Agar Overlay Method Research Articles

In vitro Analysis of XLAsp-P2 Peptide Loaded Cellulose Acetate Nanofiber for Wound Healing.

Recently, nanofiber-based wound dressings are currently a viable strategy to expedite the healing of wounds by providing a suitable microenvironment for tissue growth with active ingredients. This research study subjects the development of electrospun cellulose acetate (CA) nanofibers loaded with the XLAsp-P2, an antimicrobial peptide (AMP) that holds great potential for enhanced wound healing as a therapeutic agent. The synthesized XLAsp-P2-loaded CA nanofibers were fabricated via three loading percentages, 0.1 %, 0.2 %, and 0.3 % w/w, and characterized and evaluated their antimicrobial potential with MTT assay and Agar overlay methods as an alternative strategy. FT-IR analysis confirmed the compatibility of the peptide loaded CA nanocomposite, showing distinct peaks corresponding to the constituent materials. Scanning electron microscopy (SEM) analysis was employed to characterize the morphology of electrospun peptide CA nanocomposites and illustrate the fiber's size at the nanoscale. The in vitro release study during the 24 hrs, 87 % of the peptide was released which was approximately 5.2 mg; which was closer matched to the square root model of Higuchi at room temperature. MTT assay presented sensitive results towards Gram-positive bacteria compared to Gram Negative bacteria; which corresponded to the inhibition zones of the Agar overlay method proving that Escherichia coli (ATCC 25922) 17.66 ± 0.38 mm and Pseudomonas aeruginosa (ATCC 27853) 17.44 ± 0.38 mm exhibited moderate susceptibility, while Staphylococcus aureus (ATCC 25923)19.89 ± 0.69 mm and Bacillus cereus (ATCC 11778) 23.00 ± 0.33 mm showed promising responses. Collectively, The study's findings indicate that the XLAsp-P2 incorporated CA mat possesses an opportunity to function as an efficient platform for delivering therapeutic peptides.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates

BackgroundBacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics.ResultsUsing a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature.ConclusionThis study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Characterization and genome-informatic analysis of a novel lytic mendocina phage vB_PmeS_STP12 suitable for phage therapy pseudomonas or biocontrol.

A novel lytic bacteriophage (phage) was isolated with Pseudomonas mendocina strain STP12 (P. mendocina) from the untreated site of Sewage Treatment Plant of Lovely Professional University, India. P. mendocina is a Gram-negative, rod-shaped, aerobic bacterium belonging to the family Pseudomonadaceae and has been reported in fifteen (15) cases of economically important diseases worldwide. Here, a novel phage specifically infecting and killing P. mendocina strain STP12 was isolated from sewage sample using enrichment, spot test and double agar overlay (DAOL) method and was designated as vB_PmeS_STP12. The phage vB-PmeS-STP12 was viable at wide range of pH and temperature ranging from 4 to10 and - 20 to 70°C respectively. Host range and efficiency of plating (EOP) analysis indicated that phage vB-PmeS-STP12 was capable of infecting and killing P. mendocina strain STP6 with EOP of 0.34. Phage vB_PmeS_STP12 was found to have a significant bacterial reduction (p < 0.005) at all the doses administered, particularly at optimal MOI of 1 PFU/CFU, compared to the control. Morphological analysis using high resolution transmission electron microscopy (HR-TEM) revealed an icosahedral capsid of ~ 55nm in diameter on average with a short, non-contractile tail. The genome of vB_PmeS_STP12 is a linear, dsDNA containing 36,212bp in size with a GC content of 58.87% harbouring 46 open reading frames (ORFs). The 46 predicted ORFs encode proteins with functional information categorized as lysis, replication, packaging, regulation, assembly, infection, immune, and hypothetical. However, the genome of vB_PmeS_STP12 appeared to be devoid of tRNAs, integrase gene, toxins genes, virulence factors, antimicrobial resistance genes (ARGs) and CRISPR arrays. The blast analysis with phylogeny revealed that vB_PmeS_STP12 is genetically similar to Pseudomonas phage PMBT14, Pseudomonas phage Almagne and Serratia phage Serbin with a highest identity of 74.00%, 74.93% and 59.48% respectively. Taken together, characterization, morphological analysis and genome-informatics indicated that vB_PmeS_STP12 is podovirus morphotype belonging to the class Caudoviticetes, family Zobellviridae which appeared to be devoid of integrase gene, ARGs, CRISPR arrays, virulence factors and toxins genes, exhibiting stability and infectivity at wide range of pH (4 to10) and temperature (-20 to 70°C), thereby making vB_PmeS_STP12 suitable for phage therapy or biocontrol. Based on the bibliometric analysis and data availability with respect to sequences deposited in GenBank, this is the first report of a phage infecting Pseudomonas mendocina.

