Plate Count Agar Research Articles

Assessment of biofilm, enzyme production and antibiotic susceptibility of bacteria from milk pre- and post-pasteurization pipelines in Algeria

Bacterial biofilm is a major concern of dairy industry due to its association with milk contamination and its derived products. Algerian pasteurized milk shelf-life does not exceed one day, which may reflect the high level of contamination of this product and presence of extracellular enzymes such as lipases and proteases. This work aimed to investigate the microbial biodiversity in milk-processing surfaces of a dairy plant in Algeria. Therefore, stainless steel cylinders were placed in piping system of the dairy system before and after pasteurization of the milk, being removed after 7 days, for biofilm maturation and microorganism isolation and identification by mass spectrometry.Fifty-nine Gram-positive isolates were identified, namely Bacillus altitudinis, Bacillus cereus, Bacillus pumilus, Bacillus subtilis, Bacillus weithenstephanensis, Enterococcus casseliflavus, Enterococcus faecium, and Staphylococcus epidermidis. In addition, twenty-four Gram-negative isolates were identified, namely Acinetobacter schindleri Enterobacter cloacae, Enterobacter xiangfangensis, Leclercia adecarboxylata, and Raoultella ornithinolytica. Bacterial isolates showed ability for production of extracellular enzymes, being 49 % capable of both proteolytic and lipolytic activities. Milk isolates were tested for the ability to form biofilms on stainless steel. The cell numbers recovered on plate count agar plates from stainless steel biofilms ranged from 3.52 to 6.92 log10 CFU/cm2, being the maximum number detected for Enterococcus casseliflavus. Bacterial isolates showed intermediate and/or resistant profiles to multiple antibiotics. Resistance to amoxicillin, cefoxitin and/or erythromycin was commonly found among the bacterial isolates.

Quantitative characterization of microbial load in wild-harvested edible insects of Nagaland, India

Aim: To analyze the microbial load in raw and roasted insects in an attempt to determine the efficiency of heat treatment in removing the microbial load. Methodology: Fresh as well as roasted (over low flame for 15 min) insect samples were used to determine the microbial contaminants. Crushed insect samples were suspended in sterile half-strength nutrient broth solution and were further serially diluted 10-fold (up to 10-10) in isotonic half-strength nutrient broth solution. The total number of aerobic mesophilic microorganisms were determined on plate count agar and expressed as log Cfu g-1. Results: Differences in the number of microbial colonies were observed in fresh and roasted samples. The microbial load ranged from 6.30-8.75 log Cfu g-1 and showed that the highest microbial colonies were present in the fresh samples. The average log Cfu g-1 in fresh insect samples (7.57±0.87) was significantly higher (P<0.05) as compared to roasted samples (7.07±0.76). Interpretation: Edible insects require proper processing before consumption to reduce microbial contamination and further study is needed to identify specific microbes/food pathogens to develop microbial quality and parameters to ensure consumer safety. Key words: Alternative food, Edible insects, Food safety, Microbial contaminants, Traditional foods

Isolation and antimicrobial activity of lactic acid bacteria isolated from pineapple and watermelon

Probiotics are living microorganism which when consumed in adequate amount provide health benefit to the host, examples are lactic acid bacteria which are gram positive organisms that can withstand different environmental stress during production and storage of products containing them. This study aims to evaluate probiotic potential of lactic acid bacteria (LAB) strains isolated from pineapple and water melon. The materials used in the evaluation were properly sterilized including the different media like De-man Rogosa & Sharpe agar (MRS), MRS broth for the isolation of LAB by streaking method, nutrient agar, and Mueller Hinton agar for the antibiotic susceptibility test using Disc diffusion method. Out of 168 bacteria counted on plate count agar, a total of six strains were screened based on their cultural and biochemical characteristics. The fruit isolates were screened for antibacterial activity and for their antibiotics susceptibility; the six isolates showed antibacterial behaviour towards both gram-positive and gram-negative human pathogens (Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, and Corynebacteria spp.), while some showed no zone of inhibition. The antibiotic susceptibility test was also promising with P1 showing 31 mm against Amoxicav. Molecular identification analysis (16sRNA sequencing) revealed that the isolates were different species belonging to the same group, Lactobacillus fermentum, Lactobacillus plantarum and Weissella cibaria. Conclusively, these results demonstrate that bacteria isolated from fruit samples have interesting properties and could potentially be used as probiotics and in the preparation of functional fruit products.

