Unveiling the positive impact of biofloc culture on Vibrio parahaemolyticus infection of Pacific white shrimp by reducing quorum sensing and virulence gene expression and enhancing immunity.

This study aimed to evaluate and unveil the positive impact of biofloc culture on Vibrio parahaemolyticus infection of Pacific white shrimp by reducing quorum sensing (QS) and virulence gene expression and enhancing shrimp's immunity. The shrimp with an average body weight of 0.50 ± 0.09 g were reared in containers with a volume of 2.5 L, 21 units, and a density of 20 shrimp L-1. The shrimp were cultured for 5 days, with each treatment including biofloc system maintenance with a C/N ratio of 10 and a control treatment without biofloc, followed by a challenge test through immersion using V. parahaemolyticus at densities of 103, 105, and 107 CFU mL-1 initially. The results of the in vitro experiment showed that biofloc suspension can inhibit and disperse biofilm formation, as well as reduce the exo-enzyme activity (amylase, protease, and chitinase) of V. parahaemolyticus. Furthermore, the biofloc treatment significantly reduced the expression of the QS regulatory gene OpaR, the PirB toxin gene, and the virulence factor genes T6SS1 and T6SS2 in both in vitro and in vivo. The biofloc system also increased the expression of shrimp immunity-related genes (LGBP, proPO, SP, and PE) and the survival rate of white shrimp challenged with V. parahaemolyticus.

Phyllosphere bacteria with antiquorum sensing and antibiofilm activities against fish pathogenic bacteria.

OBJECTIVE: This research aims to quantify antiquorum sensing and antibiofilm activity of f phyllosphere bacteria against biofilm formed by pathogenic fish bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. RESULTS: Antiquorum sensing assay using Chromobacter violaceum as indicator bacteria and antibiofilm assay showed six phyllosphere bacteria have antiquorum sensing and antibiofilm activities against tested bacteria. The highest inhibition and destruction activity was showed by metabolite of JB 3B and EJB 5 F against A. hydrophila, respectively. Determination using light microscope and scanning electron microscope performed decreaing in biomass of biofilm observed after treated with metabolite from phyllosphere bacteria.

The potential of shallot skin powder and actinomycetes metabolites as antimicrobe and antibiofilm in the treatment of eel (Anguilla bicolor bicolor) infected with Aeromonas hydrophila.

BACKGROUND: Eel (Anguilla bicolor bicolor) is an Indonesian export commodity. However, it is facing a problem related to Aeromonas hydrophila, which can cause motile aeromonas septicemia (MAS) and produce biofilm formation. Problem with antibiotic resistance challenges the need of an alternative treatment. Therefore, it is important to explore a solution to treat infection and the biofilm formed by A. hydrophila. OBJECTIVES: In this study, we used shallot skin powder and actinomycetes metabolite 20 PM as antimicrobe and antibiofilm to treated eels infected with A. hydrophila. RESULTS: Shallot skin powder (6.25 g 100 g-1 feed) and Actinomycetes 20 PM metabolite (2 mL 100 g-1 feed) were found to be effective as antimicrobe and antibiofilm agent in treating eels infected with A. hydrophila. Eel treated with antibiotic, shallot skin powder, and actinomycetes metabolite had 80%, 66%, and 73% survival rates, respectively. Other indicators such as red blood cell count, hemoglobin, and hematocrit were increased, but white blood cell count and phagocytic activity were dropped. Biofilm destruction were analyzed using scanning electron microscopy to determined antibiofilm activity of actinomycetes metabolite against biofilm of A. Hydrophila. CONCLUSIONS: Shallot skin powder and actinomycetes metabolite were potential to treat infection of A. hydrophila in eel as an alternative treatment to antibiotics.

Control of pathogenic bacteria using marine actinobacterial extract with antiquorum sensing and antibiofilm activity.

