Campylobacter is the major bacterial cause of human gastroenteritis in the United States and other developed countries. Poultry are considered a main source of human Campylobacter infections. Thus, reduction of Campylobacter load in poultry is significant in food safety and public health. However, no effective measure is commercially available to prevent Campylobacter colonization in poultry to date. Antimicrobial peptides (AMPs) are short and bactericidal peptides widely present in intestine to limit bacterial infections. Recently, AMPs have been increasingly recognized as a novel class of antibiotics (peptide antibiotics) to control foodborne pathogens. Notably, several potent anti-Campylobacter bacteriocins, a group of AMPs produced by commensal bacteria, dramatically reduced C. jejuni colonization in chickens and are being directed toward on-farm control of this pathogen to protect public health. As an important strategy to evade killing by potential peptide antibiotics and by host innate defense, AMP resistance mechanisms in C. jejuni are critical to understand, but are still unknown. In this dissertation, molecular basis of Campylobacter resistance to polymyxin B, the anti-Campylobacter bacteriocins (BCNs), and a chicken host defense AMP (fowlicidin-1) was comprehensively examined using both in vitro and in vivo systems. Although polymyxin B has been successfully used as a model peptide to study AMP resistance in other Gram-negative bacteria, functional genomics examination in this study suggested that polymyxin B is not a good surrogate to study Campylobacter resistance to physiologically relevant AMPs. Campylobacter only developed low-level BCN resistance with low frequency in vitro and in vivo; the acquired BCN resistance was not stable in Campylobacter. Genomic examination of two BCN resistant mutants using DNA microarray and random transposon mutagenesis revealed that the multidrug efflux pump CmeABC contributes to both intrinsic and acquired resistance of Campylobacter to the BCNs. Random transposon mutagenesis and targeted site-directed mutagenesis identified four genes (cbrR, tig, cjaB, and cj1583c) involved in Campylobacter resistance to fowlicidin-1. These genes were also required for optimal colonization of Campylobacter in chickens. Together, the findings from this dissertation revealed uniqueness and complexity of AMP resistance in Campylobacter and will enable us to develop more sustainable peptide antibiotics and novel intervention strategies to prevent and control Campylobacter infections in humans and animal reservoirs. Key words: Campylobacter, antimicrobial peptide resistance, polymyxin B, bacteriocins, fowlicidins

A total of twenty-eight Bacillus spp. isolated from value-added surimi nuggets and their raw ingredients, were tested against each other and selected reference strains of Bacillus and Clostridium for their production of inhibitory substances using the deferred antagonism assay plating method. The isolated Bacillus strains showed inhibitory activity against all Bacillus strains, with the exception of the producer strain, as well as being effective against various strains of C. botulinum (type A, B and E). Subsequent studies showed that the inhibitory activity was detected in the culture supernatant in the late stationary phase of growth prior to sporulation. The inhibitory activity of two Bacillus strains (FN2A and FN33) were selected for further study. The inhibitory substances produced by these two strains were proteinaceous in nature, heat stable (100°C for 15min) and unaffected by organic solvents. A comprehensive study was conducted on the structural characterization of the inhibitor produced by B. subtilis FN2A using FPLC, FTIR, MS and MS/MS. Structural analysis of the inhibitor produced by B. subtilis FN2A showed that it was similar in structure to Surfactin. / Preliminary studies have shown that the Surfactin-like-compound from B. subtilis FN2A was produced in significant amounts during growth in bread with maximum production occurring in the late stationary phase (72h), at 30--35°C and at pH 6.5--7.0. Optimization studies on the production of the Surfactin-like-compound by B. subtilis FN2A in bread using a response surface methodology approach showed that temperature (33--36°C); autoclaving time (30 min); inoculum level (4%), alkali pre-treatment (0.16%), water activity (0.995) and pH 6.66 enhanced the production of the Surfactin-like-compound in bread. The compound produced under these optimal conditions also maintained its activity when subjected to various processing treatments (autoclaving, freezing and freeze drying). / Initial studies showed that low levels (1% w/w) of the Surfactin-like-compound inhibited the growth of B. cereus and proteolytic and non-proteolytic strains of C. botulinum in a model agar system. However, it had no effect on non-proteolytic strains of C. botulinum when bread, or methanol extracts of bread (1--20%), were added to formulated value-added sterile trout nuggets, with all nuggets being toxic after 28 days at 12°C. Furthermore, inoculation of B. subtilis FN2A directly into nuggets also failed to inhibit growth of non-proteolytic strains of C. botulinum. Omitting certain ingredients in the formulation failed to enhance the anti-botulinal effect of the bread or methanol extracts of the Surfactin-like-compound in the value-added nuggets. However, reducing the pH of the nuggets to ~5.5 enhanced the anti-botulinal effect of the Surfactin-like-compound. Further research is required to improve the dispersibility of the Surfactin-like-compound to inhibit the growth of C. botulinum in food systems.