Isolation and antimicrobial activity of lactic acid bacteria isolated from pineapple and watermelon

Probiotics are living microorganism which when consumed in adequate amount provide health benefit to the host, examples are lactic acid bacteria which are gram positive organisms that can withstand different environmental stress during production and storage of products containing them. This study aims to evaluate probiotic potential of lactic acid bacteria (LAB) strains isolated from pineapple and water melon. The materials used in the evaluation were properly sterilized including the different media like De-man Rogosa & Sharpe agar (MRS), MRS broth for the isolation of LAB by streaking method, nutrient agar, and Mueller Hinton agar for the antibiotic susceptibility test using Disc diffusion method. Out of 168 bacteria counted on plate count agar, a total of six strains were screened based on their cultural and biochemical characteristics. The fruit isolates were screened for antibacterial activity and for their antibiotics susceptibility; the six isolates showed antibacterial behaviour towards both gram-positive and gram-negative human pathogens (Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, and Corynebacteria spp.), while some showed no zone of inhibition. The antibiotic susceptibility test was also promising with P1 showing 31 mm against Amoxicav. Molecular identification analysis (16sRNA sequencing) revealed that the isolates were different species belonging to the same group, Lactobacillus fermentum, Lactobacillus plantarum and Weissella cibaria. Conclusively, these results demonstrate that bacteria isolated from fruit samples have interesting properties and could potentially be used as probiotics and in the preparation of functional fruit products.

Deuterium isotope probing (DIP) on Listeria innocua: Optimisation of labelling and impact on viability state.

An innovative approach, Raman microspectroscopy coupled with deuterium isotope probing (Raman-DIP), can be used to evaluate the metabolism of deuterated carbon source in bacteria and also to presume different anabolic pathways. This method requires the treatment of cells with heavy water that could affect the bacterial viability state at higher concentration. In this study, we evaluated the effect of heavy water incorporation on the viability state of Listeria innocua cells. We exposed the L. innocua suspensions to different heavy water concentrations (0%, 25%, 50% and 75%) from 30 minutes to 72 h of incubation times at 37°C. The total, viable and viable culturable populations were quantified by qPCR, PMA-qPCR and plate count agar respectively. We analyzed heavy water incorporation by Raman-DIP. The exposure of L. innocua cells to different concentrations of heavy water did not alter their cell viability to 24 h incubation time. In addition, the maximum intensity for C-D band, specific for the incorporation of heavy water, was reached after 2 h of exposure in a media containing 75% v/v D2O but an early detection of the labelling was possible at t = 1 h 30 min. In conclusion, the use of D2O as a metabolic marker was validated and can be developed for the detection of L. innocua cell viability state.

The Role of Catalase and Pyruvate on the Recovery of Cold - Shocked Bacteria

The study principally aimed at evaluating different additives to the culture medium for the recovery of cold-shocked bacteria isolated from chilled carcasses. Sixty randomly selected beef carcasses were assigned for investigation from which 630 swabs were collected for assessment of microbiological contamination of carcasses during the processes after skinning and evisceration, along the washing to the chilling stage. Obtained results were analysed by statistical package (SAS 2000, version 9.0) and revealed bacterial counts decreased significantly (p≤0.05) from skinning to chilling (3.67±0.0 to 1.23±0.10, p≤0.01). The predominant bacteria isolated from the carcasses were Staphylococcus spp. followed by Enterobacteriaceae and Pseudomonas spp. To test for recovery of cold-shocked bacteria, the bacterial load as CFU was evaluated after treatments with catalase, pyruvate and their combination. The CFU of samples from chilled carcasses increased after treatment with catalase and pyruvate (1.23±0.52 vs. 2.54±0.12 and 2.42±0.33, respectively; p≤0.05). The results showed that catalase treatment has induced higher bacterial recovery compared to pyruvate alone or the combination of pyruvate and catalase, but the bacteria died when the concentration of catalase and pyruvate was increased. Addition of catalase or pyruvate to plate count agar medium gives true estimates of the bacterial load of chilled carcasses and other cold foods.