Isolation and characterization of lytic bacteriophages from various sources in Addis Ababa against antimicrobial-resistant diarrheagenic Escherichia coli strains and evaluation of their therapeutic potential

BackgroundEscherichia coli is a common fecal coliform, facultative aerobic, gram-negative bacterium. Pathogenic strains of such microbes have evolved to cause diarrhea, urinary tract infections, and septicemias. The emergence of antibiotic resistance urged the identification of an alternative strategy. The use of lytic bacteriophages against the control of pathogenic E. coli in clinics and different environmental setups (waste and drink water management) has become an alternative therapy to antibiotic therapy. Thus, this study aimed to isolate and characterize lytic bacteriophage from various sources in Addis Ababa, tested them against antimicrobial-resistant diarrheagenic E. coli strains and evaluated their therapeutic potential under in vitro conditions.MethodsA total of 14 samples were processed against six different diarrheagenic E. coli strains. The conventional culture and plaque analysis agar overlay method was used to recover lytic bacteriophage isolates. The phage isolates were characterized to determine their lytic effect, growth characteristics, host range activity, and stability under different temperature and pH conditions. Phage isolates were identified by scanning electron microscope (SEM), and molecular techniques (PCR).ResultsIn total, 17 phages were recovered from 84 tested plates. Of the 17 phage isolates, 11 (65%) were Myoviridae-like phages, and 6 (35%) phage isolates were Podoviridae and Siphoviridae by morphology and PCR identification. Based on the host range test, growth characteristics, and stability test 7 potent phages were selected. These phages demonstrated better growth characteristics, including short latent periods, highest burst sizes, and wider host ranges, as well as thermal stability and the ability to survive in a wide range of pH levels.ConclusionsThe promising effect of the phages isolated in this study against AMR pathogenic E. coli has raised the possibility of their use in the future treatment of E. coli infections.

Anti-Staphylococcus aureus activity of Lactobacillus rhamnosus and Lactobacillus acidophilus on clinical isolates- an in vitro study

Background: This study evaluated the in vitro antimicrobial and antagonistic activity of both neat and pH-adjusted whole-cell suspensions of L. rhamnosus and L. acidophilus towards S. aureus, as an alternative to control the spread of MRSA, which is endemic in India. Materials and Methods: The study was conducted at School of Medical Education, Kerala, India, between October 2022 and May 2023. Anti-Staphylococcus aureus activity of Lactobacillus rhamnosus MTCC 1408 and Lactobacillus acidophilus MTCC 10307 grown on MRS agar, was evaluated on a total of 76 clinical isolates of S. aureus from Kerala, India, by the Agar overlay method. The study involved a comparison of probiotic activities by both neat and pH-adjusted whole-cell suspensions of L. rhamnosus and L. acidophilus. Results: The study results indicate that the mean value of inhibition zones produced by L. rhamnosus was greater than that of L. acidophilus, and statistically significant data was obtained in which the pH-adjusted whole cell suspensions of L. rhamnosus exhibited larger inhibition zones than that of neat suspensions of the same. Also, the mean value of activity by pH-adjusted L. acidophilus suspension was slightly greater than its neat suspension. Conclusions: L. rhamnosus and L. acidophilus were found to possess in vitro anti-Staphylococcus aureus activity. The antimicrobial and antagonistic activity of pH-adjusted live suspensions suggests the possible use of L. acidophilus and L. rhamnosus as probiotic treatment options in sites other than the gastrointestinal tract, supporting their use as local administration and topical applications, in particular for the treatment and decolonization of S. aureus.