No description available.

Leucocin A is a bacteriocin produced by Leucoconostoc gelidium UAL 187-22. Bacteriocin producer strains possess an immunity protein, which enables the strain to protect itself against its own bacteriocin. The immunity gene from Leucoconostoc gelidium was isolated via PCR from a recombinant clone pJF5.5. This fragment was cloned by amplifying the immunity gene from pJF5.5 and ligating it into pMALc2. The resulting recombinant plasmid pKP1 was then transformed into Escherichia coli strain JM103. The clone putative, was confirmed by DNA sequencing and southern blot hybridization using the primers EAL-2 and EAL-3. It was shown to contain an insert of 3.6 kb. Expression analysis showed the construct as an in frame malE fusion protein expressed within E. coli. The fusion construct was isolated by affinity chromatography. Leucocin A was purified to 90% purity, from the supernatant of Leucocnostoc gelidium UAL 187-22 by ion-exchange chromatography and HPLC. It was found to elute from a C18 reverse phase column at 55% actetonitrile, 0.1% TFA. Binding interaction and the stability of the immunity gene fusion protein were compared using a Biacore 2000. The supernatant and cytoplasmic extract isolated from Leucocnostoc gelidium UAL 187-22 were tested for interaction with the fusion construct. Surface Plasmon resonance studies indicated that there was no binding partner present in the supernatant which would influence the immunity process. However, a stable interaction was found between the immunity protein and an orphan ligand within the cytoplasm. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Bacteriocin leader pepides are currently receiving much attention due to their possible functions. It is predicted that these leaders prevent cytoplasmic toxicity within the producer organism by rendering the bacteriocin inactive. Leucocin A, a class IIa bacteriocin produced by Leuconostoc gelidum UAL187-22 is synthesized with a 24 amino acid leader pepide which is cleaved during extracellular translocation. The antimicrobial activity of the leucocin A precursor, pre-leucocin A, was determined to gain insight into whether, the presence of a leader peptide has an impact on anti-listerial activity. The leucocin A and pre-leucocin A genes were generated by PCR of L. gelidum UAL187-22 plasmid DNA. Recombinant plasmids, pLcaA and pPreLcaA were isolated by cloning the amplified genes into the Escherichia coli pMAL.c2 vector, and by screening transformant colonies using blue white selection methods. The malE-LcaA and malE-preLcaA fusion genes were expressed, and resulting maltose binding fusion proteins, were purified using amylose affinity chromatography. Fractions collected, contained partially pure forms of MBP-LcaA (46.433 kDa) and MBP-preLcaA (49.088 kDa) fusion proteins. Following Factor Xa digestion, the MBP affinity tag was removed; and recombinant peptides, leucocin A and pre-leucocin A were further purified by reverese phase high performance liquid chromatography. It was determined that leucocin A was eluted with a retention time of 24.893, while pre-leucocin A was eluted with a retention time of 31.447. Fractions of pure leucocin A and pre-leucocin A were thereafter assayed for activity using a deferred antagonism assay, with Listeria monocytogenes being the indicator strain. Pre-leucocin A tested positive for antimicrobial activity. However, when compared to leucocin A it was found that the leucocin A precursor inhibits Listeria to a lesser degree than leucocin A. The relative bactericidal activities of leucocin A and pre-leucocin A was calculated at 6.0 x 10⁵ AU and 4.0 x 10⁵ AU. Taking this into consideration, it was estimated that the leucocin A precursor is ~66.667 % active as mature leucocin A. Hence the presence of a leader peptide does not have an influence on leucocin A antimicrobial activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