Use of a product based on wood vinegar of Eucalyptus clone I144 used in the control of bovine mastitis

Bovine mastitis is one of the most frequent diseases in dairy cattle worldwide. The use of antiseptics in milking, if properly used, can lead to a reduction in potentially pathogenic microorganisms and their transmission between herds. Several medicinal plants have antiseptic potential, including eucalyptus (Eucalyptus spp.). Therefore, the objective of this study was to evaluate the effectiveness of wood vinegar from Eucalyptus urograndis clone GG I144 (EU) as an antiseptic in vitro and in vivo; in addition, to its cytotoxicity and antimicrobial resistance. Fifteen bovines were used, lactating females 3–6 years of age and divided into three groups of five animals each. The wood vinegar was placed in the teats of the animal for 28 days and collections of cellular debris were performed every 7 days. At the Veterinary Microbiology Laboratory (LAMIV) of UFERSA, the samples were processed and serial dilution was performed in Petri plates with plate count agar (PCA) at 37 °C. Cytotoxicity was verified based on morphological alterations and metabolic activity. Morphological changes were not observed in all cells incubated with 1 % pyroligneous extract. The in vitro data demonstrated antimicrobial activity against S. aureus, S. agalactiae, Salmonella, E. coli and P. aeruginosa. The bacteria of the genus Staphylococcus and Corynebacterium were resistant to penicillin (PEN), rifampicin (RIF), nitrofurantoin (NIT), erythromycin (ERI), and ciprofloxacin (CIP). The extract was used in vivo in the post-dipping of dairy cows, which reduced the microbiological load present in the mammary glands from 4.74 to 2.54 CFU, indicating its future use as an antiseptic.

In vitro antibacterial activity of green tea-loaded chitosan nanoparticles on caries-related microorganisms and dentin after Er:YAG laser caries removal.

This study aimed to determine the inhibitory effects of green tea (Gt), EGCG, and nanoformulations containing chitosan (Nchi) and chitosan+green tea (Nchi+Gt) against Streptococcus mutans and Lactobacillus casei. In addition, the antibacterial effect of nanoformulations was evaluated directly on dentin after the selective removal of carious lesion. At first, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against S. mutans and L. casei isolates were investigated. In parallel, dentin specimens were exposed to S. mutans to induce carious lesions. Soft dentin was selectively removed by Er:YAG laser (n=33) or bur (n=33). Remaining dentin was biomodified with Nchi (n=11) or Gt+Nchi (n=11). Control group (n=11) did not receive any treatment. Dentin scraps were collected at three time points. Microbiological analyses were conducted and evaluated by agar plate counts. Gt at 1:32 dilution inhibited S. mutans growth while 1:16 was efficient against L. casei. EGCG at 1:4 dilution completely inhibited S. mutans and L. casei growth. Independently of the association with Gt, Nchi completely inhibited S. mutans at 1:4 dilution. For L. casei, different concentrations of Nchi (1:32) and Nchi+Gt (1:8) were required to inhibit cell growth. After selective carious removal, viability of S. mutans decreased (p<0.001), without difference between bur and Er:YAG laser (p>0.05). Treatment with Nchi and Nchi+Gt did not influence the microbial load of S. mutans on dentin (p>0.05). Although variations in concentrations were noticed, all compounds showed antibacterial activity against S. mutans and L. casei. Both bur and Er:YAG laser have effectively removed soft dentin and reduced S. mutans counts. Nanoformulations did not promote any additional antibacterial effect in the remaining dentin.