Gut Microbiome Derived Lactic Acid Bacteria (GM-d-LAB) from Dwarf Goats (Capra aegagrus hircus) Inhibit the Resident Multiple Antibiotics Resistance Bacterial pathogens

Background: The global health threat posed by antimicrobial resistance (AMR) has created an urgent need for developing alternative treatment methods. Probiotics, especially Lactic Acid Bacteria (LAB), are gaining interest in this context, as they demonstrate health-enhancing effects and potent antimicrobial activities. The intestines of goats could be a potential origin for developing new probiotics applications in animal feed and human health. Hence this study was carried out to determine the antibiotic resistance profiles of LAB and pathogens within the intestines of Nigerian dwarf goats (Capra aegagrus hircus), the antimicrobial activity of LAB against resident MDR pathogens, and subsequent identification of bioactive LAB isolated from goat faeces in Nigeria as potential probiotics in animal feed and human health.&#x0D; Method: Selective isolation of the LAB was carried out using de Mann Rogosa Sharpe (MRS) agar while enteric pathogens were isolated on MacConkey agar. Preliminary identification was carried out based on Gram reaction, and morphological, colonial, and biochemical characteristics of each isolate. Antibiotic susceptibility profiles of all isolates were determined using the Kirby-Bauer disc diffusion method. The agar overlay method was used to test the most resistant LAB isolates for antimicrobial activity against enteric pathogens. Selected LAB isolates were identified by 16SrRNA sequencing. &#x0D; Results: The antibiotics susceptibility profile showed that a majority (77%) of LAB isolates and minimal (҇≤10%) of enteric pathogens demonstrated resistance to at least three classes of antibiotics, indicating a pattern of multi-drug resistance. Over half (62%) of these LAB isolates displayed significant antimicrobial activity against at least five of the resident-resistant pathogens, illustrating their potential role in controlling these pathogens. The sequencing results identified the most active LAB isolates, revealing a mix of strains including Pediococcus lolli (46%), Pediococcus pentosaceus (23%), Weissella confusa (8%), Enterococcus faecium (8%), Enterococcus hirae (8%), and Lactobacillus sanfranciscensis (8%).&#x0D; Conclusion: The discovery of a diverse range of LAB strains in goat intestines with significant antimicrobial activity against resident enteric pathogens is valuable. This finding suggests the potential use of these bacteria as natural alternatives to traditional antibiotics, especially in the context of growing AMR in animal husbandry.

Comparative Antimicrobial Activity of Lactobacillus acidophilus and Lactobacillus rhamnosus on Clinically Isolated Escherichia coli: An In vitro Study

Aim: The aim of present study is to evaluate the comparative antimicrobial activity of Lactobacillus acidophilus and Lactobacillus rhamnosus on clinical isolates of Escherichia coli.&#x0D; Study Design: Cross-sectional observational study.&#x0D; Place and Duration of Study: This study was conducted in Department of Medical Microbiology, School of Medical Education between January 2023 to September 2023.&#x0D; Methodology: A total of 100 E. coli isolates collected from various diagnostic laboratories were included in the sample population and the prevalence of XDR, MDR, and non MDR isolates among them were determined by Kirby-Bauer disk diffusion method. The inhibitory activity of untreated and treated (pH adjusted) suspension of standard strains of L. acidophilus and L. rhamnosus on E. coli were determined by agar overlay method and the data was statistically analysed using ANOVA single-factor.&#x0D; Results: The antimicrobial activity was confirmed for untreated and treated suspension of Lactobacillus spp. by measuring the zone of inhibition surrounding E. coli strains spotted on MRS medium but treated suspension exerted greater inhibitory activity than untreated suspensions of both Lactobacillus spp. and among the treated suspension L. rhamnosus exhibit greater inhibitory activity. Statistical analysis of this data using ANOVA single-factor was found to be not significant (P &gt;0.05), that is treated suspension of both L. acidophilus and L. rhamnosus has the independent activity against E. coli. While untreated suspensions of both L. acidophilus and L. rhamnosus was significant (P =0.03), that is untreated suspension of L. rhamnosus (mean inhibition of 12.19 mm) has greater inhibitory activity than untreated cell cultures of L. acidophilus (mean inhibition of 11.39 mm).&#x0D; Conclusion: This result, disclosed that even if both Lactobacillus spp. exhibit antimicrobial activity against E. coli, L. rhamnosus showed greater inhibition than L. acidophilus. The study suggested the use probiotic Lactobacillus as a biotherapeutic in antibiotic resistant E. coli infection and should be further studied for their human health benefits.

