Crude Bacteriocins Research Articles

Production of crude bacteriocins by selected lactic acid bacteria isolated from some fermented foods

Production of crude bacteriocins by selected lactic acid bacteria isolated from some fermented foods

Evaluation of the Effect of Lactobacillus acidophilus ATCC 4356 Bacteriocin against Staphylococcus aureus.

Lactobacillus acidophilus is lactic acid bacteria that produce bacteriocins. Bacteriocins are antimicrobial peptides or proteins that exhibit activity against closely related bacteria. The aim of this study was to determine the effect of L. acidophilus ATCC 4356 bacteriocin against Staphylococcus aureus. Material and Methods. We used four different phenotypic methods for antimicrobial activities against two standard strains: methicillin-resistant S. aureus (MRSA) ATCC 33591 and methicillin-susceptible S. aureus (MSSA) ATCC 25923. The methods were (1) agar well diffusion, (2) overlay soft agar, (3) paper disk, and (4) modification of punch hole. The ammonium sulfate method was used to concentrate crude bacteriocin, and ultrafiltration and dialysis tubes were used to remove ammonium sulfate from the bacteriocins. Each method was repeated in triplicate. L. acidophilus ATCC 4356 showed antimicrobial activity against both MRSA and MSSA standard strains only by the overlay soft agar method and not by the agar well diffusion, punch hole modification, and paper disk methods. No antimicrobial effects were observed in crude bacteriocins concentrated. The growth inhibition of S. aureus in overlay soft agar method may be due to the production of bacteriocin-like substances. The overlay soft agar method is a qualitative test, so there is a need for further study to optimize the conditions for the production of bacteriocin-like substances in the culture supernatant and precise comparison between the inhibitory activity and pheromone secretion of different strains.

Bifidobacterium longum subsp. infantis as widespread bacteriocin gene clusters carrier stands out among the Bifidobacterium.

Bifidobacterium is the dominant genus, particularly in the intestinal tract niche of healthy breast-fed infants, and many of these strains have been proven to elicit positive effects on infant development. In addition to its effective antimicrobial activity against detrimental microorganisms, it helps to improve the intestinal microbiota balance. The isolation and identification of bacteriocins from Bifidobacterium have been limited since the mid-1980s, leading to an underestimation of its ability for bacteriocin production. Here, we employed a silicon-based search strategy to mine 354 putative bacteriocin gene clusters (BGCs), most of which have never been reported, from the genomes of 759 Bifidobacterium strains distributed across 9 species. Consistent with previous reports, most Bifidobacterium strains did not carry or carry only a single BGC; however, Bifidobacterium longum subsp. infantis, in contrast to other Bifidobacterium species, carried numerous BGCs, including lanthipeptides, lasso peptides, thiopeptides, and class IId bacteriocins. The antimicrobial activity of the crude bacteriocins and transcription analysis confirmed its potential for bacteriocin biosynthesis. Additionally, we investigated the association of bacteriocins with the phylogenetic positions of their homologs from other genera and niches. In conclusion, this study re-examines a few Bifidobacterium species traditionally regarded as a poor source of bacteriocins. These bacteriocin genes impart a competitive advantage to Bifidobacterium in colonizing the infant intestinal tract. IMPORTANCE Development of the human gut microbiota commences from birth, with bifidobacteria being among the first colonizers of the newborn intestinal tract and dominating it for a considerable period. To date, the genetic basis for the successful adaptation of bifidobacteria to this particular niche remains unclear since studies have mainly focused on glycoside hydrolase and adhesion-related genes. Bacteriocins are competitive factors that help producers maintain colonization advantages without destroying the niche balance; however, they have rarely been reported in Bifidobacterium. The advancement in sequencing methods and bacteriocin databases enables the use of a silicon-based search strategy for the comprehensive and rapid re-evaluation of the bacteriocin distribution of Bifidobacterium. Our study revealed that B. infantis carries abundant bacteriocin biosynthetic gene clusters for the first time, presenting new evidence regarding the competitive interactions of Bifidobacterium in the infant intestinal tract.

