Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents.

INTRODUCTION: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential. METHODS: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene. RESULT: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR. CONCLUSION: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

Evolution of ColE1-like plasmids across γ-Proteobacteria: From bacteriocin production to antimicrobial resistance.

Antimicrobial resistance is one of the major threats to Public Health worldwide. Understanding the transfer and maintenance of antimicrobial resistance genes mediated by mobile genetic elements is thus urgent. In this work, we focus on the ColE1-like plasmid family, whose distinctive replication and multicopy nature has given rise to key discoveries and tools in molecular biology. Despite being massively used, the hosts, functions, and evolutionary history of these plasmids remain poorly known. Here, we built specific Hidden Markov Model (HMM) profiles to search ColE1 replicons within genomes. We identified 1,035 ColE1 plasmids in five Orders of γ-Proteobacteria, several of which are described here for the first time. The phylogenetic analysis of these replicons and their characteristic MOBP5/HEN relaxases suggest that ColE1 plasmids have diverged apart, with little transfer across orders, but frequent transfer across families. Additionally, ColE1 plasmids show a functional shift over the last decades, losing their characteristic bacteriocin production while gaining several antimicrobial resistance genes, mainly enzymatic determinants and including several extended-spectrum betalactamases and carbapenemases. Furthermore, ColE1 plasmids facilitate the intragenomic mobilization of these determinants, as various replicons were identified co-integrated with large non-ColE1 plasmids, mostly via transposases. These results illustrate how families of plasmids evolve and adapt their gene repertoires to bacterial adaptive requirements.

Assessment of the bioprotective potential of lactic acid bacteria against Listeria monocytogenes on vacuum-packed cold-smoked salmon stored at 8 °C.

Smoked salmon is a highly appreciated delicatessen product. Nevertheless, this ready-to-eat (RTE) product is considered at risk for Listeria monocytogenes, due to both the prevalence and growth potential of this bacteria on the product. Biopreservation may be considered a mild and natural effective strategy for minimizing this risk. In this study, we evaluated the following three potential bioprotective lactic acid bacterial strains against L. monocytogenes in three smoked salmon types with different physicochemical characteristics, primarily fat, moisture, phenol and acid acetic content: two bacteriocin-like producers that were isolated from smoked salmon and identified as Lactobacillus curvatus and Carnobacterium maltaromaticum and a recognized bioprotective bacteriocin producer from meat origin, Lactobacillus sakei CTC494. L. sakei CTC494 inhibited the growth of L. monocytogenes after 21 days of storage at 8 °C in all the products tested, whereas L. curvatus CTC1742 only limited the growth of the pathogen (<2 log increase). The effectiveness of C. maltaromaticum CTC1741 was dependent on the product type; this strain limited the growth of the pathogen in only one smoked salmon type. These results suggest that the meat-borne starter culture, L. sakei CTC494, may potentially be used as a bioprotective culture to improve the food safety of cold-smoked salmon.

Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Accelerating emergence of antimicrobial resistance among food pathogens and consumers' increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein-one of the primary self-assembly proteins in milk with a high polymeric film production capability-as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time-kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm-2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g-1 ). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Lanthipeptides: chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production.

Lanthipeptides (also called lantibiotics for those with antibacterial activities) are ribosomally synthesized post-translationally modified peptides having thioether cross-linked amino acids, lanthionines, as a structural element. Lanthipeptides have conceivable potentials to be used as therapeutics, however, the lack of stable, high-yield, well-characterized processes for their sustainable production limit their availability for clinical studies and further pharmaceutical commercialization. Though many reviews have discussed the various techniques that are currently employed to produce lanthipeptides, a direct comparison between these methods to assess industrial applicability has not yet been described. In this review we provide a synoptic comparison of research efforts on total synthesis and in vivo biosynthesis aimed at fostering lanthipeptides production. We further examine current applications and propose measures to enhance product yields. Owing to their elaborate chemical structures, chemical synthesis of these biomolecules is economically less feasible for large-scale applications, and hence biological production seems to be the only realistic alternative.

Time-dependent fermentation control strategies for enhancing synthesis of marine bacteriocin 1701 using artificial neural network and genetic algorithm.