Phage-antibiotic synergism against Salmonella typhi isolated from stool samples of typhoid patients.

Typhoid fever is a fatal disease in humans that is caused by Salmonella typhi. S. typhi infections need immediate antibiotic therapy, and their extensive use has led to multidrug-resistant (MDR) pathogens. The use of bacteriophages is becoming a new way to treat these resistant bacteria. This research was directed to bacteriophage isolation against S. typhi and to determine phage-antibiotic synergism. To isolate bacteriophages targeting S. typhi, the causative agent of typhoid fever, and investigate their potential synergistic effects when combined with antibiotics. A cross-sectional study. The Widal test was positive; twenty diarrheal stool samples were taken, and for confirmation of S. typhi, different biochemical tests were performed. The disc-diffusion technique was used to determine antimicrobial resistance, and the double agar overlay method was used for bacteriophage isolation from sewage water against S. typhi. To test antibiotic-phage synergism, the S. typhi bacteria was treated by phages together with varying antibiotic concentrations. Eleven samples were positive for S. typhi with black colonies on SS-agar. These were catalase and MR positive with alkali butt on TSI. Clear plaques were observed after the agar overlay. Isolated phages were stable at various pH and temperature levels. Synergism was observed on agar plate. The zone was enlarged when phages were combined with bacterial lawn culture and ciprofloxacin disk. Bacterial growth inhibition had a significant p-value of 0.03 in titration plates, with the phage-ciprofloxacin combination being more effective than the phage and antibiotic alone. The study highlights the synergistic effects of isolated bacteriophages with antibiotics, which are not only effective against S. typhi infection but also decrease antibiotic resistance.

Isolation and characterization of bacteriophages from soil against food spoilage and foodborne pathogenic bacteria

Microbial food spoilage and foodborne disease are the main challenges in the food industry regarding food shelf life. Current preservation methods are frequently associated with changes in organoleptic characteristics and loss of nutrients. For this reason, bacteriophage offers an alternative natural method as a biocontrol agent that can reduce bacterial contamination in food without altering the organoleptic properties. This study was conducted to isolate and characterize bacteriophage from soil to control food spoilage bacteria, such as Bacillus cereus and Bacillus subtilis, and foodborne pathogenic bacteria, such as enterotoxigenic Escherichia coli (ETEC) and enterohemorrhagic E. coli (EHEC). Isolation was done by agar overlay assay method, and phages BC-S1, BS-S2, ETEC-S3, and EHEC-S4 were recovered. The host range of all isolated phages tended to be narrow and had high specificity towards the specific bacteria. The phage efficiency were measured where ETEC-S3 showed no effectivity against B. cereus and EHEC-S4 showed low efficiency against Enteropathogenic E. coli (EPEC). Morphology analysis was conducted for phage BC-S1 and ETEC-S3 with Transmission Electron Microscopy (TEM), and it is shown to belong to the Caudovirales order. Phages BC-S1 and BS-S2 significantly reduced the host bacteria when applied to the cooked rice and pasteurized milk samples with miMOI of 0.1. While phage ETEC-S3 at miMOI of 0.001 and phage EHEC-S4 at miMOI of 1 also showed a significant reduction when applied to chicken meat and lettuce samples at storage temperatures of 4 °C and 28 °C. The highest bacterial reduction of 100% was shown by phage BC-S1 on pasteurized milk samples and reduction up to 96.06% by phage ETEC-S3 on chicken meat samples at 28 °C incubation.

In Vitro Evolution to Increase the Titers of Difficult Bacteriophages: RAMP-UP Protocol.

