Biomedical subjects

M L Chikindas

Publications and source records attributed to M L Chikindas.

At least 19 recordsLinked to original sources

PubMed2005

Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832.

Pediocin PD-1 is a ribosomally synthesized antimicrobial peptide produced by Pediococcus damnosus NCFB1832. It inhibits the growth of several food spoilage bacteria, including malolactic bacteria isolated from wine. Pediocin PD-1 is 2866.87+/-0.4 Da in size, has an isoelectric point (pI) of ca. 9.0 and, on amino acid composition, has partial homology to the lantibiotic plantaricin C. The highest activity of pediocin PD-1 against cells of Oenococcus oeni was observed at an external pH of 5.0 and at 25 degrees C. The primary mode of action of pediocin PD-1 is most probably due to pore formation, as indicated by the efflux of K+ from metabolically active cells of O. oeni. In the presence of 10 mM gadolinium (Gd3+), pediocin PD-1 did not affect cells of O. oeni. This suggests that the mode of action of pediocin PD-1 relies on a net negatively charged cell surface. In comparison to nisin, pediocin PD-1 is less active against non-growing cells of O. oeni.

Amino Acid Sequence↗

PubMed2003

Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae.

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.

Amino Acid Sequence↗

PubMed2003

Cold and carbon dioxide used as multi-hurdle preservation do not induce appearance of viable but non-culturable Listeria monocytogenes.

AIMS: To study whether the exposure to cold (4 degrees C) and carbon dioxide which results in the elongation of Listeria cells, induces a viable but nonculturable (VBNC) state. METHODS AND RESULTS: When cold and CO2 stressed L. monocytogenes were observed under a fluorescence microscope, using the LIVE/DEAD BacLight bacteria viability kit (Molecular Probes, Eugene, OR, USA), the healthy, mildly injured, and the putative VBNC cells accounted for 31.0% of the stressed cell population. By using the selective plate count, 31.4% of the same stressed cell population was found to be healthy and mildly injured (putative VBNC cells not included). If there were VBNC state cells present, we should have observed a significant difference between the above two numbers. In fact, there was no significant difference between the results obtained from those two methods. CONCLUSIONS: There were no VBNC state cells observed in the stressed cell population. We conclude that cold and CO2 do not induce L. monocytogenes to enter a VBNC state. SIGNIFICANCE AND IMPACT OF THE STUDY: Cold and modified atmospheres are widely used in fresh muscle food and fruit preservation. Whether they would induce L. monocytogenes into a VBNC state is of a great concern for microbial food safety.

Adenosine Triphosphate↗

PubMed2001

Bacteriocins: safe, natural antimicrobials for food preservation.

Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of other bacteria. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is currently the only bacteriocin widely used as a food preservative. Many bacteriocins have been characterized biochemically and genetically, and though there is a basic understanding of their structure-function, biosynthesis, and mode of action, many aspects of these compounds are still unknown. This article gives an overview of bacteriocin applications, and differentiates bacteriocins from antibiotics. A comparison of the synthesis. mode of action, resistance and safety of the two types of molecules is covered. Toxicity data exist for only a few bacteriocins, but research and their long-time intentional use strongly suggest that bacteriocins can be safely used.

Anti-Bacterial Agents↗

PubMed2001

Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136.

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (DeltaPsi) and the intracellular ATP pool of the indicator organism, the pH gradient (DeltapH) component of the proton motive force (Deltap) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.

Adenosine Triphosphate↗

PubMed2001

Inhibition of Saccharomyces cerevisiae by slow release of propyl paraben from a polymer coating.

The inhibition of Saccharomyces cerevisiae by slow release of propyl paraben from a styrene-acrylate copolymer coating was examined and compared with the inhibition by direct addition of propyl paraben. Under slow release condition, a slow but continuous microbial inhibition was observed, whereas a cell outgrowth was observed under direct addition condition after a sufficient incubation time. S. cerevisiae cells isolated from the culture under direct addition condition were more tolerant of propyl paraben than cells isolated from the culture under slow release condition. The equilibrium propyl paraben concentrations under both conditions were 150 ppm. The release kinetics of propyl paraben from the coating at 30 degrees C was found to be controlled by Fickian diffusion within the coating. The estimated partition and diffusion coefficients were 4.65 x 10(-3) and 2.01 x 10(-10) cm2/s, respectively.

Food Contamination↗

PubMed2000

Sensitivity of nisin-resistant Listeria monocytogenes to heat and the synergistic action of heat and nisin.