Quality assessment of smoke-dried &lt;i&gt;Clarias gariepinus&lt;/i&gt; and &lt;i&gt;Chrysichthys nigrodigitatus&lt;/i&gt; stored in two storage facilities

The quality of smoke-dried fish can be enhanced by storing the products in good storage facilities. This research was carried out to determine the proximate composition, microbiological quality and sensory properties of smoke-dried Clarias gariepinus and Chrysichthysis nigrodigitatus stored in refrigerator and kitchen cabinet during a 6-week storage period. Sensory properties were assessed using panel of assessors. Bacterial load was done using plate count agar spread plate technique. Fish were purchased alive, washed, sacrificed and smoked for 72 hours. Percentage weight loss after smoking C. gariepinus and C. nigrodigitatus were 57.42% and 65.91%, respectively. Results for crude protein, moisture and lipid content of smoke-dried C. gariepinus stored in refrigerator ranged between 67.80-68.31%, 8.32-9.00% and 12.15-12.34% while the values for those stored in kitchen cabinet ranged from 67.04-68.30%, 8.30-8.49% and 12.15-12.34, respectively; for smoke-dried C. nigrodigitatus the ranges of the nutrients were 61.75-62.20%, 7.40%-8.00, 16.15-16.28% and 60.89-62.19%, 7.40-7.49%, 16.15-16.28% for those stored in refrigerator and kitchen cabinet, respectively. The results showed no significant difference (p&gt;0.05) in proximate composition of the samples in the two storage facilities. Microbes isolated from the smoke-dried fish samples were Staphyloccus aureus, Bacillus subtilis and Escherichia coli. Total bacterial counts (TBC) at week 6 for C. gariepinus were 0.17×10-6 and 0.25×10-6; C. nigrodigitatus, 0.24×10-6 and 0.31×10-6 in refrigerator and kitchen cabinet, respectively, and the values were not significantly different (p&gt;0.05) in the two storage facilities. No fungal growth was present throughout the six-week storage period. The sensory quality ratings ranged between 8.00±0.00 for flavour and 4.67±0.58 for overall acceptability. Sensory quality was significantly different (p&lt;0.05) in flavour, texture, appearance and aroma at week 2 and 6. The findings of this study show that the storage facilities retained good nutritional and sensory qualities as well as overall acceptability of the smoke-dried fish throughout storage.

Cold atmospheric plasma: Its time-dependent effects on the elimination of bacterial colony on periodontal manual scalers.

This in vitro study investigated the time-dependent bactericidal effects of cold atmospheric argon plasma treatment of periodontal hand scalers as well as the scanning electron microscopic view of the scaler tip surfaces before and after plasma treatment. The study used 34 periodontal hand scalers which were divided into test and control groups. The scaler tips were inoculated with Escherichia coli and Staphylococcus aureus bacteria, following which the scalers in the control and test groups were subjected to conventional sterilization and argon plasma sterilization, respectively. Varying exposure times of plasma treatment were done on the test group samples to evaluate the minimum time required for complete sterilization. Subsequently, streaks were made on plate count agar using each of these instruments. The agar plates were then kept in an incubator for 24 h, following which bacterial colony count was assessed (colony-forming units/mL). Furthermore, the scanning electron microscopic (SEM) view of the scaler tip was studied before and after plasma treatment. A complete elimination of bacterial load (Gram-positive as well as Gram-negative) from the instrument surface was achieved by the plasma exposure time of 15-20 s. SEM analysis did not show a significant difference before and after plasma treatment as not many organic residues were present on the scaler tip. Cold atmospheric pressure plasma is an efficient and time-saving method of sterilization, capable of destroying both Gram-positive and Gram-negative bacteria.