Effects of Bacteriocin Extracted from Lactobacillus rhamnosus on Cutaneous Wound Healing in Mice

Aim: This study evaluates the effects of bacteriocin produced by Lactobacillus rhamnosus on cutaneous wound healing in mice.
 Study Design: Randomized experimental design was employed in the study.
 Methodology: Probiotic L. rhamnosus was isolated from nono milk using MRS agar media and identified based on morphological and biochemical characteristics. The potent bacterium was subjected to bacteriocin production. Antimicrobial activity of the bacteriocin was carried out against wound pathogenic bacteria and wound healing effects of the bacteriocins were assessed on mice. 2 mm cutaneous wound was induced on the mice and crude bacteriocin was topically applied on the wounds. White blood cell count was carried out after days 3, 5, 9, and 14. Histopathological analyses were performed after day 14.
 Results: Crude bacteriocin was effective against wound pathogenic bacteria such as S. aureus, P. aeruginosa and E. coli. Topical application of bacteriocin showed a faster and better wound healing when compared to positive control groups in P. aeruginosa infected wound model. Histology examination revealed an increase wound healing time in L. rhamnosus bacteriocin treated mice. The crude bacteriocins demonstrated excellent wound-healing activity in skin excision albino mice model.
 Conclusion: L. rhamnosus bacteriocin had various beneficial actions on wounds. It improved wound healing, inhibited wound pathogens and increase wound healing time.

Antibacterial Activity of Crude Bacteriocins Produced by Lactobacillus Species Isolated from Nono (Fermented Milk)

Bacteriocins are newly discovered exometabolites of lactic acid bacteria (LAB) with antimicrobial activity and biopreservative potential; however, studies investigating the potential of LAB from locally fermented milk (Nono) in Northern Nigeria for bacteriocin production and their antimicrobial effects on foodborne pathogenic bacteria are still insufficient. The present study aims to evaluate the antibacterial activity of bacteriocins produced by Lactobacillus spp isolated from Nono against foodborne pathogens. A total of 30 samples of Nono were purchased from Bayero University Kano old campus and K/Wambai market Kano State. Nono’s physicochemical parameters (pH and Titratable acidity) and Lactic acid Bacteria (LAB) counts were determined according to standard methods. The samples were further screened for Lactobacillus spp based on routine cultural characteristics, general morphological, biochemical tests and API 50CHL techniques. Crude bacteriocins were extracted and evaluated for antibacterial activity by Agar well diffusion method. The analyses revealed that Nono had pH values ranging from 2.84- 4.08, titratable acidity (1.16- 1.34%) and LAB counts ranged from 8.60x106 – 3.00x108. Thirty – eight (38) Lactobacillus spp based on distinct morphology were isolated from Nono. The different species of the genus Lactobacillus were identified phenotypically based on their carbohydrates fermentation profiles (API 50 CHL) as Lactobacillus plantarum 1, Lactobacillus fermentum 1 , Lactobacillus pentosus, Lactobacillus fermentum 2 and Lactobacillus delbrueckii ssp lactis 1. Ten (10) out of the 38 isolates were potential bacteriocin producers. Extracted crude bacteriocins exhibited broad-spectrum activity against E. coli (9.5+0.3 – 20 +0.8mm) and S. aureus (9.47+0.6 -18.3 + 0.6mm). Bacteriocins produced by Lactobacillus spp isolated from Nono could be used to solve the problem of contaminating microorganisms in food industries.

Complete Genome Sequencing Revealed the Potential Application of a Novel Weizmannia coagulans PL-W Production with Promising Bacteriocins in Food Preservative.

