Progress 07/01/07 to 09/30/12
Outputs
OUTPUTS: The following are the results of screening food and environment for antimicrobial peptides with potential application against foodborne pathogens. TASK 1. Coproduction of subtilosin, surfactin and plipastatin by Bacillus atrophaeus OSY-7LA for inhibition of foodborne and plant pathogens. (Intorduction and objectives) Bacillus strains have been employed to inhibit bacterial and fungal pathogens. Understanding of the antagonistic mechanism will help for the rational use of these strains in agricultural applications. Bacillus atrophaeus OSY-7LA exhibits strong antagonistic activity against Rhizopus arrhizus and many foodborne pathogens. The purpose of this study was to characterize the antimicrobials produced by OSY-7LA. TASK 2. Isolation of a new Paenibacillus strain and its broad-spectrum lipopeptide antibiotic (Intorduction and objectives) The emerging resistance of pathogenic bacteria to antibiotics poses a serious health challenge. Resistance of Staphylococcus aureus to methicillin, Pseudomonas aeruginosa and Clostridium difficile to fluoroquinolones, and Salmonella serovars to multiple drugs are examples of this challenge. The discovery and approval of new antimicrobial agents do not match the rate at which the currently-used antibiotics lose efficacy. This discrepancy makes it urgent to search for new potent and safe antimicrobial agents. Environment remains an important reservoir for microorganisms capable of producing potent antimicrobials. Advances in sensitivity testing, material separation, and chemical structure elucidation facilitate the discovery of novel antimicrobials from natural sources. This research was initiated in pursuit of novel antimicrobial compounds produced by food or environmental microorganisms. PARTICIPANTS: Yaoqi, Guo: Food Science & Technology, OSU En, Huang: Food Science & Technology, OSU Chunhua, Yuan: Nuclear Magnetic Resonance Facility, Campus Chemical Instrument Center, OSU Liwen Zhang: Mass Spectrometry and Proteomics Facility, Campus Chemical Instrument Center, OSU Ahmed E. Yousef: Department of Food Science & Technology, and Department of Microbiology,The Ohio State University, Columbus, Ohio 43210 Jianrong Li: Food Science & Technology, OSU TARGET AUDIENCES: - Food processors (e.g., Kraft Food, Nestle, Unilever, General Mills) - Pharmaceutical industry OSU commercialization unit has attempted to contact potential users of these technologies but their efforts have not succeeded yet. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
TASK 1. Coproduction of subtilosin, surfactin and plipastatin by Bacillus atrophaeus OSY-7LA for inhibition of foodborne and plant pathogens. (Methods and outcomes) The antimicrobial agents were harvested by solvent extraction from OSY-7LA culture supernatant, followed by purification using high performance liquid chromatography (HPLC). Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) were performed to identify these compounds. A protonated ion at m/z 3401.414 matched the molecular mass of the lantibiotic subtilosin; and the identity of the antimicrobial agent was further confirmed by polymerase chain reaction (PCR) amplification of the subtilosin gene (sbo). Sodiated ions at m/z 1030.553, 1044.642 and 1058.701 were identified as C13, C14 and C15 lipopeptide surfactin. Additionally, production of the antifungal lipopeptides, plipastatin A and B, were proven by LC/MS. Coproduction of subtilosin, surfactin and plipastatin contributes to the antagonistic function of OSY-7LA against foodborne and plant pathogens. Utilization of PCR and MS facilitate the identification of both bacteriocin and lipopeptides for strain OSY-7LA. TASK 2. Isolation of a new Paenibacillus strain and its broad-spectrum lipopeptide antibiotic (Methods and outcome) A new bacterial strain, designated as OSY-SE, which produces a unique and potent antimicrobial agent was isolated from soil. The isolate was identified as Paenibacillus spp. through cultural, biochemical, and genetic analyses. The antimicrobial compound was extracted from OSY-SE cells with acetonitrile, and purified using liquid chromatography. After analyses by mass spectrometry (MS) and nuclear magnetic resonance (NMR), the antimicrobial compound was determined to be a cyclic lipopeptide consisting of a C15 fatty acyl (FA) chain and thirteen amino acids. The deduced sequence is: FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-Val-Lys-Ile. The carboxyl terminal Ile is connected with Thr by ester linkage. The new compound, designated as paenibacterin, showed antagonistic activities against most Gram-positive and Gram-negative bacteria tested, including Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, Escherichia coli O157: H7, and Salmonella enterica serovar Typhimurium. Paenibacterin is resistant to trypsin, lipase, α-glucosidase and lysozyme. Its antimicrobial activity was lost after digestion by pronase and polymyxin acylase. Paenbacterin is readily soluble in water, and fairly stable to exposure to heat and a wide range of pH values. The new isolate and associated antimicrobial agent are currently investigated for usefulness in food and medical applications.
