in the viable plate count compared to the control group. Confocal imaging and crystal violet staining verified the CFU data. Moreover, the expression of icaACDR was reduced on three different orthopaedic implant materials, and the expression of clfA was also inhibited on titanium alloy coupons exposed to the iodophor.ConclusionsOur results indicated that exposure to an iodophor for 5 min could significantly eliminate biofilms. When Staphylococcus aureus that had adhered to these three materials, which were used for artificial joints, was treated with an iodophor for 5 min, the expression of icaACDR was significantly reduced. This provides strong evidence for clinically clearing periprosthetic joint infections without removing the artificial joints.

Chemical, microbiological, textural, and sensory characteristics of pilot-scale Caciofiore cheese curdled with commercial Cynara cardunculus rennet and crude extracts from spontaneous and cultivated Onopordum tauricum

The aim of this study was the chemical, microbiological, textural, and sensory characterization of pilot-scale prototypes of an Italian ewe’s raw milk cheese (Caciofiore) curdled with commercial Cynara cardunculus rennet, used as a control, and crude extracts obtained from flowers of either spontaneous or cultivated Onopordum tauricum. Hence, the control and experimental cheese prototypes produced in two rounds of cheesemaking trials were assayed, at the end of their 60-day maturation, for the following features: pH, titratable acidity, dry matter, fat, total and soluble nitrogen (TN and SN, respectively), ash, salt, protein, lactose, viable plate counts and composition of the bacterial and fungal populations, color, texture, volatile organic compounds (VOCs), and olfactory attributes by sensory analysis (the latter for the sole prototypes curdled with the commercial rennet and the extract obtained from cultivated O. tauricum). The data overall collected showed a very low impact of the type of thistle rennet on the analyzed cheese traits, with significant differences being exclusively found for SN/TN%, titratable acidity, color, and adhesiveness. By contrast, a higher impact of the cheesemaking round was seen, with significant differences being observed for salt content, load of presumptive lactobacilli, thermophilic cocci, and Escherichia coli, and levels of the following VOCs: 2,3-butanedione, 2-pentanone, 1-butanol, 2-heptanone, 3-methyl-1-butanol, 2-heptanol, 2-nonanone, dimethyl trisulfide, 2-methyl propanoic acid, butanoic acid, and 3-methyl butanoic acid. Sensory analysis revealed a strong ewe’s cheese odor, accompanied by other olfactory notes, such as pungent, sour curd, sweet, and Parmesan cheese-like notes, in all the analysed cheese prototypes. Moreover, key odor active compounds, including butanoic acid, ethyl butanoate, 2,3-butanedione, 1-octen-3-one, and dimethyl trisulfide, were identified by GC-olfactometry analysis. Regarding the odor attributes as determined by sensory analysis, again the type of rennet had an almost negligible impact, with significant differences being only perceived for 1 or 2 out of 20 odor attributes, depending on the analytical conditions applied. Although some aspects deserve further investigation, the results herein collected confirm that O. tauricum can be regarded as an alternative source of thistle rennet for the manufacture of Caciofiore cheese, and more in general, Mediterranean ewe’s milk cheeses.

Evaluation of Solutions Containing Fluoride, Sodium Trimetaphosphate, Xylitol, and Erythritol, Alone or in Different Associations, on Dual-Species Biofilms.

Although the association of polyols/polyphosphates/fluoride has been demonstrated to promote remarkable effects on dental enamel, little is known on their combined effects on biofilms. This study assessed the effects of solutions containing fluoride/sodium trimetaphosphate (TMP)/xylitol/erythritol on dual-species biofilms of Streptococcus mutans and Candida albicans. Biofilms were grown in the continuous presence of these actives alone or in different associations. Quantification of viable plate counts, metabolic activity, biofilm biomass, and extracellular matrix components were evaluated. Overall, fluoride and TMP were the main actives that significantly influenced most of the variables analyzed, with a synergistic effect between them for S. mutans CFUs, biofilm biomass, and protein content of the extracellular matrix (p < 0.05). A similar trend was observed for biofilm metabolic activity and carbohydrate concentrations of the extracellular matrix, although without statistical significance. Regarding the polyols, despite their modest effects on most of the parameters analyzed when administered alone, their co-administration with fluoride and TMP led to a greater reduction in S. mutans CFUs and biofilm biomass compared with fluoride alone at the same concentration. It can be concluded that fluoride and TMP act synergistically on important biofilm parameters, and their co-administration with xylitol/erythritol significantly impacts S. mutans CFUs and biomass reduction.