Microbiological Analysis of Street Foods Sold in Kathmandu, Nepal

Objectives: To investigate whether the street foods are potential source of pathogens or not. Methods: A total of 36 samples of different street foods were aseptically collected from different areas of Kathmandu Valley and was transported to laboratory. The bacterial load was enumerated using pour plate technique on plate count agar. Fungal load was enumerated using pour plate technique on potato dextrose agar and coliform load was also calculated using pour plate technique on VRBA. While Staphylococcus aureus was selectively detected on Mannitol Salt Agar. Escherichia coli was detected by observation of metallic sheen on Eosin Methylene Blue Agar. Results: The highest bacterial load was found on pani-puri with 70.4x103 cfu/ml and highest fungal load was found on samosa with 34.5x103 cfu/ml whereas the presence of coliforms was only detected in panipuri and chana chatpate. The food samples consisted of E. coli and S. aureus. E.coli was only detected from chana-chatpate. While the average Staphylococcal load was found to be highest in pani-puri (91.6x103 cfu/ml) and lowest in mo: mo (1.7x103 cfu/ml). Conclusion: This study concludes that E. coli and coliforms are the most common contaminant in channa chatpate sold in streets. Panipuri and channa chatpate sold in streets have the highest microbial loadand hence more chances of harboring potential pathogens. Street food sample in Kathmandu is mostly contaminated with the S. aureus.

Bacteriological evaluation of instant noodles stored under sub-optimal conditions from selected markets in Sagamu- Ibafo axis of Ogun State, Nigeria

This study evaluated 8 brands of 5 packets each of instant noodles for bacterial load, clinical status, and resistance of the isolates to conventional antibiotics, in addition to pH. These brands were cultured on plate count agar for bacterial enumeration while isolates were challenged on selective media for bacteria of clinical status. The antibiogram was determined using standard methods. The pH varied from acidic (4.92) as shown in GPJ 3 to closely neutral (6.94) as recorded in HWJ 2. TTJ1 had the lowest average bacteria count of 1.6 x 103 while BFJ5 with 6.3 x 105 counts was the highest. Prevalence of isolates of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa varied in the ratio 40:30:30. Of the 40 samples, 8 of the 16 Escherichia coli were resistant to gentamicin, ciprofloxacin, and Augmentin, 7 of the 12 Staphylococcus aureus elicited resistance to ampicillin and cotrimoxazole while 8 of the Pseudomonas aeruginosa were resistant to meropenem, cotrimoxazole, Augmentin, and cephalexin. Presence of bacteria of clinical status, in counts that exceeded recommended safe limits for ready-to-eat noodles and higher numbers of resistant isolates to the antibiotics appropriated, could be attributed to product storage under sub-optimal conditions which portend a public health risk.

Development of PP/EPDM thermoplastic elastomer by doped with 2-hydroxypropyl- 3-piperazinyl-quinoline carboxylic acid methacrylate based neusilin® for sanitary applications

The effect of the addition of 2-Hydroxypropyl-3-Piperazinyl-Quinoline carboxylic acid Methacrylate (HPQM) in Neusilin® into thermoplastic vulcanizates (TPV) Polypropylene/ethylene propylene diene monomer (PP/EPDM) type on the mechanical and antibacterial properties were studied. Ethylene propylene diene monomer compounds were mixed with Zinc dimethacrylate (ZDMA), Dicumyl peroxide (DCP), and paraffin oil by a two-roll mill machine. Polypropylene and HPQM in Neusilin® were extruded by varies HPQM content 0, 1250, 2500, 3750 and 5000 ppm. Polypropylene added antibacterial agent and EPDM compounds were mixed by twin-screw extruded weight ratios of 100:0, 75:25, 50:50, and 25:75 w/w with varied screw speeds of 60, 80, and 100 r/min. Polypropylene/Ethylene propylene diene monomer compounds were produced by compression moulding to form specimens. The mechanical properties were tested by tensile test and hardness test. It was found that screw speed did not affect the mechanical properties. Disk diffusion test and plate count agar (PCA) method were used for antibacterial properties. For both methods, the specimens contained an antibacterial agent against Escherichia coli ( E. coli ATCC 25922) and Staphylococcus aureus ( S. aureus ATCC 25923). The result of the disk diffusion test suggests that the more HPQM in Neusilin® content, the more the inhibition radius of only E. Coli bacteria. Plate count agar method proceeded by varying the contact time at 0, 1, 2, 3 and 4 h. Viable cell counts of E. coli decreased significantly at HPQM content 2500 ppm, while the viable cell count of S. aureus remained unchanged at 3750 ppm. Moreover, the rubber ingredients in EPDM, which are DCP and ZDMA, affect the release of antibacterial agents by hydrophobic characteristics and compatible conditions.