The disease listeriosis is caused by Listeria monocytogenes. This common food-borne disease has been responsible for about 0.1 to 10 cases per million inhabitants per year. However, this disease is serious with its high fatality rates of 20% - 30%, and 40% of all cases reported have been in pregnant women suffered from a foetal abortion. Recently the organism has acquired resistance to antibiotic treatment and the development of an alternative treatment is necessary. Class IIa bacteriocins such as leucocin A have been shown to be active against L. monocytogenes. However, the leucocin A receptor molecule responsible for growth inhibition within L. monocytogenes remains unclear. Various studies have implicated the mannose PTS permease (EIIt Man) of L. monocytogenes as the putative receptor for class IIa bacteriocins. The results from studies reviewed indicate that the EIIt Man of L. monocytogenes could be the chiral receptor needed for bacteriocin interaction at the surface of targeted cells. Specifically, the membrane associated IIDMan and IICMan subunits were implicated in direct interaction with class IIa bacteriocins. Our study focused on cloning, expression and purification of the subunits of the mannose PTS permease of L. monocytogenes EGD. Primers were designed to amplify the subunit genes of the mptACD operon. The mptC, mptD and mptAB genes which were then successfully cloned into pET28a expression vector and transformed into E. coli JM109(DE3) host strain. Recombinant plasmids were screened using colony PCR. Subsequently recombinant pET28-C, pET28-D and pET28-AB was once again transformed and expressed in the E. coli BL21(DE3) pLysS expression host strain. After an induction at 30°C for 5 hours, IICMan and IIDMan were found to be expressed in the cell membrane, whilst IIABMan was expressed in the cytosol of the host expression strain. Membrane proteins His-IICMan, His- IIDMan, and cytosol associated His-IIABMan were purified using Ni2+-NTA affinity chromatography. Results for His-IICMan yielded a 28 kDa protein and a 55 kDa co-purified protein. Results for His-IIDMan yielded a 31 kDa protein and a 60 kDa co-purified protein. Results for His-IIABMan yielded a 35 kDa protein and a 68 kDa co-purified protein. A western blot analysis revealed that all proteins purified carried an attached His-tag as detected by an anti-mouse peroxidase conjugate anti-His-tag antibody. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

ABSTRACT CHARACTERIZATION AND IDENTIFICATION OF BACTERIOCINS FROM TWO LACTOCOCCUS LACTIS SUBSP. LACTIS STRAINS Ak&ccedil / elik, Oya M.S., Department of Biotechnology In this study, bacteriocins from two L. lactis subsp. lactis isolates of Turkey origin designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after &amp / #61537 / -amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of partially purified bacteriocins were determined by SDS-polyacrylamide gel electrophoresis. PCR amplification was carried out with different primers for the detection of structural genes of lactococcal bacteriocins. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Association of the bacteriocin production with plasmid DNA was examined by using acriflavine as a plasmid curing agent. Plasmid profiles of the wild type and its non-bacteriocin producing mutants were determined by using the alkali lysis method followed by agarose gel electrophoresis. The genetic nature of industrially important characteristics of Lactococcus lactis strains were investigated through gene transfer studies via conjugation. According to the results of plasmid curing and conjugal transfer trials, it was concluded that in Lactococcus lactis subsp. lactis OC1 strain a 39,7 kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity. In Lactococcus lactis subsp. lactis OC2 strain, on the other hand, a 16 kb plasmid appeared to be responsible for lacticin 481 production and lactose fermentation.

The objectives of this study were to determine the microbial ecology of “blown-pack” fresh beef obtained from a federally inspected facility and to use biopreservation to prevent spoilage of vacuum packaged chilled beef. Organic acids and alcohols in the purge obtained from the “blown” beef packages were detected using HPLC. PCR analysis indicated that Clostridium spp. were present in the purge of the commercial sample. Biochemical tests, RFLP and 16S rDNA sequencing were used to identify organisms isolated from the meat. Out of 66 isolated strains, 26 isolates were strict anaerobes and RFLP indicated that all were clonal isolates. Based on sequence analysis, the isolate was identified as Clostridium putrefaciens. The isolate caused “blown-pack” spoilage and produced butyric and propionic acids when inoculated onto fresh meat and Carnobacterium maltaromaticum UAL307 prevented the production gas and obvious signs of spoilage. / Food Science and Technology