Weizmannia coagulans is an important potential probiotic with dual characteristics of Bacillus and Lactobacillus. This study describes a novel Weizmannia coagulans PL-W with excellent antibacterial activity isolated from Mongolian traditional cheese, in which safety and probiotic potential were evaluated by complete genome sequencing. The crude bacteriocins of W. coagulans PL-W showed antibacterial activity against various foodborne pathogens, including Listeria monocytogenes CMCC 54,004, Bacillus cereus ATCC 14,579, and Staphylococcus aureus ATCC 25,923. Moreover, the crude bacteriocins have outstanding stability against pH, temperature, surfactants, and are sensitive to protease. The complete genome sequencing revealed W. coagulans PL-W consists of 3,666,052-base pair (bp) circular chromosomes with a GC content of 46.24% and 3485 protein-coding genes. It contains 84 tRNA, 10 23S rRNA, 10 16S rRNA, and 10 5S rRNA. In addition, no risk-related genes such as acquired antibiotic resistance genes, virulence, and pathogenic factors were identified, demonstrating that W. coagulans PL-W is safe to use. Furthermore, the presence of gene clusters involved in bacteriocin synthesis, adhesion-related genes, and genes contributing to acid and bile tolerance indicate that W. coagulans PL-W is a potential candidate probiotic. Thus, antimicrobial activity and genome characterization of W. coagulans PL-W demonstrate that it has extensive potential applications as a food protective culture.

Characterization and antibacterial modes of action of bacteriocins from Bacillus coagulans CGMCC 9951 against Listeria monocytogenes

As one of the conventional probiotics, our previous findings have shown that Bacillus coagulans CGMCC 9951 had good antibacterial activity against various pathogenic bacteria. However, the action of the bacteriocins on antibacterial mode remained unclear. Based on the genomic DNA analysis of CGMCC 9951, two bacteriocins including Circularin A with 7736.14 Da and Amylocyclicin with 6371.57 Da were predicted and analyzed to simply simulate their structure. The crude bacteriocins mainly inhibited the growth of gram-positive bacteria, and the MIC against Listeria monocytogenes was 62.5 μg mL−1. The extracted bacteriocins showed good stability against pH, heat and surfactants, sensitivity to protease and weak hemolysis toxicity. The leakage of lactic dehydrogenase, nucleic acids and proteins, the bacterial uptake of propidium iodide, the observation of bacterial cell integrity, and gel retardation of genomic DNA demonstrated that the bacteriocins can destroy firstly cell membrane due to the presence of pore, and then lead to the leakage of cell contents and the inactivation of genomic DNA, affecting finally the normal growth and reproduction of indicator bacteria. The discovery of the bacteriocins and the investigation of antibacterial mechanism were expected to contribute to the development of a natural food preservative for the application of food industry.

Antimicrobial activity of bacteriocin like inhibitory substance (BLIS) and lactic acid bacteria (LAB) isolated from traditional fermented buffalo milk from West Sumatra, Indonesia

Food preservation by chemical preservatives addition is not recommended in large quantities. However, lactic acid bacteria (LAB) metabolites can be used as a safe alternative. LAB can produce a type of antimicrobial peptide known as bacteriocin. One source of LAB that can be used as a bacteriocin-producing candidate is dadih, a traditional fermented buffalo milk product from West Sumatra, Indonesia. In this study, eight bacterial isolates isolated from dadih were identified using the catalase test and Gram straining and obtained 6 isolates that were taken as LAB. An antimicrobial activity test was carried out using the diffusion method on LAB liquid isolates suspension and crude bacteriocins from the six isolates. The isolates with the highest antimicrobial activity were DK8, the diameter of inhibiting zone of liquid isolates was 15.75 mm (Salmonella sp.), 14.25 mm (Escherichia coli), and 12.25 mm (Staphylococcus aureus). Meanwhile, the diameter of crude bacteriocin inhibition of DK 8 isolates was 8 mm (Salmonella sp.). 8 mm (E. coli), and 8.5 mm (S. aureus). Furthermore, 16S rRNA sequencing confirmed DK8 as Lactobacillus pentosus.