Atmosferic pressure non-thermal plasma: Preliminary investigation.

Antibacterial activity of atmosferic pressure non-thermal plasma (APNTP) was assessed for bacterial, yeast and mold strains. This investigation is to be considered preliminary: a second step is envisaged in which the efficacy of the technique and the device will be assessed directly on food of animal and plant origin. The strains (ATCC or wild type) of Listeria innocua, Escherichia coli, Salmonella thyphimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis (bacteria); Alternaria alternata, Aspergillus flavus, Cladosporium herbarum, Fusarium graminearum, Geotrichum candidum, Penicillium roqueforti, Rhizopus nigricans (moulds); Candida parapsilosis and Candida albicans (yeasts) were subjected to plasma plume generated by the action of electric fields with a gas mixture (oxygen and helium) delivered for 5 min at a distance of 2 cm. Types of experiments were listed as following: microorganism at concentration 1×10^8 and 1×104 cfu on PCA (Plate Count Agar); Listeria innocua and Salmonella thiphymurium at concentration 1×10^4 cfu on semi-synthetic and synthetic medium; mycetes (moulds and yeasts) at concentration 1×10^8 and 1×10^4 cfu on SDA (Sabouraud Dextrose Agar). The results obtained on the bacteria subjected to atmospheric cold plasma were evident on all the strains tested except for Proteus mirabilis (1×10^8 cfu), most evident at a concentration of 1×10^4 cfu, not only on culture media PCA but also on semi-synthetic medium and jelly meat-PCA medium. In spite of bacterial results, treatment with plasma plume did not decrease or inhibit of fungal growth. That means plasma plume was neither fungicidal nor fungistatic activities.

Graphene oxide coated aluminium as an efficient antibacterial surface

Antimicrobial coatings on metallic surfaces are rapidly emerging to combat bacterial pathogens. The excess use of conventional antibiotics increases the number of resistant strains at an alarming rate, which in turn leads to detrimental implications in the healthcare sector. Hence, it is of great importance in developing a new class of material with inherent bactericidal activity and good biocompatibility. In the present study, we have modified the aluminium surfaces by a coating of graphene oxide (GO) due to its excellent physicochemical properties, water dispersity and low cytotoxicity. Coatings were developed through facile and enviornment-friendly transfer method. The antimicrobial properties of GO coated aluminium are investigated against Gram-negative strain E. coli through agar plate counting and ‘Live/Dead’ fluorescence staining. Further, to shed light into the mechanism of antibacterial activity of GO at the molecular level, we have performed X-ray reflectivity (XRR) study considering a phospholipid multilayer as a model system to mimic bacteria cell membrane. Results show a significant bactericidal activity of the GO coatings compared to uncoated aluminium with lower concentration showing slightly better antibacterial property due to higher roughness. The obtained results may pave the way for engineering graphene-based antimicrobial coatings on a material surface using an easy, environment-friendly, cost-effective and straight forward processing route. • Antibacterial GO coating on aluminium surfaces were developed. • A facile, cost-effective and eco-friendly method was used for coating. • Sharp edges of GO sheets at lower concentration shows maximum bactericidal effect. • Rupture of cell membrane is considered to be dominant in antibacterial activity. • Molecular mechanism is confirmed by X-ray scattering studies.