A One-Year Environmental and Microbiological Monitoring of Storage Areas and Production Room of a Pharmaceutical Industry from Northern Nigeria

The degradation and reduction of the potency of both raw materials and finished pharmaceutical products (FPPs) are of great concern to pharmaceutical industries, making the environmental monitoring of storage areas and production rooms a must. In this study, a one year monthly data for environmental and microbiological monitoring for storage areas (raw materials quarantine room, raw materials approved room, finished product quarantine room and finished product approved room) and production room were collected and analyzed using IBM SPSS Statistics Version 23. The results revealed that the lowest temperature (21.01oC) was obtained from the raw materials approved room. At the same time, the highest (29.50oC) was recorded from the finished product quarantine room. In contrast, the lowest relative humidity of 11.11% was obtained from raw materials quarantine room while raw materials approved room had the highest relative humidity of 33.33%. The results obtained for microbial loads showed that Escherichia coli, Staphylococcus aureus, Salmonella typhimurium and Pseudomonas aeruginosa recorded 0.00cfu/ml each. In contrast, the value obtained for total viable aerobic mesophilic bacteria plate count ranges from 0.00-28.67 cfu/ml and fungi range from 0.67-5.00 cfu/mL respectively. The significant difference was determined at p&lt;0.05. However, the results obtained for the temperature, relative humidity and microbial loads were within the stated specifications. This shows that the temperature and relative humidity of the storage areas and the production room were controlled and well monitored in line with the current good manufacturing practice which will eventually positively impact the quality, marketability and stability of raw materials and drug products. Environmental monitoring of storage areas and production room should be highly encouraged in pharmaceutical industries to curb the menace of speedy degradation and loss of potency experienced in raw materials and finished pharmaceutical products.

Comparative Analysis Using Raman Spectroscopy of the Cellular Constituents of Lacticaseibacillus paracasei Zhang in a Normal and Viable but Nonculturable State.

This study aimed to investigate the molecular composition of a viable but nonculturable (VBNC) state of a probiotic strain, Lacticaseibacillus paracasei Zhang (L. paracasei Zhang), using single-cell Raman spectroscopy (SCRS). Fluorescent microcopy with live/dead cell staining (propidium iodide and SYTO 9), plate counting, and scanning electron microscopy were used in combination to observe bacteria in an induced VBNC state. We induced the VBNC state by incubating the cells in de Man, Rogosa, and Sharpe broth (MRS) at 4 °C. Cells were sampled for subsequent analyses before VBNC induction, during it, and up to 220 days afterwards. We found that, after cold incubation for 220 days, the viable plate count was zero, but active cells could still be observed (as green fluorescent cells) under a fluorescence microscope, indicating that Lacticaseibacillus paracasei Zhang entered the VBNC state under these conditions. Scanning electron microscopy revealed the altered ultra-morphology of the VBNC cells, characterized by a shortened cell length and a wrinkled cell surface. Principal component analysis of the Raman spectra profiles revealed obvious differences in the intracellular biochemical constituents between normal and VBNC cells. Comparative analysis of the Raman spectra identified 12 main differential peaks between normal and VBNC cells, corresponding to carbohydrates, lipids, nucleic acids, and proteins. Our results suggested that there were obvious cellular structural intracellular macromolecular differences between normal and VBNC cells. During the induction of the VBNC state, the relative contents of carbohydrates (such as fructose), saturated fatty acids (such as palmitic acid), nucleic acid constituents, and some amino acids changed obviously, which could constitute a bacterial adaptive mechanism against adverse environmental conditions. Our study provides a theoretical basis for revealing the formation mechanism of a VBNC state in lactic acid bacteria.

Bacteriological Investigations on the Quality of Herbal Medicinal Concoctions Vended in Gombe Markets.