OBJECTIVE: The objectives of this research were to screen the anti-quorum sensing and antibiofilm activity of marine actinobacteria, isolated from several aquatic environments in Indonesia against several pathogenic bacteria, such as Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Vibrio cholerae, Salmonella Typhimurium, and Pseudomonas aeruginosa. RESULTS: Ten out of 40 actinobacteria were found to have anti-quorum sensing activity against wild-type Chromobacterium violaceum (ATCC 12472); however, the validation assay showed that only eight of 10 significantly inhibited the quorum sensing system of Chromobacterium violaceum CV026. The crude actinobacteria extracts inhibited and disrupted biofilm formation produced by pathogens. The highest antibiofilm inhibition was discovered in isolates 11AC (90%), 1AC (90%), CW17 (84%), TB12 (94%), 20PM (85%), CW01 (93%) against Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Vibrio cholerae, Salmonella Typhimurium, and Pseudomonas aeruginosa, respectively. The highest biofilm destruction activity was observed for isolate 1AC (77%), 20PM (85%), 16PM (72%), CW01 (73%), 18PM (82%), 16PM (63%) against Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Vibrio cholerae, Salmonella Typhimurium, and Pseudomonas aeruginosa, respectively. Actinobacteria isolates demonstrated promising anti-quorum and/or antibiofilm activity, interfering with the biofilm formation of tested pathogens. Appropriate formulations of these extracts could be developed as effective disinfectants, eradicating biofilms in many industries.

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.

Application of BI-EHEC and BI-EPEC bacteriophages to control enterohemorrhagic and enteropathogenic escherichia coli on various food surfaces.

OBJECTIVES: The purposes of this study were to determine the Efficiency of Plating (EOP) value of Bacteriophage BI-EHEC and BI-EPEC and to evaluate the application of these bacteriophages in reducing population of EHEC and EPEC on various food samples. RESULTS: In this study, we used bacteriophage BI-EHEC and BI-EPEC, which were isolated from previous study. Both phages were tested with other multiple pathotypes of intestinal pathogenic E. coli to determine the efficiency of plating. BI-EHEC had high efficiency toward ETEC with an EOP value of 2.95 but low efficiency toward EHEC with an EOP value of 0.10, while BI-EPEC had high efficiency toward EHEC and ETEC with EOP values of 1.10 and 1.21, respectively. As biocontrol agents, both bacteriophages able to reduce CFU of EHEC and EPEC in several food samples using 1 and 6-days incubation times at 4 [Formula: see text]. BI-EHEC reduced the number of EHEC with an overall percentage of bacterial reduction value above 0.13 log10, while BI-EPEC reduced number of EPEC with reduction value above 0.33 log10.

Antibiofilm activity from endophyte bacteria, Vibrio cholerae strains, and actinomycetes isolates in liquid and solid culture.

BACKGROUND: Biofilm-associated infections are a global threat to our economy and human health; as such, development of antibiofilm compounds is an urgent need. Our previous study identified eleven environmental isolates of endophyte bacteria, actinomycetes, and two strains of Vibrio cholerae as having strong antibiofilm activity, but only tested crude extracts from liquid culture. Here we grew the same bacteria in solid culture to induce the formation of colony biofilms and the expression of genes that may ultimately produce antibiofilm compounds. This research aimed to compare antibiofilm inhibition and destruction activities between liquid and solid cultures of these eleven environmental isolates against the biofilms of representative pathogenic bacteria. RESULTS: We measured antibiofilm activity using the static antibiofilm assay and crystal violet staining. The majority of our isolates exhibited higher inhibitory antibiofilm activity in liquid media, including all endophyte bacteria, V. cholerae V15a, and actinomycetes strains (CW01, SW03, CW17). However, for V. cholerae strain B32 and two actinomycetes bacteria (TB12 and SW12), the solid crude extracts showed higher inhibitory activity. Regarding destructive antibiofilm activity, many endophyte isolates and V. cholerae strains showed no significant difference between culture methods; the exceptions were endophyte bacteria isolate JerF4 and V. cholerae B32. The liquid extract of isolate JerF4 showed higher destructive activity relative to the corresponding solid culture extract, while for V. cholerae strain B32 the solid extract showed higher activity against some biofilms of pathogenic bacteria. CONCLUSIONS: Culture conditions, namely solid or liquid culture, can influence the activity of culture extracts against biofilms of pathogenic bacteria. We compared the antibiofilm activity and presented the data that majority of isolates showed a higher antibiofilm activity in liquid culture. Interestingly, solid extracts from three isolates (B32, TB12, and SW12) have a better inhibition or/and destruction antibiofilm activity compared to their liquid culture. Further research is needed to characterize the activities of specific metabolites in solid and liquid culture extracts and to determine the mechanisms of their antibiofilm actions.