Spatio-temporal variation of bacteria of the Vibrio and Pseudomonas genera in some underground water points developed in the locality of Ombessa (Department of Mbam-et-Inoubou, Center-Cameroon)

A study aimed at evaluating the seasonal variation of Vibrio and Pseudomonas in groundwater in the city of Ombessa, Center region of Cameroon was conducted from January to July 2021. The microorganisms sought were Aerobic Mesophilic Bacteria BHAMs, bacteria of the Vibrio and Pseudomonas genera. These bacteria were isolated from 10 groundwater points by the surface spreading technique for BHAM and the membrane filter method for Vibrio and Pseudomonas. Plate count agar was used for BHAMs, TCBS medium for Vibrio and Cetrimide agar for Pseudomonas. Some abiotic parameters such as temperature, pH, were measured using the usual techniques. The data obtained was analyzed using the appropriate software. Correlation and comparison tests between the variables were carried out. It shows that the water studied has a pH of 5.94 UC to 7.9 UC, the content of dissolved O2 reached 5 mg/L, and there are positive and significant correlations between the density of isolated bacteria and the levels of physicochemical variables. Two species of the genus Pseudomonas (aeruginosa and fluorescens) and four of the genus Vibrio (cholerea, alginolyticus, vulnificus and parahaemolyticus) were isolated, with variable abundance rates ranges from 194 to 898 CFU/100mL for Pseudomonas and from 58 to 683 CFU/100mL for Vibrio. The presence of these germs in groundwater can be explain by the proximity of sources of pollution. This water would be unfit for human consumption without prior treatment according to the World Health Organization standard.

Viability droplet digital polymerase chain reaction accurately enumerates probiotics and provides insight into damage experienced during storage.

Probiotics are typically enumerated by agar plate counting (PC) techniques. PC has several limitations including poor specificity, high variability, inability to enumerate dead cells, viable but non-culturable cells and cells in complex matrices. Viability droplet digital polymerase chain reaction (v-ddPCR) is an emerging enumeration technique with improved specificity, precision, and the ability to enumerate cells in varying states of culturability or in complex matrices. Good correlation and agreement between v-ddPCR and PC is well documented, but not much research has been published on the comparison when enumerating freeze-dried (FD) probiotics during storage. In this study, v-ddPCR utilizing PE51 (PE51-ddPCR), a combination of propidium monoazide (PMA) and ethidium monoazide (EMA), was evaluated as alternative enumeration technique to PC on blends of four FD probiotic strains over the course of a 3-month storage study with accelerated conditions. When PMA and EMA are combined (PE51), this study demonstrates agreement (bias = 7.63e+9, LOA = 4.38e+10 to 5.9e+10) and association (r = 0.762) between PC and v-ddPCR, at or above levels of an accepted alternative method. Additionally, v-ddPCR with individual dyes PMA and EMA provide insight into how they individually contribute to the viable counts obtained by PE51-ddPCR and provide a more specific physiological understanding of how probiotics cope with or experience damage during storage.

Improvement of degraded agricultural soil in an arid zone following short- and long-term treated municipal wastewater application: A case study of Gabes perimeter, Tunisia

This work aimed to study the short, medium, and long-term influences of treated wastewater (WWTP) as a soil irrigation technique on physical, chemical, and microbiological properties. Sampled soil plots were irrigated with WWTP for 1, 7, and 15 years. Rain-fed regime soil has been used as a control. This study was conducted in El Hamma of Gabes (southeast of Tunisia), an arid zone, during the dry season. Three soil profiles at 0–20, 20–40, and 40–60 cm were sampled. Physical and chemical analyzes, including pH, electrical conductivity, total organic carbon, total nitrogen, cation exchange capacity, and active and total CaCO3 were evaluated. Soil microbial biomass C and N were performed according to the fumigation extraction method. Soil microbial contamination was assessed by enumerating total, Fecal Coliforms, E. coli, and Fecal Streptococci with the most probable number (MPN) method and plate medium count. Bacterial and fungal enumeration were evaluated by the plate count agar method. In addition, the DGGE molecular analysis was also assessed in the different soil samples. The obtained physical and chemical evaluation proved that the WWTP irrigation practice improves soil fertility. Microbial biomass C and N increased by increasing the time of WWTP irrigation. In addition, the sanitary quality of the soil is affected by the increase of pathogens. We have observed an increase in microbial biomass as demonstrated by an increase in heterotrophic bacteria and filamentous fungi in the soil irrigated with WWTP compared to the control. Long-term WWTP irrigation has increased the activities of the soil microorganisms and then soil fertility. Meanwhile, the same pathogens and bacteria could pose a serious threat to sanitary soil quality and a limiting factor for the use of WWTP in agriculture areas without tertiary treatment.