The artificial neural network (ANN) and genetic algorithm (GA) were combined to optimize the fermentation process for enhancing production of marine bacteriocin 1701 in a 5-L-stirred-tank. Fermentation time, pH value, dissolved oxygen level, temperature and turbidity were used to construct a "5-10-1" ANN topology to identify the nonlinear relationship between fermentation parameters and the antibiotic effects (shown as in inhibition diameters) of bacteriocin 1701. The predicted values by the trained ANN model were coincided with the observed ones (the coefficient of R(2) was greater than 0.95). As the fermentation time was brought in as one of the ANN input nodes, fermentation parameters could be optimized by stages through GA, and an optimal fermentation process control trajectory was created. The production of marine bacteriocin 1701 was significantly improved by 26% under the guidance of fermentation control trajectory that was optimized by using of combined ANN-GA method.

Biotechnological valorization of low-cost sugar-based media for bacteriocin production by Leuconostoc mesenteroides E131.

The aim of the present study was to evaluate the suitability of low-cost carbon sources for bacteriocin production by Leuconostoc mesenteroides strain E131. For this purpose, inexpensive sugars derived from a sugar refinery plant (glucose, fructose and sucrose) as well as waste molasses were utilized as carbon sources in submerged shake-flask experiments and the kinetic response of the microorganism was evaluated. Interestingly, in the case of molasses, non-negligible decolorization-detoxification (up to ∼27%) of the residue was performed together with the production of bacteriocin. In all instances the initial concentration of sugars employed was adjusted at 20 and 30 g/L, therefore the effect of both the nature and the initial quantity of sugar upon the growth of the microorganism was assessed. All media proved to be suitable for both biomass and bacteriocin production by L. mesenteroides, whereas variable quantities of lactate, acetate and ethanol were detected into the medium. Employment of fructose, sucrose or molasses as carbon sources resulted in the accumulation of mannitol (in some cases in significant quantities) into the medium; remarkable portion thus of the available or released fructose acted as electron acceptor instead of carbon source by the microorganism. The highest bacteriocin production achieved (=640 AU/mL) was obtained when initial glucose at 30 g/L was used as substrate. Finally, utilization of waste molasses as carbon source by L. mesenteroides resulted in satisfactory bacteriocin production (up to 320 AU/mL) besides the decolorization of the residue.

Native and heterologous production of bacteriocins from gram-positive microorganisms.

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.

Rapid quantifiable assessment of nutritional parameters influencing pediocin production by Pediococcus acidilactici NRRL B5627.

A direct plate bioassay procedure was applied for rapid and quantifiable assessment of the influence of various nutritional parameters on pediocin production by Pediococcus acidilactici NRRL B5627. Solid-state cultivation of the microorganism was done on MRS-based media over 3-and 6-hours incubation periods. Nutritional parameters assessed included the carbon source (glucose, sucrose, fructose, galactose, glycerol), and various salts (NH(4)PO(4), CaCl(2), KH(2)PO(4), MnSO(4).H(2)O). Glucose was found to be the optimal carbon source while glycerol exhibited the most suppressive effect. Using glucose as the carbon source, addition of various salts, in amounts used in liquid media commonly applied in the cultivation of the pediococci, was assessed with respect to bacteriocin production on a per cell basis. Experimental data obtained showed that several nutritional parameters repress pediocin production by P. acidilactici, while the direct plate assay proved to be a good pilot assay prior to conducting more intensive kinetic analysis in liquid cultivation.

Safety assessment of dairy microorganisms: coagulase-negative staphylococci.

The genus Staphylococcus is made up of 36 validated species which contain strains that are pathogenic, saprophytic, or used as starter cultures for the food industry. Staphylococci species used in cheese-making are novobiocin-resistant, coagulase-negative and are not usually identified at species level by routine laboratories. A bibliographic survey was conducted to assess safety status of CNS used in fermented dairy foods. Commercial kits based on phenotypic discrimination can't reliably identify these species because of the variable expression of some phenotypic traits. Several molecular targets, including 16S rRNA, hsp60, tuf, SodA and rpoB genes can be used for identifying species of the Staphylococcus genus. No coagulase-negative staphylococci isolated from milk or dairy products has ever been involved in a case of food poisoning or human pathology following ingestion of dairy products. Nevertheless, a few case of nosocomial infection caused by some species (S. caprae, S. capitis, S. sciuri) have been reported in patients with depressed immune systems or who have undergone long severe hospital treatments, or in the presence of an indwelling catheter or foreign materials such as cardiac prostheses. They may therefore be regarded as exceptional opportunistic pathogens in certain clinical situations.