English

This study evaluated the preservative ability of protein-like cell free supernatants produced by lactic acid bacteria (LAB) isolates from samples of Ictalurus punctatus (Cat fish) and slurry of fermented Zea mays (Ogi). The LAB strains were separately isolated from understudied samples using De Man, Rogosa and Sharpe (MRS) media at 37°C for 48 h. The isolated strains were characterized with Gram staining, oxidase and catalase tests, microscopy study, carbohydrate fermentation, acid production and NaCl tolerance. Thereafter, the protein concentrations of crude bacteriocin supernatants from the Gram positive, rod shaped, oxidase and catalase negative strains were studied. Also, the growth inhibition of Bacillus subtilis, Staphyloccocus aureus and Escherichia coli, heat stability, pH tolerance, effect of proteolytic enzyme and biopreservation efficiency of protein-like cell free supernatants (crude bacteriocins) were determined.  Biopreservative efficiency of the crude bacteriocin samples was also determined in orange (Citrus sinenses) and Titus fish (Scomber scombrus). The isolates from intestine of I. punctatus and fermented Z. mays fermented carbohydrate, and grew optimally at 3% NaCl, and 10 and 37°C, respectively. They inhibited the multiplication of E. coli at various extents, but more effective on different strains. The bacteriocins from slurry of fermented Z. mays on the other hand, were more potent in E. coli (22.7 ± 0.8 mm) than S. aureus (7.9 ± 0.1 mm). The biopreservative efficiency of crude bacteriocin from I. punctatus was greater than that of Z. mays. The LAB obtained from the selected samples produced protein-like substances in form of bacteriocins with potent antibacterial and biopreservative proficiencies through the growth inhibition of tested pathogens and low colony counts on tested food samples, respectively. Bacterial isolates obtained from samples of I. punctatus and Z. mays can be successfully used in the preservation of food and vegetables.   Key words: Ictalurus punctatus, Zea mays, bacteriocin, protein-like substances, biopreservative ability.

Antimicrobial Activity and Characteristics of Bacteriocin Producing <i>Bacillus subtilis</i> against Mastitis Pathogens

The use of drugs and antibiotics has increased the resistance of pathogenic bacteria in both animals and humans. This has been a significant problem and therefore triggers the investigation of novel antimicrobial agents produced by a bacterial strain of low virulence and having antimicrobial activity with a wide range of clinical significance. The use of bacteriocin has been extensively used in food industries, animals, and pharmaceutical industries. This is because it has been linked to antimicrobial activity, which has specific self-protection mechanisms. This study sought to evaluate antimicrobial activity and characteristics of bacteriocin producing Bacillus subtilis against Mastitis pathogens. For the screening of the isolates for bacteriocin properties against mastitis pathogens, antimicrobial activity was done using well diffusion methods on the nutrient agar. The results were obtained after 24hours and 48hours. Physiochemical characterization of the bacteriocin from Bacillus subtilis was determined at different temperatures of 60°C to 121°C for 15 minutes and monitor the effect of the temperature. The bacteriocin was also prepared at different pH (3-9) and incubated at room temperature; each sample's residual activity was determined against the indicator organisms. Metal ions (Cu2+, Zn2+, and Fe2+) on crude bacteriocin activity were determined to assess the residual antimicrobial activity by agar well diffusion assay. The results showed that bacteriocins from Bacillus subtilis were effective against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumonia. Physiochemical characterization showed that bacteriocin from different isolates had no inhibition from pH 3-5 and varied inhibition from pH 6-9 across the test organisms' isolates. On the temperature, crude bacteriocins at a temperature of 50°C to 60°C showed no activity loss based on initial activity. As temperature increases to 70°C to 80°C, there is reduced the bacteriocin activity of about 20%. 100°C had a 40% loss of the bacteriocin activity and 121°C with more than 50% loss of the activity. On metal ions, Cu2+, Fe2+, Zn2+ had a varied effect on bacteriocin activity against test organisms.