Bacteriophages are becoming increasingly important in the race to find alternatives to antibiotics. Unfortunately, bacteriophages that might otherwise be useful are sometimes discarded due to low titers making them unsuitable for downstream applications. Here, we present two distinct approaches used to experimentally evolve novel New Zealand Paenibacillus larvae bacteriophages. The first approach uses the traditional agar-overlay method, whereas the other was a 96-well plate liquid infection protocol that improved phage titers in as little as four days. We also used a mathematical model to probe the parameters and limits of the RAMP-UP approach to rapidly select mutants that improve bacteriophage titers. Both experimental approaches resulted in an increase in plaque-forming units (PFU/mL). The liquid infection approach developed here, which we call RAMP-UP for Rapid Adaptive Mutation of Phage - UP, was significantly faster and simpler, and allowed us to evolve high titer bacteriophages in as little as four days. Titers were increased from 100-100,000-fold relative to their ancestors. The resultant titers were sufficient to extract and sequence DNA from these bacteriophages. An analysis of these phage genomes is provided. The RAMP-UP protocol is an effective method for experimentally evolving previously intractable bacteriophages in a high-throughput and expeditious manner.

Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria.

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7-151.6 nM against M. tuberculosis H37RvTin vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.

Promising biotherapeutic prospects of different probiotics and their derived postbiotic metabolites: in-vitro and histopathological investigation

BackgroundProbiotics and their derived postbiotics, as cell-free supernatants (CFS), are gaining a solid reputation owing to their prodigious health-promoting effects. Probiotics play a valuable role in the alleviation of various diseases among which are infectious diseases and inflammatory disorders. In this study, three probiotic strains, Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, and Pediococcus acidilactici, were isolated from marketed dietary supplements. The antimicrobial activity of the isolated probiotic strains as well as their CFS was investigated. The neutralized CFS of the isolated probiotics were tested for their antibiofilm potential. The anti-inflammatory activity of the isolated Lactobacillus spp., together with their CFS, was studied in the carrageenan-induced rat paw edema model in male Wistar rats. To the best of our knowledge, such a model was not previously experimented to evaluate the anti-inflammatory activity of the CFS of probiotics. The histopathological investigation was implemented to assess the anti-inflammatory prospect of the isolated L. plantarum and L. rhamnosus strains as well as their CFS.ResultsThe whole viable probiotics and their CFS showed variable growth inhibition of the tested indicator strains using the agar overlay method and the microtiter plate assay, respectively. When tested for virulence factors, the probiotic strains were non-hemolytic lacking both deoxyribonuclease and gelatinase enzymes. However, five antibiotic resistance genes, blaZ, ermB, aac(6’)- aph(2”), aph(3’’)-III, and vanX, were detected in all isolates. The neutralized CFS of the isolated probiotics exhibited an antibiofilm effect as assessed by the crystal violet assay. This effect was manifested by hindering the biofilm formation of the tested Staphylococcus aureus and Pseudomonas aeruginosa clinical isolates in addition to P. aeruginosa PAO1 strain. Generally, the cell cultures of the two tested probiotics moderately suppressed the acute inflammation induced by carrageenan compared to indomethacin. Additionally, the studied CFS relatively reduced the inflammatory changes compared to the inflammation control group but less than that observed in the case of the probiotic cultures treated groups.ConclusionsThe tested probiotics, along with their CFS, showed promising antimicrobial and anti-inflammatory activities. Thus, their safety and their potential use as biotherapeutics for bacterial infections and inflammatory conditions are worthy of further investigation.

Thin Layer Chromatography Bio-autography guided identification of Antibacterial constituents of leaf extract of Stereospermum kunthianum Cham. (Bignoniaceae)