Bacteriophage DW-EC with the capability to destruct and inhibit biofilm formed by several pathogenic bacteria.

Biofilm formation by pathogenic bacteria is a major challenge in the food industry. Once a biofilm is established, such as on food processing equipment, it becomes more difficult to eradicate. Although physical and chemical treatments are often used to control biofilm formation, these treatments can have significant drawbacks. Alternative biofilm treatments are needed. Phage DW-EC was isolated from dawet, an Indonesian traditional Ready-To-Eat food, which has high specificity for Enterohaemorrhagic Escherichia coli (EHEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC). Phage DW-EC produces several enzymes that can prevent the development of biofilm and biofilm eradication. Depolymerase enzymes break down the polysaccharides layer on the biofilms can lead to biofilm damage. On the other hand, endolysin and putative like-T4 lysozyme will lyse and kill a bacterial cell, thereby preventing biofilm growth. This research aims to determine the capability of previously identified phage DW-EC to inhibit and destroy biofilms produced by several foodborne pathogens. Phage DW-EC formed plaques on the bacterial lawns of EHEC, EPEC, and ETEC. The efficiency of plating (EOP) values for EHEC, EPEC, ETEC, and Bacillus cereus were 1.06, 0.78. 0.70, and 0.00, demonstrating that DW-EC was effective in controlling pathogenic E. coli populations. Furthermore, phage DW-EC showed anti-biofilm activity against foodborne pathogenic bacteria on polystyrene and stainless-steel substrates. DW-EC biofilm inhibition and destruction activities against pathogenic E. coli were significantly higher than against B. cereus biofilms, which was indicated by a lower density of the biofilm than B. cereus. Microscopic visualization verified that bacteriophage DW-EC effectively controlled EHEC, EPEC, and ETEC biofilms. The results showed that DW-EC could inhibit and destroy biofilm, making it promising to be used as an anti-biofilm candidate for polystyrene and stainless steel equipment in the food industry.

Antibiofilm properties of bioactive compounds from Actinomycetes against foodborne and fish pathogens.

In nature, bacteria can form biofilms, multi-layered structures that adhere microbial populations to solid surfaces by exopolysaccharides, proteins, and nucleic acids. In addition to causing foodborne infections, biofilms can be a major problem in aquaculture. Actinomycetes extracts have previously demonstrated antibiofilm activity against multiple foodborne and fish pathogens, and further characterization of these extracts is needed. In this study, we identified the chemical structures and antibiofilm properties of four extracts and determined the genetic similarity of the isolates to known Streptomyces isolates. We found that several extracts contained multiple antibiofilm compounds, and the antibiofilm activities of all extracts were most stable at pH 6. Furthermore, the antibiofilm inhibition and destruction activities of the isolates were stable at different temperatures. All of crude extracts demonstrated activity against biofilms formed by foodborne and fish pathogens on the surface of stainless-steel coupons as well as polystyrene that commonly used in industrial equipment. Using PCR 16S-rRNA gene and DNA sequencing analysis, the four Actinomycetes isolates were found to be 99% (1 AC), 97% (20 PM), 95% (16 PM), and 85% (18 PM) similar to Streptomyces. Biofilm structure were analyzed using Scanning Electron Microscopy coupled with Energy-Dispersive Spectrometry analysis. Coniine/(S)-2-propylpiperidine was the most active fraction of the crude extracts of the 1 AC, 20 PM, and 16 PM isolates, and piperidine, 2-(tetrahydro-2-furanyl) was most active in the 18 PM isolate.