Isolation and Identification of Bacteriocin-Producing Lactic Acid Bacteria from Urutan, Balinese Traditional Fermented sausage

This study aimed to isolate and identify lactic acid bacteria (LAB) that could produce bacteriocins from urutan. Urutan is a Balinese sausage that is traditionally produced and naturally fermented. Urutans used in this study were obtained from several traders in the Baturiti, Mengwi, and Tegallalang region, Bali. More than 300 LAB colonies were isolated and underwent inhibition test against the growth of the indicator bacteria. Crude bacteriocins from two isolates, namely J2 and J6, showed inhibition zones against the growth of Lactobacillus plantarum E12.1 and Escherichia coli ATCC 8739. The J6 isolate also inhibited the growth of Enterococcus faecalis ATCC 29212. Both J2 and J6 were gram-positive, bacilli shaped, and showed negative catalase test. Based on the results of the 16S rDNA gene amplification, the two isolates were identified as Lactobacillus plantarum.
 Keywords: Lactic Acid Bacteria, Bacteriocin, Lactobacillus plantarum, Urutan

Sourcing new potential bacteriocin-producing bacteria from dangke, ethnic cheese of Enrekang, Indonesia

Bacteriocins are bioactive peptides produced by microorganisms. Sourcing of new bacteriocin-producing bacteria is essential as bacteriocin production depends on the microbial strain One of potential source for bacteriocin-producing microorganism is Dangke—a traditional cheese of Enrekang, South Sulawesi, Indonesia. Dangke contains compounds that can support the growth of lactic acid bacteria (LAB)—one of the well-known bacteriocin-producing bacteria. Specifically, this study focused on finding new strains of bacteriocin-producing LAB from dangke and screening its activity against pathogenic bacteria. LAB were isolated from cow and buffalo dangke using MRSA-CaCO3 1% media. The bacterial isolates obtained were identified based on 16S rRNA analysis. Fermentation was done in MRSB media in 37°C for 72 hours then concentrated with ammonium sulphate 80%, followed by dialysis. Crude bacteriocins were tested against Escherichia coli and Staphylococcus aureus. LAB strain B3B1 and S2A1 were successfully isolated from Dangke and were identified as Lactobacillus fermentum strain NBRC 15885 (identities 99%) and Weissella confusa strain JCM 1093 (identities 99%). Crude bacteriocin of L. fermentum strain NBRC 15885 was active against S. aureus with inhibition zone of 11.34 ± 2.97 cm. Based on the result, isolate B3B1 identified as Lactobacillus fermentum from buffalo dangke has potential in producing bacteriocin. Further studies of production, optimization, characterization and screening of other bioactivity of the bacteriocin are suggested

Characterization of a bacteriocin as biopreservative synthesized by indigenous lactic acid bacteria from dadih soya traditional product used in West Sumatra, Indonesia

Abstract. Retnaningrum E, Yossi T, Nur'azizah R, Sapalina F, Kulla PDK. 2020. Characterization of a bacteriocin as biopreservative synthesized by indigenous lactic acid bacteria from dadih soya traditional product used in West Sumatra, Indonesia. Biodiversitas 21: 4192-4198. A total of 4 isolates of lactic acid bacteria (strain BDL08, BDL11, BDL12, and BDL13) isolated from dadih soya were investigated for the ability to produce bacteriocin based on their antibacterial activities against Listeria monocytogenes ATCC 7644. Based on comparative 16S rDNA sequencing analysis, isolates BDL 11, BDL12, and BDL13 were identified as Lactobacillus plantarum while the isolates BDL 08 was identified as Leuconostoc mesenteroides. All crude bacteriocins producing strains revealed broad antibacterial spectrum against 7 different indicator bacteria, including Gram-positive and Gram-negative bacteria. L. plantarum BDL11 displayed the highest bacteriocin activity relative to others. The bacteriocin produced by the strain was not affected by pH, heating, and NaCl concentration but was sensitive to proteolytic enzymes. This research indicated that the bacteriocin in the food industry has the potential to be used as a biopreservative.