Nisin, a bacteriocin produced by some strains of Lactococcus lactis, acts against foodborne pathogen Listeria monocytogenes. A single exposure of cells to nisin can generate nisin-resistant (Nisr) mutants, which may compromise the use of nisin in the food industry. The objective of this research was to compare the heat resistance of Nisr and wild type (WT) Listeria monocytogenes. The synergistic effect of heat-treatment (55 degrees C) and nisin (500 IU ml-1) on the Nisr cells and the WT L. monocytogenes Scott A was also studied. When the cells were grown in the absence of nisin, there was no significant (alpha = 0.05) difference in heat resistance between WT and Nisr cells of L. monocytogenes at 55, 60 and 65 degrees C. However, when the Nisr cells were grown in the presence of nisin, they were more sensitive to heat at 55 degrees C than the WT cells. The D-values at 55 degrees C were 2.88 and 2.77 min for Nisr ATCC 700301 and ATCC 700302, respectively, which was significantly (alpha = 0.05) lower than the D-value for WT, 3.72 min. When Nisr cells were subjected to a combined treatment of heat and nisin, there was approximately a four log reduction during the first 7 min of treatment.

Culture Media↗

PubMed2000

Nisin depletes ATP and proton motive force in mycobacteria.

This study examined the inhibitory effect of nisin and its mode of action against Mycobacterium smegmatis, a non-pathogenic species of mycobacteria, and M. bovis-Bacill Carmette Guerin (BCG), a vaccine strain of pathogenic M. bovis. In agar diffusion assays, 2.5 mg ml(-1) nisin was required to inhibit M. bovis-BCG. Nisin caused a slow, gradual, time- and concentration-dependent decrease in internal ATP levels in M. bovis-BCG, but no ATP efflux was detected. In mycobacteria, nisin decreased both components of proton motive force (membrane potential, Delta Psi and Delta pH) in a time- and concentration-dependent manner. However, mycobacteria maintained their intracellular ATP levels during the initial time period of Delta Psi and Delta pH dissipation. These data suggest that the mechanism of nisin in mycobacteria is similar to that in food-borne pathogens.

Adenosine Triphosphate↗

PubMed2000

Carbon dioxide and nisin act synergistically on Listeria monocytogenes.

This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis(r)) cells grown in broth at 4 degrees C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis(r) cells. Wild-type cells grown in 100% CO(2) were two to five times longer than cells grown in air. Nisin (2.5 microg/ml) did not decrease the viability of Nis(r) cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO(2). There was a quantifiable synergistic action between nisin and CO(2) in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 microg of nisin per ml increased from 3.1 to 12.5 microg/ml over 35 days, but this increase was markedly delayed for cultures in CO(2). This synergism between nisin and CO(2) was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO(2) and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO(2) atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4 degrees C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30 degrees C. Cells grown in the presence of 100% CO(2) and those grown at 4 degrees C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO(2) is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.

Anti-Bacterial Agents↗

PubMed1999

Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis.

Nisin is a bacteriocin produced by many strains of Lactococcus lactis. This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium. Nisin had a minimum inhibitory concentration of 8.0 micrograms ml-1 and a minimum inhibitory dose of 7.5 micrograms ml-1 against Myco. smegmatis. Treatment with 25.0 micrograms ml-1 nisin caused partial inhibition of Myco smegmatis; the survivors were nisin-sensitive when tested in a separate experiment. Mycobacterium smegmatis cells exposed to 50.0 micrograms ml-1 of nisin, lost their viability. the effect of nisin on the growth of Myco. smegmatis was both time- and concentration-dependent. Nisin (10.0 micrograms ml-1) caused 97.7 +/- 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco. smegmatis cells after several hours of treatment. These data suggest that nisin inhibits Myco. smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.

Adenosine Triphosphate↗

PubMed1998

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum.

Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 degrees C, but loses 50% of its activity after 60 min at 100 degrees C and 75% of its activity after autoclaving (121 degrees C, 15 min). Plantaricin 423 remains active after incubation at pH 1-10 and is inactivated when treated with pepsin, papain, alpha-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3.5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1.0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.

Bacteria↗

PubMed1997

Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832.