Publications
- Guo Y, Huang E, Yuan C, Zhang L, and Yousef AE. 2012. Isolation of a strain of Paenibacillus sp. and structural elucidation of its broad-spectrum lipopeptide antibiotic. Appl. Environ. Microbiol. 78:3156-3165.
- Huang E, and Yousef AE. 2012. Draft genome sequence of Paenibacillus polymyxa OSY-DF that coproduces a lantibiotic, paenibacillin, and polymyxin E1. J. Bacteriol. 194:4739-4740.
- Huang E, Guo Y, and Yousef AE. 2012. Draft genome sequence of Paenibacillus sp. OSY-SE, a bacterium produces a novel broad-spectrum lipopeptide antibiotic, Paenibacterin. J. Bacteriol. 194:6306-6306.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Subtitle: Identification and characterization of a new Bacillus atrophaeus strain producing multiple antimicrobial agents. Aims: This work was conducted to address the shortage of novel antimicrobial agents against emerging foodborne pathogens. Methods and results: A new bacterial strain, OSY-7LA, was isolated from "Fa Cai," a Chinese delicacy food. The isolate exhibited strong antagonistic activity against Listeria innocua and was identified as Bacillus atrophaeus by morphological, physiological, and biochemical properties and genetic relatedness. The culture supernatant has antimicrobial activities against the Gram-positive pathogens tested, namely, L. monocytogenes, B. cereus and methicillin-resistant Staphylococcus aureus. Additionally, the culture supernatant was active against the tested fungal species, Rhizopus arrhizus. The antimicrobial agents were harvested by solvent extraction of OSY-7LA culture supernatant and were purified by high performance liquid chromatography (HPLC). Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and tandem mass spectrometry (MS/MS) were performed to identify these compounds. A protonated ion at m/z 3401.414 corresponded to the molecular mass of subtilosin, and the identity of the antimicrobial agent was confirmed by amplification of subtilosin gene (sbo) from isolate's genomic DNA. Sodiated ions at m/z 1030.553, 1044.642 and 1058.701 were identified as C13, C14 and C15 surfactin. Production of plipastatin A and B were proven by LC/MS. PARTICIPANTS: Visiting scholar, Yaoqi Guo, was the researcher who ran the experiments En Huang helped with experimental design and research execution. TARGET AUDIENCES: Food industry is the primary beneficiary of the project outcome. Feed industry may also benefit from the study. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Conclusions: B. atrophaeus OSY-7LA was isolated from food and showed good antimicrobial activity against foodborne pathogens, antibiotic resistant strain, and a fungus. This bacterium produces multiple antimicrobial agents, subtilosin, surfactin and plipastatin. Significance and impact of the study: The new strain and related antimicrobials are potentially useful in biocontrol of foodborne microorganisms or in medical application.