Prevalence and characterization of Ice Nucleation Active (INA) bacteria from rainwater in Indonesia.

BACKGROUND: Ice nucleation active (INA) bacteria are a group of microorganisms that can act as biological nucleator due to their ice nucleation protein property. Unfortunately, little is known about their prevalence and characteristics in tropical areas including Indonesia. Here, we monitor the presence of INA bacteria in rainwater and air samples collected from Jakarta, Tangerang and several areas in Western Java, Indonesia for one year. We further identify and characterize selected Class A of INA bacteria isolated from these areas. RESULTS: Most of the INA bacteria were isolated from rainwater samples collected during March-August 2010, particularly from Jakarta, Bandung, and Tangerang. A total of 1,902 bacterial isolates were recovered from these area. We found a limited number of bacterial isolates from air sampling. From ice nucleation activity assays, 101 INA isolates were found active as ice nucleator at a temperature above -10 °C. A large majority (73 isolates) of them are classified as Class C (active below -8 °C), followed by Class A (26 isolates; active at -2 to -5 °C) and Class B (two isolates; active at -5 to -8 °C). We sequenced the 16S rRNA gene of 18 Class A INA isolates and identified 15 isolates as Enterobacteriaceae, while the remaining three as Pseudomonadaceae. The vast majority of our Class A INA isolates were likely Pantoea spp. with several isolates were deduced as either Pseudomonas, Cronobacter, and Klebsiella. We found that these 18 Class A INA isolates had acquired resistance to antibiotics erythromycin and ampicillin, which are considered two critically important antibiotics. CONCLUSIONS: Our results showed that the prevalence of INA bacterial population varies across locations and seasons. Furthermore, our isolates were dominated by Class A and C INA bacteria. This study also cautions regarding the spread of antibiotic resistance among INA bacteria.

Isolation, characterization, molecular analysis and application of bacteriophage DW-EC to control Enterotoxigenic Escherichia coli on various foods.

Among food preservation methods, bacteriophage treatment can be a viable alternative method to overcome the drawbacks of traditional approaches. Bacteriophages are naturally occurring viruses that are highly specific to their hosts and have the capability to lyse bacterial cells, making them useful as biopreservation agents. This study aims to characterize and determine the application of bacteriophage isolated from Indonesian traditional Ready-to-Eat (RTE) food to control Enterotoxigenic Escherichia coli (ETEC) population in various foods. Phage DW-EC isolated from Indonesian traditional RTE food called dawet with ETEC as its host showed a positive result by the formation of plaques (clear zone) in the bacterial host lawn. Transmission electron microscopy (TEM) results also showed that DW-EC can be suspected to belong to the Myoviridae family. Molecular characterization and bioinformatic analysis showed that DW-EC exhibited characteristics as promising biocontrol agents in food samples. Genes related to the lytic cycle, such as lysozyme and tail fiber assembly protein, were annotated. There were also no signs of lysogenic genes among the annotation results. The resulting PHACTS data also indicated that DW-EC was leaning toward being exclusively lytic. DW-EC significantly reduced the ETEC population (P ≤ 0.05) in various food samples after two different incubation times (1 day and 6 days) in chicken meat (80.93%; 87.29%), fish meat (63.78%; 87.89%), cucumber (61.42%; 71.88%), tomato (56.24%; 74.51%), and lettuce (46.88%; 43.38%).

Genomic characterization of bacteriophage BI-EHEC infecting strains of Enterohemorrhagic Escherichia coli.