Pediocin PD-1, produced by Pediococcus damnosus NCFB 1832, is inhibitory to several food spoilage bacteria and food-borne pathogens. However, pediocin PD-1 is not active against other Pediococcus spp. and differs in this respect to other pediocins produced by Pediococcus acidilactici and Pediococcus pentosaceus. Production of pediocin PD-1 starts during early growth and reaches-a plateau at the end of exponential growth. Pediocin PD-1 was partially purified and its size was determined by tricine-SDS-PAGE as approximately 3.5 kDa. The isoelectric point (pI) of pediocin PD-1 is approximately 3.5, as determined with the Rotofor electrofocusing cell (BioRad). Pediocin PD-1 is heat-resistant (10 min at 121 degrees C) and remains active after 30 min of incubation at pH 2-10. Pediocin PD-1 is resistant to treatment with pepsin, papain, alpha-chemotrypsin and trypsin, but not Proteinase K. Pediocin PD-1 is bactericidal against sensitive cells of Oenococcus oeni (previously Leuconostoc oenos).

Bacteriocins↗

PubMed1997

Rapid and Efficient Purification Method for Small, Hydrophobic, Cationic Bacteriocins: Purification of Lactococcin B and Pediocin PA-1.

The bacteriocins lactococcin B and pediocin PA-1 were purified by ethanol precipitation, preparative isoelectric focusing, and ultrafiltration. The procedure reproducibly leads to high final yields in comparison to the generally low yields obtained by column chromatography. Specifically, during isoelectric focusing no loss of activity occurs. The method, in general, should be applicable to small, hydrophobic, cationic bacteriocins.

Journal Article↗

PubMed1995

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme.

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

ATP-Binding Cassette Transporters↗

PubMed1995

Expression of lactococcin A and pediocin PA-1 in heterologous hosts.

Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0. The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes. Here, expression of the pediocin operon in Lactococcus lactis is reported, which could only be achieved by placing it under control of a lactococcal promoter. Expression of the lactococcin A operon in Pediococcus is also described: recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA-1 and lactococcin A.

Anti-Bacterial Agents↗

PubMed1995

Mutacin II, a bactericidal antibiotic from Streptococcus mutans.

Mutacin II is an antibiotic that is produced by group II Streptococcus mutans. It inhibits the growth of other streptococci as well as many other gram-positive microorganisms by a hitherto unknown mechanism. Mutacin II possess bactericidal activity against susceptible cells. It transiently depolarizes the transmembrane electrical potential (delta psi) and the transmembrane pH gradient (delta pH) and partially inhibits amino acid transport. However, it rapidly depletes the intracellular ATP pool in glucose-energized cells and prevents the generation of ATP. It is concluded that mutacin II does not belong to the group of pore-forming antibiotics (type A) or to the type B antibiotics, which inhibit phospholipases or interfere with peptidoglycan biosynthesis. Mutacin II acts by inhibiting essential enzyme functions at the level of metabolic energy generation, an activity that has not yet been classified for antibiotics.

Adenosine Triphosphate↗

PubMed1994

Functional organization of the riboflavin biosynthesis operon from Bacillus subtilis SHgw.

We have sequenced 6006 bp DNA of a region from the Bacillus subtilis SHgw chromosome known to contain riboflavin biosynthesis genes (rib gene cluster, 210 degrees on the B. subtilis genetic map). Five of the seven open reading frames found within the sequence are shown to represent the genes ribG, ribB, ribA, ribH and ribTD. The calculated molecular masses for the putative translation products are 39,305, 23,481, 44,121, 16,287 and 14,574 daltons respectively. The five rib genes are transcribed as a polycistronic 4277 nucleotide messenger RNA. The steady-state level of the transcript is negatively regulated by riboflavin. A cis-acting element necessary for regulation was mapped by analysis of constitutive mutations within the 5' untranslated region of the operon. The element is at least 48 bp in length and does not bear obvious similarity to well defined prokaryotic regulatory elements. The molecular mechanism of regulation remains unknown, but the data presented argue against regulation by attenuation.

Bacillus subtilis↗

PubMed1993

Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.

Pediocin PA-1 is a bacteriocin which is produced by Pediococcus acidilactici PAC1.0. We demonstrate that pediocin PA-1 kills sensitive Pediococcus cells and acts on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, pediocin PA-1 dissipates the transmembrane electrical potential and inhibits amino acid transport in sensitive cells. Pediocin interferes with the uptake of amino acids by cytoplasmic membrane vesicles derived from sensitive cells, while it is less effective with membranes derived from immune cells. In liposomes fused with membrane vesicles derived from both sensitive and immune cells, pediocin PA-1 elicits an efflux of small ions and, at higher concentrations, an efflux of molecules having molecular weights of up to 9,400. Our data suggest that pediocin PA-1 functions in a voltage-independent manner but requires a specific protein in the target membrane.

Amino Acids↗