Background: The use of medicinal plant products for the treatment of diseases is still highly valued globally due to several rationales,&nbsp; especially in low-income countries. The recognized potency of herbal medicines based on their vast pharmacological functions has&nbsp; enabled their uses to produce modern pharmaceutical drugs. However, the unregulated production and widespread use of nonsterile&nbsp; pharmaceutical medicinal products like herbal medicinal concoctions have instigated public health experts to question&nbsp; their quality and wholesomeness for consumption. This prompted the study aimed at investigating the quality of herbal medicinal&nbsp; concoctions vended in Gombe markets.&nbsp; Methods: A total of sixty (60) herbal medicinal concoctions were randomly sampled from four different vending outlets or markets for&nbsp; this study. Microbiological culturing techniques which include ten-fold serial dilution and spread-plating method were adopted for the&nbsp; isolation of bacteria in herbal medicinal concoction samples, and viable plate counts were established in colony-forming units per&nbsp; mL&nbsp; according to standard practice. Identification of bacterial isolates was achieved by colony morphology determination/macroscopy,&nbsp; gram staining and microscopy, while the identified bacteria were further confirmed using several biochemical analyses.&nbsp; Results: The&nbsp; mean viable plate count for all the different concoctions sampled was high and above the safe level of the established&nbsp; standards for non-sterile herbal preparations. Consequently, this initially signifies the unsafe status of the herbal concoctions for&nbsp; public consumption. Also, eight (8) bacteria genera/species among which are pathogenic strains were isolated and identified from the&nbsp; herbal concoctions. These bacterial contaminants with different percentages of occurrences include Enterobacter spp., Escherichia coli,&nbsp; Staphylococcus aureus, Shigella spp., Staphylococcus epidermidis, Klebsiella pneumoniae, Citrobacter freundii, Salmonella typhi.&nbsp; Conclusion: Herbal or medicinal concoctions vended in different markets within the Gombe metropolis where samples were collected&nbsp; were grossly contaminated beyond the established allowable limits for non-sterile pharmaceuticals, thus depicting the unsafe quality&nbsp; status of the medicinal products for consumption.&nbsp;&nbsp

Anti-Biofilm Efficacy of Commonly Used Wound Care Products in In Vitro Settings

Considering the prevalence and pathogenicity of biofilms in wounds, this study was designed to evaluate the anti-biofilm capabilities of eight commercially available wound care products using established in vitro assays for biofilms. The products evaluated included dressings with multiple delivery formats for ionic silver including nanocrystalline, gelling fibers, polyurethane (PU) foam, and polymer matrix. Additionally, non-silver-based products including an extracellular polymeric substance (EPS)-dissolving antimicrobial wound gel (BDWG), a collagenase-based debriding ointment and a fish skin-based skin substitute were also evaluated. The products were evaluated on Staphylococcus aureus and Pseudomonas aeruginosa mixed-species biofilms grown using colony drip flow reactor (CDFR) and standard drip flow reactor (DFR) methodologies. Anti-biofilm efficacy was measured by viable plate counts and confocal scanning laser microscopy (CSLM). Four of the eight wound care products tested were efficacious in inhibiting growth of new biofilm when compared with untreated controls. These four products were further evaluated against mature biofilms. BDWG was the only product that achieved greater than 2-log growth reduction (5.88 and 6.58 for S. aureus and P. aeruginosa, respectively) of a mature biofilm. Evaluating both biofilm prevention and mature biofilm disruption capacity is important to a comprehensive understanding of the anti-biofilm efficacy of wound care products.

Detection of microbial growth in aseptic food products using non-invasive Tunable Diode Laser Absorption Spectroscopy (TDLAS)

The sensitivity and reliability of Tunable Diode Laser Absorption Spectroscopy (TDLAS) for monitoring the growth of microorganisms in real food matrices was explored. The rapid and non-destructive TDLAS equipment uses laser light to monitor carbon dioxide changes due to microbial growth in the container head space which has the additional advantage of detecting contamination in products and packaging where visual inspection is not possible. TDLAS growth detection of Bacillus fengqiuensis, Candida albicans, Lactococcus lactis, Microbacterium luteolum, Paenibacillus chitinolyticus and Staphylococcus pasteuri was studied in various Ready To Feed (RTF) infant formula products. Detection of growth was correlated with cell numbers and the reliability of detection was tested using multiple inoculum levels. Overall, detection varies based on the matrices and the characteristics of the test organisms. TDLAS was capable of detecting growth of L. lactis within 20 h and S. pasteuri in 55 h when foods were contaminated with as low as ∼100 CFU/ml. However, the spore former B. fengqiuensis was not detected after 72 h in three matrices when inoculated at low levels. The lowest cell density detected at 4.47 CFU/ml was for the yeast (C. albicans) after 28.99 ± 1.82 h and the highest at 8.53 CFU/mL was for the actinomycete (M. luteolum) at 37.02 ± 1.84 h in RTF infant formula matrices. A strong linear relationship (R2 value ≥ 0.827) between initial inoculum and Time To Detection (TTD) for multiple inoculum levels was observed and growth rates (μ) calculated from TTD data was comparable with viable plate count methods for P. chitinolyticus but showed higher variability for L. lactis. Therefore, the TDLAS equipment was shown to be reliable with some specific limitations in identifying microbiological contamination by typical spoilage microbes in commercially sterile dairy beverage products.