OBJECTIVE: The aims of this research were to determine the genomic properties of BI-EHEC to control Enterohemorrhagic Escherichia coli (EHEC), which was isolated from previous study. Genomic analysis of this phage is essential for the assessment of this bacteriophage for further application as food preservatives. RESULTS: Genome of BI-EHEC was successfully annotated using multiPhATE2. Structural and lytic cycle-related proteins such as head, tail, capsid, and lysozyme (lysin) were annotated. The phylogenetic tree of tail fiber protein and BRIG results showed that BI-EHEC was similar to phages of the same host in the bacteriophage genome database. There were no indications of virulence properties, antibiotic resistance genes and lysogenic protein among annotated genes which implied BI-EHEC followed a lytic life cycle. PHACTS analysis was done to confirm this notion further and yielded a lytic cycle result. Further analysis using CARD found that BI-EHEC does not contain residual ARGs per recommended parameter. Furthermore, BI-EHEC confirmed as lytic bacteriophage, making it a good candidate for biocontrol agent.

Microbial analysis and virulence genes detection of milk preserved using heat-assisted pulsed electric field.

OBJECTIVE: Microbial analysis in milk preserved using heat-assisted Pulsed Electric Field (PEF) need to be assessed. In this study we analyze the microbial quality and virulence-associated genes in milk samples preserved using heat-assisted PEF from several producers in Indonesia. RESULTS: Milk samples were collected consisting of raw milk, milks taken after the heating, PEF, mixing, cooling, and packaging. Microbiological and Polymerase Chain Reaction (PCR) detection for virulence genes were performed. Heat-assisted PEF treatment gave 2.7-7.47 log reduction for TPC; 1.6-2.56 log reduction for MPN number; 3.13-6.48 log reduction for S. aureus; and for B. cereus there was an increase of 0.76 log and a reduction of 0.46 log. While milk samples from thermal pasteurization gave log reduction numbers of TPC, MPN, and S. aureus respectively 5.28; 2.56; and 4.73, for B. cereus was increasing 2.4 log. Producer C performed the best results with significant reduction compared with others (p < 0.005). There were no colonies of L. monocytogenes found in all of the samples. PCR results showed that milk samples possessed virulence genes 17.5% (10/57) of invA genes, 54.4% (31/57) of nheA genes, 68.4% (39/57) of cytK genes, 38.6% (22/57) of nuc genes, 63.2% (36/57) of ileS genes, while hly and actA genes were not detected.

Application of bacteriophage as food preservative to control enteropathogenic Escherichia coli (EPEC).

OBJECTIVES: This study was conducted to characterize lytic bacteriophages infecting enteropathogenic Escherichia coli (EPEC) on several types of food and analyze their ability as phage biocontrol to be used as a food preservative. Characterization was done for bacteriophage morphology and stability, along with the determination of minimum multiplicity of infection (miMOI), and application of bacteriophage in the food matrix. RESULTS: Out of the five samples, BL EPEC bacteriophage exhibited the highest titer of 2.05 × 109 PFU/mL, with a wide range of pH tolerance, and high thermal tolerance. BL EPEC also showed the least reduction after 168 h of incubation, with a rate of 0.90 × 10-3 log10 per hour. Bacteriophages from BL EPEC and CS EPEC showed an ideal value of miMOI of 0.01. As a food preservative, BL EPEC bacteriophage was able to reduce bacteria in food samples with a reduction above 0.24 log10 in lettuce and approximately 1.84 log10 in milk. From this study we found that BL EPEC bacteriophage showed the greatest potential to be used as phage biocontrol to improve food safety.

Induction of amylase and protease as antibiofilm agents by starch, casein, and yeast extract in Arthrobacter sp. CW01.