Background: There is an incessant quest for the discovery of novel antibacterial agents.&nbsp;Stereospermum kunthianum Cham.&nbsp; Bignoniaceae) is used in traditional medicine in Nigeria and other&nbsp;parts of Africa in the treatment of venereal diseases e.g., syphilis,&nbsp; wounds, refractory cough, malaria,&nbsp;tooth ache leprosy etc. The present work investigates the antimicrobial activity and phytochemical&nbsp;constituents of the ethanolic leaf extract of the plant.&nbsp; Methods: Ethanolic leaf extract of S.Kunthianum was obtained by macerations method. Agar well&nbsp;diffffusion method was used to carry&nbsp; out preliminary antimicrobial susceptibility testing. The test was&nbsp;carried out at extract concentration of 100, 200 and 400 mgmL-1 . The&nbsp; agar dilution method was used to&nbsp;determine the Minimum Inhibitory Concentration (MIC) of the extracts. Phytochemical screening&nbsp; was&nbsp;conducted using standard procedures. Thin layer chromatography bioautography analysis was carried&nbsp;out using the agar overlay&nbsp; method. Six micro-organisms including two gram positive- Bacillus subtilis&nbsp;and Staphylococcus aureus, two gram negative Salmonella&nbsp; typhi , and Pseudomonas aeruginosa, two&nbsp;fungi, Aspergillus niger and Candida albicans were used as test organisms.&nbsp; Results: The ethanol extracts of S.kunthianum leaves showed antimicrobial activity against B.subtilis,&nbsp;S.aureus and S.typhi with zones of&nbsp; Inhibition ranging from 13±1.41 to 29.5 ± 1.00 mm and MIC values&nbsp;ranging from 16 -64 mgmL-1 . Phytochemical screening revealed the&nbsp; presence of saponins, flflavonoids&nbsp;and terpenoid compounds&nbsp; Conclusion: The study showed that the antibacterial activities of the leaf extract are probably due to&nbsp;polyphenolic and steroidal&nbsp; compounds.&nbsp; &nbsp

Antibacterial activity of lactic acid bacteria isolated from Etsako Osuemegbe rice.

Background: Probiotics have antibacterial effects against pathogenic bacteria in the gut while maintaining the balance of intestinal flora. This study aimed to experiment the antibacterial activity of two Lactobacillus species against pathogenic bacteria. Methods: Two different species of Lactobacillus lactiplantibacillus plantarum (CIP 103151) and Limosilactobacillus fermentum (CIP 102980) that were previously isolated from fermented Etsako Osuemegbe rice were investigated for their inhibitory ability against three (3) organisms (Escherichia coli, Staphylococcus aureus and Salmonella). Agar overlay method was employed and plates were examined for clear zone of inhibition. Results: The lactiplantibacillus plantarum inhibited the growth of E. coli, S. aureus, and Salmonella sp with diameter zone of inhibition range between 15.00+0.57 and 18.00+0.77 mm, while Limosilactobacillus fermentum inhibited the growth of E. coli, S. aureus, and Salmonella sp with diameter zone of inhibition range between 14.00+1.00 and 18.00+0.50 mm. Conclusion: The findings in this study suggest that LAB species can be used as alternate antibiotic if explored due to their ability to inhibit the growth of pathogenic organisms.

Antibiofilm activities of crude extract and supernatant of Actinobacteria against biofilm formed by foodborne pathogens and food spoilage bacteria

Bacteria form a biofilm that attaches the microbial population to a solid surface, thus acting as a barrier to protect external stresses from the bacterial community. In the food industry, biofilms are believed to be responsible for food-borne diseases and food spoilages. This research was conducted to characterize anti-quorum sensing (QS), anti-biofilm activity of crude extract, and supernatant from Actinobacteria isolates against biofilm of foodborne, and food spoilage bacteria. Actinobacteria is the group of filamentous spore-forming Gram-positive bacteria, which is well recognized as a source for novel secondary metabolites, such as anti-biofilm and anti-quorum sensing. Two isolates of Actinobacteria utilized in this study were recovered from the Indonesian marine environment in the previous study. Both isolates demonstrated anti-QS activity on early detection assay using the agar overlay method. The Actinobacteria isolates were fermented, and the crude extracts were obtained through extraction using 100 % ethyl acetate and further evaporation, while the supernatant was collected directly following fermentation. The entire Actinobacteria isolates displayed capability as an anti-biofilm agent in crude extracts form at a concentration of 20 mg.mL-1, alongside when in its supernatant form. The Actinobacteria isolates in both extracts and supernatants form demonstrated anti-QS activity based on the N-Hexanoyl-L- homoserine lactone (HHL) validation assay and were also categorized as non-toxic based on Brine Shrimp Lethality Assay (BSLA).