Publications
- Guo, Y, E. Huang, L. Zhang, R. Zhang and A.E. Yousef. 2011. Isolation and Identification of a new Bacillus atrophaeus strain producing multiple antimicrobial agents that are potentially useful as antimicrobial additives. Oral presentation at the International Association of Food Protection, August 3rd., Milwaukee, WI (Session: T11)
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The following studies were completed: a. Investigating the genetics of biosynthesis of paenibacillin, a natural antimicrobial peptide produced by a foodborne bacterial isolate. b. Feasibility of inactivation of Listeria monocytogenes in meat products by paenibacillin. This information is being disseminated through the following presentations: 1. Ahmed Yousef. 2010. Searching for novel antimicrobial peptides. Presentation at the Faculty of Science, Alexandria University, Egypt, February 11. 2. En Huang, Jing He, Ahmed Yousef. 2011. The biosynthesis of paenibacillin and its use against Listeria monocytogenes in meat products. Abstract submitted to the annual meeting of the International Association of Food Protection, Milwaukee, WI, USA. 3. Yaoqi Guo, En Huang, Liwen Zhang, Rijun Zhang, Ahmed Yousef. 2011. Isolation and identification of a new Bacillus atrophaeus strain producing multiple antimicrobial agents that are potentially useful as antimicrobial additives. Abstract submitted to the annual meeting of the International Association of Food Protection, Milwaukee, WI, USA. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Food processors who seek novel natural additives to control pathogens in food. These include companies such as Nestle and Unilever. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The following information has been generated as a result of this project: Partial identification of the gene cluster encoding paenibacillin has been accomplished. In an eight-kb fragment, putative lantibiotic dehydratase (paenB), lantibiotic cyclase (paenC) and ABC transporter (paenT) genes were identified. However, the structural gene encoding paenibacillin precursor has not been found within this fragment. Currently, experiments are designed to target the structural gene. We also have proved the feasibility of using the new lantibiotic (i.e., paenibacillin) to control L. monocytogenes in meat. Crude extract (CE) of paenibacillin was prepared from the producer culture by ion-exchange chromatography and solid-phase extraction using Sep-Pak C18 cartridges. Adding 3.3% (v/w) CE to Vienna sausage inhibited the growth of L. monocytogenes when the product was incubated for 24 h at 25C. Under similar conditions, the population of Listeria increased from 5.0 logs to 8.7 logs in the untreated control. Similarly, paenibacillin inhibited the growth of L. monocytogenes in irradiated ground beef while being held at 4 C for 7 days. In conclusion, the novel lantibiotic (paenibacillin) has been proven useful in food applications to control L. monocytogenes.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The purpose of the study is to isolate and characterize a natural additive that may be useful in controlling disease-causing bacteria in food. The additive under investigation is an antimicrobial compound produced by a food bacterium (called Enterococcus faecalis OSY-RM6); this compound will be referred to as Enterocin RM6. We developed a protocol to purify Enterocin RM6. The protocol employs cation-exchange chromatography, multiple Sep-Pak C18 cartridges and reverse phase HPLC. The purification of Enterocin RM6 allowed us to determine its molecular weight precisely. Using advanced chromatography and mass spectroscopy techniques, we determined that the molecular weight of Enterocin RM6 was 7146. Additionally, we were able to determine the structure of this compound with reasonable accuracy. Enterocin RM6 turned out to be a cyclic compound that is made of 70 amino acids. The new compound has a broad antimicrobial activity against dangerous pathogens including Listeria monocytogenes, Bacillus cereus and the famous methicillin resistant Staphylococcus aureus (MRSA). We tested Enterocin RM6 against Listeria monocytogenes in cottage cheese during temperature abuse at 35C for 26 hours. Addition of 10% of crude extract of Enterocin RM6 killed 99.99% of the population of Listeria monocytogenes in cottage cheese within 30 min. After 26 hours of treatment with Enterocin RM6, the pathogen was not detectable in the cheese. PARTICIPANTS: Participants include: Ahmed Yousef (PI): Manages the project. Eh Huang (Graduate student): Carries out laboratory research Yaoqi Guo (visiting scholar): Assist with lab research Partner organization: Kraft Food is sponsoring some of the project's ativities TARGET AUDIENCES: Food processors who seek novel natural additives to control pathogens in food. These include companies such as Nestle and Unilever. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We successfully identified a new compound, produced by a harmless microorganism, that can be useful in improving the safety of food and in protecting consumers against diseases. The new compound was found to protect cottage cheese against a dangerous bacterium called Listeria monocytogens. There is a shortage of compounds that are as effective as the one we found in this study. It is possible the new compound would be applied to food by some segments of the food industry.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: In search for food-grade bacteriocin-producing bacteria that are active against spoilage and pathogenic microorganisms, various commercial food products were screened and fifty-one promising Gram-positive isolates were studied. Among them, fourteen food isolates with antimicrobial activity against food-borne pathogenic bacteria, Listeria monocytogenes and Bacillus cereus, were chosen for further study. Based on 16S ribosomal RNA gene sequence analysis, fourteen food isolates were identified as Enterococcus faecalis, and these enterococcal isolates were investigated for the presence of virulence factors and antibiotic resistance through genotypic and phenotypic screening. Results indicated that isolates encoded some combination of virulence factors. The esp gene, encoding extracellular surface protein, was not detected in any of the isolates. Phenotype of antibiotic resistance indicated that all isolates were resistant to kanamycin. Some isolates were also resistant to tetracycline. All isolates were sensitive to ampicillin, erythromycin, and chloramphenicol. Bacteriocins produced by E. faecalis OSY-RM1 and E. faecalis OSY-3E1, referred to as enterocins OSY-RM1 and OSY-3E1, respectively, were chosen for further characterization due to their strong antimicrobial activity against selected indicators. Results indicated that both enterocins were relatively heat stable, sensitive to protease, and stable over a wide pH range. Application of enterocin OSY-RM1 to inactivate selected spoilage and food-borne pathogenic bacteria was investigated. Enterocin OSY-RM1 (44.7 nisin-equivalents IU/ml), extracted by ammonium sulfate precipitation, had a bactericidal effect on L. monocytogenes Scott A and bacteriostatic effect on B. cereus ATCC 11778 in sterile milk. Although enterocin OSY-RM1 was not active against Escherichia coli K12, combination treatment of high pressure processing and enterocin OSY-RM1 caused significant inactivation of E. coli K12 in phosphate buffer saline. Through polymerase chain reaction (PCR) amplification and direct sequencing, the structural gene of enterocin OSY-RM1 was identified with similarity to published enterocin EJ-97. Unlike enterocin EJ-97 which is encoded on a plasmid, enterocin OSY-RM1 is likely encoded on bacterial chromosome since plasmid-cured E. faecalis OSY-RM1 retained its antimicrobial activity. Gene encoding enterocin OSY-3E1 was also identified by PCR amplification and direct sequencing and result indicted that its structural gene was similar to that of enterocin AS-48. Unlike enterocin AS-48 that is active against Gram-negative bacteria (i.e. E. coli and Shigella sonnei), enterocin OSY-3E1 has no inhibitory activity against Gram-negative bacteria tested. Enterocin OSY-3E1 was further applied to inactivate spoilage bacterium in food system. Result showed that enterocin OSY-3E1 (25,600 AU/ml), obtained from modified MRS broth containing 5% glucose, can effectively inactivate the spoilage bacterium, Leuconostoc mesenteroides ATCC 14935, in sterile milk. In conclusion, bacteriocins produced by food isolates, E. faecalis OSY-RM1 and OSY-3E1 have potential uses to inactivate spoilage and pathogenic bacteria. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Audience who would be most interested in the findings of this research: - Research & Development divisions of food processing companies, who are looking for new preservatives. - Pharmaceutical companies who are searching for potentially useful drugs. - Feed companies who are searching for new and potent additives. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This project aims at discovering new natural preservatives for use in food and beyond. There is a world-wide shortage of effective preservatives, particularly those of natural origin. This study addresses this crises. Our research revealed several preservatives produced by bacteria that live in fermented foods such as kimchi, sausage, cheese and milk. The potential for applying the findings of these discoveries is great. The Ohio State University, Technology Licensing & Commercialization office is currently patenting one of these discoveries. Commercial partners are sough for adaptation.
Publications
- He, Z., Yuan, C., Zhang, L., and Yousef, A. E. 2008. N-terminal acetylation in paenibacillin, a novel lantibiotic. FEBS Lett. 582:2787-2792
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