Lactobacillus xylosus isolated from butter showed potentiality to be used as probiotic and biopreservative

Today, in an era of antibiotic-resistant pathogens and other looming microbial threats, the value of prevention of infection is recognized. To circumvent the indiscriminate use of antibiotics and emerging resistance to them, to reduce the use of chemical preservatives and to abate abdominal, gastrointestinal and urogenital disorders- probiotics and bacteriocins are getting paramount priority in recent times. We investigated the probiotic and bacteriocinogenic potentiality of Lactobacillus isolated from milk products. 15 lactic acid bacteria (LAB) were isolated from milk product samples using De Man Rogosa Sharpe (MRS) medium. Among them, only one (6%) isolate showed potential antibacterial activity against Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa and Bacillus subtilis in agar well diffusion method. Following conventional methods, genus and species of the isolate were confirmed as Lactobacillus xylosus. The isolate exhibited growth competency at wide range of temperatures (27-45°C), pH (2-9), NaCl (1-7%), bile salt (0.5-2%) and could produce bacteriocin or BLS, thus implying its potential probiotic nature. Bacteriocins or BLS produced by Lactobacillus xylosus inhibited E. coli and S. aureus and could retain their antibacterial activity at wide range of temperatures (37°C to 100°C) and pH (2–9) treatments. These crude bacteriocins or BLS of 5% concentration reduced the initial bacterial load of cheese and milk up to 41% and 43% respectively, after 48 h of preservation at room temperature. The experimental data revealed that our study isolate Lactobacillus xylosus could be used as probiotics and their bacteriocin or BLS could be used as bio-preservatives.
 Asian J. Med. Biol. Res. March 2020, 6(1): 27-37

Antibacterial Effect of Bacteriocinogenic Enterococci from Different Sources on Listeria monocytogenes

In this study, antimicrobial activity of partially purified enterocins and crude bacteriocins from Enterococcus isolates with different sources was investigated against Listeria monocytogenes by disk diffusion assay. Totally 70% of enterococcal isolates (Enterococcus faecalis and Enterococcus faecium from food and clinical sources) were found as potential bacteriocinogenic strains. Both of food and clinical enterococcal isolates also exhibited antimicrobial properties against L. monocytogenes. Additionally, the present study detected that inhibitory activity was strain-specific. Both crude bacteriocins and partially purified enterocins from E. faecium isolates showed lower antimicrobial activity against L. monocytogenes than E.faecalis isolates. The inhibition diameters obtained withcrude enterocins and partially purified enterocins were respectively ranging from 12.33 mm to 13.25 mm and from 8.66 mm to 9.25 mm. Crude bacteriocins retained antibacterial activity after heat treatment except 120 oC and also remained functional at pH values between 3 and 11. Asa result, it was considered that enterocins could be benefit in heated and acidic or basic food products as biopreservative.

Screening of bacteriocin-producing lactic acid bacteria in Chinese homemade pickle and dry-cured meat, and bacteriocin identification by genome sequencing