Dissertation (PhD)--University of Stellenbosch, 2009. / Bibliography. Also available via the Internet.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2006. / Kefir is a traditional fermented milk that is carbonated, has a sharp acidic taste, yeasty flavour and contains a low percentage alcohol (less than 2% (v/v)). The beverage is manufactured by fermenting milk with Kefir grains, comprised of microorganisms, polysaccharides and milk proteins. The microbial population of Kefir grains primarily include lactic acid bacteria (LAB), namely lactococci and lactobacilli, yeasts, Acetobacter and filamentous fungi. Kefir exhibits antimicrobial activity in vitro against some fungi, and Grampositive and Gram-negative bacteria. Although the exact cause of this inhibition in Kefir is not known, the ability of LAB to inhibit the growth of closely related bacteria is well known. This inhibition of pathogenic and spoilage microbes may be due to the production of organic acids, hydrogen peroxide, acetaldehyde, diacetyl, carbon dioxide or bacteriocins. Acid is not the only contributor to the antimicrobial activity of Kefir and Kefir grains, and bacteriocins may play a role in the inhibitory activity. The bacteriocin producer Lactobacillus plantarum ST8KF, isolated from Kefir and Kefir grains, produces a bacteriocin 3.5 kDa in size. The mode of activity of bacteriocin ST8KF (bacST8KF) is thought to be bacteriostatic in exponential cultures of Enterococcus faecalis E88, Lactobacillus casei LHS, Lactobacillus curvatus DF38, Lactobacillus sakei DSM 20017, Lactobacillus salivarius 241 and Listeria innocua F and LMG 13568. The peptide is sensitive to proteolytic enzymes and does not adsorb to the surface of the producer cell. The bacteriocin is stable between pH 2.0 and 10.0, and for 20 min at 121°C. Maximum bacteriocin activity was observed in modified MRS medium supplemented with glucose or saccharose, meat extract, KH2PO4, glycerol, thiamine or cyanocobalamin, or in modified MRS medium without tri-ammonium citrate. Maximum levels of adsorption of bacST8KF (80%) to Lb. casei LHS and Lb. sakei DSM 20017 were recorded. Adsorption (80%) of the bacteriocin to Lactobacillus paraplantarum ATCC 700211T and Streptococcus caprinus ATCC 700066, which are not sensitive to the bacteriocin was also recorded. Optimal adsorption to E. faecalis E88 was recorded at 25°C at pH 2.0, and to L. innocua LMG 13568 at 4°C, 10°C and 25°C at pH 6.0. Potassium ions, MgCl2, Tris, NH4- citrate, Na-acetate, Na2CO3, EDTA and SDS led to decreased adsorption to both sensitive strains, while NaCl and mercaptoethanol resulted decreased adsorption to E. faecalis E88, but not to L. innocua LMG 13568. Methanol resulted in lower levels of adsorption to L. innocua LMG 13568 but not to E. faecalis E88. Triton X-100 and Triton X-114 increased the adsorption of bacST8KF by 40%, and ethanol and chloroform had no effect on bacteriocin adsorption. The growth of Lb. plantarum ST8KF and L. innocua LMG 13568 in a mixed culture resulted in an increase of bacST8KF production. Cells treated with bacST8KF secreted DNA and galactosidase. As bacST8KF remains stable under a variety of conditions, the bacteriocin may have application, if awarded GRAS (generally regarded as safe) status, in various food products as a natural additive or preservative. The genes encoding bacteriocin production are located on a 3.9 kilo base (kb) plasmid. Curing of the plasmid resulted in a mutant strain of Lb. plantarum ST8KF, and the Lb. plantarum strains ST8KF(+) and ST8KF(-) differed with regards to antibiotic resistance and carbohydrate fermentation reactions. The wild type and the cured strain were incorporated into Kefir grains during mass cultivation. The survival of the bacST8KF sensitive Enterococcus mundtii ST4SA added to the milk during Kefir production using the enriched mass cultured grains was monitored using fluorescent in situ hybridization. Enterococcus mundtii ST4SA was present in higher numbers in the ST8KF(-) Kefir system when compared to the ST8KF(+) system. It can, therefore, be concluded that Lb. plantarum ST8KF(+) contributes to the antimicrobial activity of Kefir through the production of bacteriocin ST8KF.