BACKGROUND: In unfavourable environment, such as nutrient limitation, some bacteria encased themselves into a three dimensional polymer matrix called biofilm. The majority of microbial infections in human are biofilm related, including chronic lung, wound, and ear infections. The matrix of biofilm which consists of extracellular polymeric substances (EPS) causes bacterial colonization on medical implanted device in patients, such as catheter and lead to patient's death. Biofilm infections are harder to treat due to increasing antibiotic resistance compared to planktonic microbial cells and escalating the antibiotic concentration may result into in vivo toxicity for the patients. Special compounds which are non-microbicidal that could inhibit or destroy biofilm formation are called antibiofilm compounds, for example enzymes, anti-quorum sensing, and anti-adhesins. Arthrobacter sp. CW01 produced antibiofilm compound known as amylase. This time our preliminary study proved that the antibiofilm compound was not only amylase, but also protease. Therefore, this research aimed to optimize the production of antibiofilm agents using amylase and protease inducing media. The five types of production media used in this research were brain heart infusion (BHI) (Oxoid), BHI with starch (BHIS), casein with starch (CS), yeast extract with starch (YS), and casein-yeast extract with starch (CYS). Biofilm eradication and inhibition activities were assayed against Pseudomonas aeruginosa (ATCC 27,853) and Staphylococcus aureus (ATCC 25,923). RESULTS: The results showed that different production media influenced the antibiofilm activity. Addition of starch, casein and yeast extract increased the production of amylase and protease significantly. Higher amylase activity would gradually increase the antibiofilm activity until it reached the certain optimum point. It was shown that crude extracts which contained amylase only (BHI, BHIS and YS) had the optimum eradication activity against P. aeruginosa and S. aureus biofilm around 60-70 %. Meanwhile, CS and CYS crude extracts which contained both amylase and protease increased the biofilm eradication activity against both pathogens, which were around 70-90 %. CONCLUSIONS: It was concluded that the combination of amylase and protease was more effective as antibiofilm agents against P. aeruginosa and S. aureus rather than amylase only.

Screening and characterization of cellulolytic molds from empty fruit bunches and soils in palm oil plantation area in Indonesia.

OBJECTIVE: This research was aimed to isolate cellulolytic molds in empty fruit bunches of oil palm (EFBOP) and soils from palm oil plantation area and identify their enzyme activities to digest EFBOP. RESULTS: A total of seven molds were successfully isolated and screened for their enzyme activities from EFBOP and the soils. The enzymes from each isolate were produced in submerged culture using Mineral Mandels and 3% of alkali pretreated pollard in triplicates. The results indicated that all of the isolates were able to hydrolyze Carboxymethyl Cellulose (CMC), Whatmann No. 1 filter paper, and also EFBOP to sugars with reducing ends that reacted to 3,5-Dinitrosalicylic acid (DNS). The CMCase activity of isolate X showed the highest while the lowest was found for isolate MT8. Filter paperase (FPase) activity of isolate X performed the highest wile the lowest were found from isolate MT3 and MT6. The saccharification activity of isolate P showed the highest while MT6 performed the lowest activity.

Extract from phyllosphere bacteria with antibiofilm and quorum quenching activity to control several fish pathogenic bacteria.

OBJECTIVE: The objective of this research were to screen quorum quenching activity compound from phyllosphere bacteria as well as antibiofilm activity against several fish pathogen bacteria such as Aeromonas hydrophila, Streptococcus agalactiae, and Vibrio harveyi. RESULTS: We found eight phyllosphere bacteria isolates with potential quorum quenching activity to inhibit Chromobacterium violaceum as indicator bacteria. Crude extracts (20 mg/mL) showed various antibiofilm activity against fish pathogenic bacteria used in this study. Isolate JB 17B showed the highest activity to inhibit biofilm formation of A. hydrophila and V. harveyi, meanwhile isolate JB 3B showed the highest activity to inhibit biofilm of S. agalactiae. From destruction assay, isolate JB 8F showed the highest activity to disrupt biofilm of A. hydrophila isolate JB 20B showed the highest activity to disrupt biofilm of V. harveyi, isolate JB 17B also showed the highest activity to disrupt biofilm of S. agalactiae.

Screening and quantification of anti-quorum sensing and antibiofilm activity of Actinomycetes isolates against food spoilage biofilm-forming bacteria.