Evaluation of Antibacterial Activity of Lactic Acid Bacteria Isolated from Kunun Zaki against Selected Bread Rope Spoilage Bacteria

In line with increasing demand by consumers for safe and minimally processed food products, biopreservation has been advocated over the traditional use of chemical additives in combating food spoilage. This study evaluated the antibacterial activity of four lactic acid bacteria (LAB) (Lactobacillus fermentum, Lactococcus lactis, Lactobacillus brevis and Lactobacillus plantarum) isolated from kunun-zaki against the bread rope spoilage bacteria, Bacillus subtilis and Bacillus cereus. Antibacterial activity was determined using the dual agar overlay method with subsequent measurement of zones of inhibition. Titrimetric methods were used to determine the abilities of the isolated LAB to produce antimicrobials including lactic acid, hydrogen peroxide and diacetyl. Lactobacillus fermentum brought about the highest inhibition zone (20 mm) against Bacillus cereus. The smallest zone of inhibition (6 mm) was recorded for L. plantarum against B. cereus. Whereas L. plantarum produced the highest amount of lactic acid (0.540 g/L), L. brevis produced the lowest lactic acid amount of 0.348 g/L. L. fermentum produced the highest amount of hydrogen peroxide (0.020 g/L) whereas L.brevis recorded the lowest hydrogen peroxide production of 0.010 g/L. L. lactis and L. fermentum had the highest and lowest values of 0.649 g/L and 0.219 g/L respectively for diacetyl production. Differences in the antibacterial activities and antimicrobials production by the LAB were statistically significant (P &lt; .05). The findings show that the LAB isolates possessed antibacterial activity against the selected spoilage bacteria and as such could be exploited as potential biocontrol agents against the selected bread rope spoilage bacteria.

An elucidative study of the anti-biofilm effect of selenium nanoparticles (SeNPs) on selected biofilm producing pathogenic bacteria: A disintegrating effect of SeNPs on bacteria

In the present study, the antibacterial and anti-biofilm activity of selenium nanoparticles (SeNPs) produced by Bacillus subtilis BSN313 was investigated. Agar diffusion and microdilution methods did not reveal significant antibacterial activity of SeNPs against P. aeruginosa ATCC 9027, S. typhi ATCC 14028, and S. aureus ATCC 25923 due to the larger size of SeNPs and higher electrophoretic mobility, zeta potential, and/or poor diffusion properties in agar solids. Therefore, the agar overlay method was introduced, and significant antibacterial activity was observed with this method at concentrations (SeNPs) of 200 µg/mL, 150 µg/mL, and 150 µg/mL against P. aeruginosa ATCC 9027, S. typhi ATCC 14028, and S. aureus ATCC 25923, respectively. Atomic force spectroscopy (AFM) results, elucidated the disintegrating effect of SeNPs against Gram-negative (P. aeruginosa ATCC 9027) and Gram-positive (S. aureus ATCC 25923) pathogens. The disintegrating effect of SeNPs was caused by the destruction of the bacterial cell (biofilm producing) by rupturing the cell membrane. The hydrophobicity and zeta potential of the planktonic and biofilm populations were measured. Higher values of hydrophobicity and zeta potential heterogeneity were found for the biofilm population than for the planktonic population. The SeNPs showed significant anti-biofilm activity against biofilm-producing pathogenic strains. The ability of SeNPs to disperse biofilms was greatest at a concentration of 700 µg/mL within 15 min against P. aeruginosa ATCC 9027 (85.7%), S. typhi ATCC 14028 (89.6%), and S. aureus ATCC 25923 (78.3%). While the positive control (ascorbic acid, 32 µg/mL) showed an average dispersion of 95.1% against the biofilms of the strains studied within 90 min. Based on these results, we propose SeNPs as suitable candidates for the treatment of biofilm-producing pathogens.