Effect of Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum isolated from food and human origin on reduction of IgE-dependent hypersensitivity in Balb/c mice

Capacity and ability of Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus probiotic strains isolated from some human and food sources were evaluated for modulating and regulating the immune system against hypersensitivity type 1 (dependent on IgE). In this study, given the probiotic properties of the strains and the use of mouse models, the strains were orally administered (1×109 cfu/ml) during 45 days with gavage needles. Levels of total IgE antibodies, cytokines TGF-β, INF-ɤ, and IL-4 were measured by ELISA and compared with control groups. In addition, the nasopharyngeal lavage (total and differential count) was evaluated. Among the strains, Lactiplantibacillus plantarum a7 and Lacticaseibacillus rhamnosus M1 had the highest suppression of IL-4 cytokine production in splenocytes from ovalbumin-sensitized mice in vitro. Also, a7 and M1 had a significant (P < 0.0001) effect on reducing total IgE levels compared to the control group (OVA). Further, Lactiplantibacillus plantarum M8, a7 and M1 enhanced the production of INF-ɤ and TGF-β cytokines. Examination of nasal lavage results revealed that the total cell count in the a7, M1 and Lacticaseibacillus rhamnosus RHM groups and eosinophil cells in Lactiplantibacillus plantarum LF57, a7, and M1 significantly decreased compared to the control group. It was observed oral feeding of the isolated strains had a beneficial effect on reducing the total count on Plate Count Agar (PCA) and Mac Conkey Agar in mice feces during 7 days. M1 and a7 showed the highest level of Lactic Acid Bacteria at 7.28 and 6.9 Log cfu/g on day14, respectively. In conclusion, the animals that received the strains M1 and a7 (isolated from cow's milk and infant's small intestine) had the highest count of lactic acid bacteria in the mice gastrointestinal tract on day 14 (7.28 and 6.9 Log cfu/g), respectively. In addition, the mentioned strains can modulate immune system of mice by suppressing IL-4 production, increasing INF-ɤ and TGF-β cytokines, and reducing total IgE levels.

Measuring Single Bacterial Viability in Optical Traps with a Power Sweeping Technique.

Assessing bacterial viability is crucial in public health, food safety, environmental microbiology, and other relevant fields. The classical agar plate counting method and the popular dye-based assays have shown their strengths, but they also have limitations including high time consumption, relatively complex sample preparations, and cytotoxicity. In this work, we present a new bacterial viability assay based on optical tweezers utilizing a power sweeping strategy. By monitoring and analyzing bacterial nanomotion in optical traps under different trapping laser powers, the slope of the proportionality between the quantified extent of motion and the trapping laser power was defined as the mobility restriction coefficient (MRC) to quantify bacterial viability. We first established a firm correlation between the viability and MRC by measuring alive and dead Escherichia coli and Photobacterium phosphoreum. Then the capability of real-time long-term characterization of the assay was validated by measuring the viability of individual P. phosphoreum while regulating the viability with an inactivation light. Notably, a 'spinning-induced stabilization' mechanism was proposed to explain the surprising increase of apparent bacterial mobility after inactivation. Overall, the assay was proved to be a reliable label-free bacterial viability assay at a single-cell level, which holds potential in antibiotic susceptibility testing, drug screening, and rapid diagnostics.