BACKGROUND: Biofilms can form in many industries, one of them is the food industry. The formation of biofilms in this industry could cause immense economic losses and endanger public health. Biofilms formation is mainly triggered by quorum sensing. Therefore, inhibition of quorum sensing could be an innovative approach to inhibit the formation of biofilms. One way to inhibit quorum sensing is by using anti-quorum sensing compounds. Actinomycetes are a group of bacteria that is acknowledged to produce these compounds. RESULTS: There were eight crude extracts of Actinomycetes isolates that showed promising anti-quorum sensing activity against Chromobacterium violaceum. The concentration of the crude extracts was 20 mg/mL. All the crude extracts showed no antibacterial activity against food spoilage bacteria, except for crude extracts of isolate 18 PM that showed antibacterial activity against Bacillus subtilis. They also showed various antibiofilm activity, both inhibition and destruction. The highest inhibition and destruction activity sequentially was done by crude extracts of isolate 12 AC with 89.60% against Bacillus cereus and crude extracts of isolate SW03 with 93.06% against Shewanella putrefaciens. CONCLUSIONS: Actinomycetes isolates that isolated from different regions in Indonesia can be used as potential candidates to overcome biofilms formed by food spoilage bacteria using their ability to produce anti-quorum sensing compounds.

Shotgun metagenomic analysis reveals new insights into bacterial community profiles in tempeh.

OBJECTIVE: Amplicon sequencing targeting 16S ribosomal RNA (rRNA) has been widely used to profile the microbial community from fermented food samples. However, polymerase chain reaction (PCR) steps on amplicon sequencing analysis and intragenomic heterogeneity within 16S rRNA are believed to contribute to bias in estimating microbial community composition. As potential paraprobiotics sources, a comprehensive profiling study of tempeh microbial ecology could contribute to tempeh product development. This study employed a shotgun metagenomic approach, where metagenome fragments from tempeh samples were sequenced directly for taxonomic and functional profiling analysis. RESULTS: Taxonomic profiling showed that Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla from the shotgun metagenomic analysis in all tempeh samples. In terms of composition, this shotgun metagenomic study revealed that Proteobacteria was the most abundant phylum. Functional profiling showed that iron complex outer-membrane recepter protein (KEGG ID: K02014) was the most transcribed gene based on this metagenomic analysis. The metagenome-assembled genomes (MAGs) results from the binning pipeline could reveal almost complete whole genome sequence of Lactobacillus fermentum, Enterococcus cecorum, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii.

Screening of antibiofilm and anti-quorum sensing activty of Actinomycetes isolates extracts against aquaculture pathogenic bacteria.

BACKGROUND: Indonesia is the third largest producer of fish and other aquaculture products in the world, making this industry a major contributor in the economy of Indonesia. However, this industry continually overcome challenges, one of them are bacterial outbreaks. In addition, the emergence of these bacterial outbreaks were worsen due to the biofilm produced by many significant pathogenic bacteria and the impact of increased antibiotic resistance. These issues have become a global concern, because antibiotics are currently one of the main treatments available to overcome this problems. Therefore, studies aimed at finding and characterizing bioactive compounds to combat these issues. In this study actinomycetes isolates were screened and characterized for their bioactive compounds produced which have inhibitory and destructive activity and also QS inhibitors against biofilm structure of aquatic pathogenic bacteria, such as Vibrio harveyi, A. hydrophila, and S. agalactiae. RESULT: Extracts (20 mg/mL) produced by sixteen Actinomycetes isolates showed anti-quorum sensing activity towards reporter stain Chromobacterium violaceum wild-type. Most of these extracts showed better inhibitory activity on all of the pathogenic bacteria biofilm structure tested than the destructive activity on the preformed of those biofilm structure. Subsequently, we also performed characterization of bioactive compound and found that in this study, polysaccharide is the most common antibiofilm agents, which were responsible to their antibiofilm activity. Finally, we found that the value of LC50 of all extracts tested were more than 1 mg/mL, thereby all of extracts tested did not show cyto-toxic effect against Artemia salina. CONCLUSION: All of the extracts of Actinomycetes isolates showed promising inhibitory activity towards biofilm structure of pathogenic bacteria tested. So far, all of the extracts are potential to be QS inhibitors and antibiofilm agents of all pathogenic bacteria tested.