596. Anti-Biofilm Activity of Isolated Bacteriophages Targeting Pseudomonas aeruginosa and Staphylococcus aureus

Abstract Background A rise in the prevalence of antibiotic-resistant infections has prompted a search for alternative methods to combat bacteria in the human body. People with cystic fibrosis are especially vulnerable because their lung environment provides a habitable environment for chronic infection, biofilm formation, and antibiotic-resistant mutations. The difficulty associated with finding bacteriophages targeted to a specific infection is one reason the treatment is used relatively infrequently, even when antibiotics have failed. This study aimed to isolate phage species for strains of Pseudomonas aeruginosa and Staphylococcus aureus, the most common causes of chronic infections in patients with cystic fibrosis. Methods Phages targeting multidrug-resistant strains of S. aureus and P. aeruginosa were isolated from samples of fecal matter and soil; stocks were enumerated with the double agar overlay method. The potency of the Pseudomonas-hosted isolates in eradicating biofilms was compared to the efficacy of an antibiotic cocktail and the enzyme cellulase through an in vitro resazurin cell viability assay. Each treatment was performed in sextuplicate. Metabolic reduction of resazurin to resorufin was quantified at 24 and 48 hours of growth using a microplate reader. Results Six phage stocks (four targeting Pseudomonas bacteria [from human &amp; feline fecal matter and from soil], two targeting Staphylococcus [from human &amp; feline fecal matter]) contained bacteriophages at concentrations ranging from 3.9x104 pfu/mL to 2.8x107 pfu/mL. The most effective treatments in the assay were found to be cellulase alone and the three inhibitors together. Conclusion Feline fecal matter is a potential source of Pseudomonas and Staphylococcus bacteriophages. Enzymes were found to be a critical component of biofilm treatment. The isolated bacteriophages were not the most potent treatment alone, but when combined with cellulase and antibiotics, bacterial growth was markedly slowed. Disclosures All Authors: No reported disclosures.

Bacteriophage efficacy in controlling swine enteric colibacillosis pathogens: An in vitro study.

Swine enteric colibacillosis caused by Escherichia coli is a major problem in the swine industry, causing diarrhea among swine and resulting in substantial financial losses. However, efforts to counter this disease are impeded by the increase in antimicrobial resistance (AMR) worldwide, so intensive research is being conducted to identify alternative treatments. This study isolated, characterized, and evaluated the efficacy of bacteriophages to control pathogens causative of swine enteric colibacillosis. Five sewage samples were collected from different areas of a swine farm in Suphanburi province, Thailand and the bacteriophages were enriched and isolated, followed by purification by the agar overlay method using E. coli RENR as the host strain. The selected phages were characterized by evaluating their morphology, while their specificity was verified by the host range test. The efficiency of plating and multiplicity of infection (MOI) were also determined. Four selected phages, namely, vB_Eco-RPNE4i3, vB_Eco-RPNE6i4, vB_Eco-RPNE7i1, and vB_Eco-RPNE8i3, demonstrated different patterns of host range and phage efficiency. They significantly decreased E. coli concentration at the tested MOIs (0.01-100) from 1 h onward. However, bacterial regrowth was observed in all phage treatments. This study shows the potential of using phages as an alternative treatment for swine enteric colibacillosis. The obtained results demonstrated that the selected phages had a therapeutic effect against pathogens causative of swine enteric colibacillosis. Therefore, phages could be applied as an alternative treatment to control specific bacterial strains and reduce AMR arising from the overuse of antibiotics.

Antibiofilm activities of crude extract and supernatant of Actinobacteria against biofilm formed by foodborne pathogens and food spoilage bacteria

Bacteria form a biofilm that attaches the microbial population to a solid surface, thus acting as a barrier to protect external stresses from the bacterial community. In the food industry, biofilms are believed to be responsible for food-borne diseases and food spoilages. This research was conducted to characterize anti-quorum sensing (QS), anti-biofilm activity of crude extract, and supernatant from Actinobacteria isolates against biofilm of foodborne, and food spoilage bacteria. Actinobacteria is the group of filamentous spore-forming Gram-positive bacteria, which is well recognized as a source for novel secondary metabolites, such as anti-biofilm and anti-quorum sensing. Two isolates of Actinobacteria utilized in this study were recovered from the Indonesian marine environment in the previous study. Both isolates demonstrated anti-QS activity on early detection assay using the agar overlay method. The Actinobacteria isolates were fermented, and the crude extracts were obtained through extraction using 100 % ethyl acetate and further evaporation, while the supernatant was collected directly following fermentation. The entire Actinobacteria isolates displayed capability as an anti-biofilm agent in crude extracts form at a concentration of 20 mg.mL-1, alongside when in its supernatant form. The Actinobacteria isolates in both extracts and supernatants form demonstrated anti-QS activity based on the N-Hexanoyl-L- homoserine lactone (HHL) validation assay and were also categorized as non-toxic based on Brine Shrimp Lethality Assay (BSLA).