The control of foodborne pathogens in raw/processed foods is urgently needed. Lactic acid bacteria (LAB) bacteriocins are promising natural food preservatives. In this study, 81 antibacterial LAB strains were isolated from pickles and dry-cured meats, among which 38 LAB isolates inhibited both Staphylococcus aureus and Escherichia coli. Broad antibacterial LAB strains consisted of Lactobacillus, Pediococcus, Staphylococcus, and Weissella after identification according to 16S rDNA sequences. Antimicrobial activity of crude bacteriocins of the 38 strains was tested against multiple foodborne pathogens and L. pentosus DZ35 was selected to investigate further. L. pentosus DZ35 started to produce bacteriocin at 36 h during fermentation. The bacteriocin of L. pentosus DZ35 had good thermostability and broad pH adaption ranging from pH 2 to pH 11. Its antibacterial activity was partially lost after treated by proteinases. Genome of L. pentosus DZ35 was sequenced using Illumina sequencing and MinION nanopore sequencing platform with a size of 3,962,622 bp, including nine circular plasmids. Two novel bacteriocins, pentocin DZ1 and pentocin DZ2, were identified using BAGEL4. The present study indicates that pickle has abundant bacteriocin-producing LAB and the probiotic L. pentosus DZ35 has promising potentials to control pathogens in food.

Bacteriocin production and distribution of bacteriocin-encoding genes in enterococci from dogs

Many enterococcal strains produce bacteriocins, which could be useful as natural food preservatives through inhibition of pathogenic and spoilage microorganisms. There is little knowledge of the distribution and spectrum of bacteriocin activity and the distribution of bacteriocin-encoding genes in enterococci isolated from dogs. Therefore, we subjected 160 enterococcal isolates (E. faecium n=92, E. faecalis n=35, E. hirae n=28, E. casseliflavus n=3, E. mundtii n=2) from 105 samples of dog faeces to polymerase chain reaction (PCR) detection of genes for enterocin A, P, B, L50A, L50B, AS-48, and bac31 and to screening for bacteriocin activity. The results showed the presence of at least one of the tested genes in 54/160 isolates, with E. faecium the most common gene-possessing species. The most frequently occurring gene for production of enterocin A was observed in combination with enterocin P and B. Bacteriocin activity was observed in 76/160 isolates against at least one of 5 indicator bacteria from the genus Listeria, Enterococcus, Streptococcus and Staphylococcus. Four selected strains (IK25, Bri, I/Dz, P10) were active mostly against different species of Enterococcus (in the range 400-25 600 AU/mL) and Listeria sp. (800-12 800 AU/mL) but no Gram-negative bacteria were inhibited. Protein character, thermostability (up to 121°C) and stability at different pH values (3.0–10.0) were confirmed for crude bacteriocins of these four strains. The antimicrobial substance of E. faecium IK25 strain was identified as enterocin B using molecular weight detection and the presence of genes.

Efficient Exploitation of Multiple Novel Bacteriocins by Combination of Complete Genome and Peptidome.

Backgroud: The growing emergence of antibiotic-resistant pathogens including the most dangerous superbugs requires quick discovery of novel antibiotics/biopreservatives for human health and food safety. Bacteriocins, a subgroup of antimicrobial peptides, have been considered as promising alternatives to antibiotics. Abundant novel bacteriocins are stored in genome sequences of lactic acid bacteria. However, discovery of novel bacteriocins still mainly relies on dubious traditional purification with low efficiency. Moreover, sequence alignment is invalid for novel bacteriocins which have no homology to known bacteriocins in databases. Therefore, an efficient, simple, universal, and time-saving method was needed to discover novel bacteriocins.Methods and Results: Crude bacteriocins from both cell-related and culture supernatant of Lactobacillus crustorum MN047 fermentation were applied to LC-MS/MS for peptidome assay, by which 131 extracellular peptides or proteins were identified in the complete genome sequence of L. crustorum MN047. Further, the genes of suspected bacteriocins were verified by expressed in Escherichia coli BL21 (DE3) pLysS. Thereafter, eight novel bacteriocins and two nonribosomal antimicrobial peptides were identified to be broad-spectrum activity against both Gram-positive and Gram-negative bacteria, including some multidrug-resistant strains. Among them, BM1556 located within predicted bacteriocin gene cluster. The most active bacteriocin BM1122 had low MIC values of 13.7 mg/L against both Staphylococcus aureus ATCC29213 and E. coli ATCC25922. The BM1122 had bactericidal action mode by biofilm-destruction, pore-formation, and membrane permeability change.Conclusions: The combination of complete genome and peptidome is a valid approach for quick discovery of novel bacteriocins without/with-low homology to known ones. This method will contribute to deep exploitation of novel bacteriocins in genome of bacteria submitted to GenBank.

The anti- Leishmaniasis activity of Purified Bacteriocin Staphylococcin and Pyocin Isolated from Staphylococcus aureus and Pseudomonas aeruginosa

Tropical illnesses caused by parasites proceed to cause socioeconomic devastation that reverberate worldwide protozoan parasites, like Leishmania. This parasite has an enormous public health problem in many countries. There is a growing requisite for new control methods for many of these illnesses due to the increasing drug resistance showed by the parasites and problems with drug poisonousness. In this study, fifty-five patients (burns and wounds) were collected from patients from Al-Yarmouk Hospital and Teaching Baghdad Hospital during the period from November, 2015 to January, 2016. Cultural and morphological characteristic examination, biochemical tests were conducted and confirmed the diagnosis by antibiotics sensitivity test and Vitek-2 system. The results identified thirty-three Staphylococcus aureus and thirty Pseudomonas aeruginosa from skin burn and wounds. Vitek 2 system gave confirmation of positive results for both strains with a probability 98-99%. The S. aureus isolate (S3) and P. aeruginosa (P 5) was chosen among bacterial isolates as a good producer for crude both bacteriocins according to their widest inhibition zone by well diffusion assay WDA. Two steps method extraction were used for bacteriocin purification, first via ammonium sulphate at 70% and next step by Sephadex G-50 gel filtration chromatography. The two new drugs staphylococcin and pyocin at different concentrations was used for the treatment of L. tropica and L. donvani . All concentration of Staphylococcin showed no inhibitory activity on promastigotes of L. tropica and L. donvani . While the concentration of 32.5 µgmL -1 of pyocin showed the maximum cytotoxic effect on promastigotes of L. tropica and L. donovani, where the inhibition rate (IR%) were 87.1% and 87.9% respectively. As part of the research objectives is the discovery of new treatments against leishmaniasis also benefit from improved models.

Effect of Bacteriocin-like Inhibitory Substances Produced by <i>Vaginal Lactobacilli</i> on Group B Streptococcus

Reduction of vaginal Lactobacillus population leads to overgrowth of opportunistic organisms such as Streptococcus agalactiae (Group B Streptococcus, GBS), which causes life threatening neonatal infections. The activities of bacteriocin-like inhibitory substances (BLIS) produced by Lactobacillus species isolated from the vagina of pregnant women were tested on GBS. Crude Bacteriocins were produced from seven Lactobacillus isolates using de Man Rogosa Sharpes media. Partial purification was done using ammonium sulphate saturation (90%) and dialysis methods. The antibacterial effects of the crude and partially purified BLIS were carried out by disc Agar diffusion method. The BLIS from strains R5 and L2 gave the highest and lowest inhibition zone diameter (IZD) of 16.9 mm and 12.1 mm, respectively, for the crude extracts and 18.5 mm and 14.2 mm, respectively, for the partially purified extracts. All the BLIS had their strongest activity at 30°C and decreased with increase in temperature. BLIS from strains L3 and L4 lost their activities at 100°C. The activities of all the BLIS were higher at acidic pH. The IZD of the combined crude extract was 19.06 mm, while that of the combined partially purified extracts was 21.07 mm. The combined BLIS had higher activity than each of the single compound. There were no statistical difference between the sensitivities of the partially purified BLIS and the crude BLIS (P > 0.05). The findings of this study show that BLIS produced by vaginal Lactobacilli spp. can be used as probiotics for the control of GBS and that combining the BLIS may be more effective.Keywords: Antibacterial, Group B Streptococci, Vaginal Lactobacillus