Safety assessment of dairy microorganisms: Streptococcus thermophilus.

Streptococcus thermophilus is a major dairy starter used in yogurt and cheese production. In Streptococcus genus, S. thermophilus is the only one food species among commensal and opportunistic pathogen species. Comparative genomics suggest that this species recently emerged and evolved by combination of loss-of-function and horizontal gene transfer events. These gene transfer events detected in S. thermophilus have originated from other dairy species and might contribute to its adaptation to the milk environment.

Rings, radicals, and regeneration: the early years of a bioorganic laboratory.

This Perspective provides an overview of the progress in two of the original programs in my research group focused on the biosynthesis of the antibiotics nisin, lacticin 481, fosfomycin, and bialaphos. The path from start-up funds to tenure and beyond offers insights into the opportunities realized and missed along the road.

Assessment of a bacteriocin-producing Lactobacillus strain in the control of spoilage of a cereal-based African fermented food.

As a part of a program to develop starter cultures aiding in the spoilage control and sanitation of African fermented foods, a cereal-based food ('ogi' and its solid form 'agidi' or 'eko') was prepared using a bacteriocin-producing Lactobacillus strain as the starter culture. The survival of an enterotoxigenic Escherichia coli strain was investigated in the naturally fermented food and in food fermented with the starter bacteriocin-producing Lactobacillus strain. An inhibition of E. coli was observed within 2 h of incubation in 'ogi' fermented with the bacteriocin producing strain. After 6 h, the viable count of E. coli in locally fermented 'ogi' was log 6.41 (2.54 X 10(6) CFU/mL), whereas in 'ogi' fermented with the bacteriocin producer it was reduced to log 1.70 (0.5 x 10(2) CFU/mL). Comparison of the shelf life of 'agidi' prepared from the naturally fermented food with that bacteriocin-producing starter culture showed that the latter had a better shelf life (kept for 11 d before spoilage occurred as compared with 7 d for the natural one). The results are discussed in terms of the potential of bacteriocin-producing cultures in the control and retardation of spoilage and food-forne infections in some African fermented foods.

The assessment and application of a bacteriocin typing scheme for Clostridium perfringens.

A collection of 50 bacteriocins was assembled and used to type 802 isolates of Clostridium perfringens from food poisoning outbreaks and a variety of other sources. It was found that strains of the same serotype within an outbreak showed similar patterns of susceptibility to bacteriocins, and the use of "one difference' rule is proposed for interpretation of the typing patterns of epidemiologically related strains. Isolates of different serotype or of the same serotype isolated from different sources produced many variations in bacteriocin susceptibility patterns. Two computer programs were developed to assist in the interpretation of bacteriocin typing patterns. Their use showed that related and unrelated strains formed different clusters and enabled a range of the 20 most discriminatory bacteriocins to be selected. Isolates of C. perfringens from a wide range of sources were screened for their ability to produce bacteriocins. A much greater proportion of the strains from food poisoning outbreaks was bacteriocinogenic than were isolates from human and animal infections, various foods and the environment. The relevance of these findings to the occurrence of C. perfringens food poisoning is discussed.

The development and assessment of a bacteriocin typing method for Klebsiella.

Klebsiellas are generally typed by the method of capsular serotyping but, although this is a reliable method, it is time consuming, requires the production of a large number of antisera and is not generally available. For this reason another method for typing klebsiellas was sought. A bacteriocin typing method involving mitomycin C induction was developed and the cultural conditions giving optimum klebecin production and the best methods of testing the sensitivity of the organisms to klebecins were determined. Of 190 klebsiella strains screened for bacteriocinogeny, only 68 (35.8%) produced klebecin and after calculation of similarity values by computer analysis, a typing set of 15 producers was selected. This typing set allowed over 96% of klebsiella strains to be typed and tests of the reproducibility of the method and the variability of typing patterns in natural populations of klebsiella indicated that results of acceptable accuracy could be obtained, while retaining good discrimination if two or more differences were required between patterns before they were regarded as distinct. A complete set of capsular antisera were prepared, enabling the results obtained from klebecin typing to be compared with those from serotyping. There was generally close agreement between the results from the two typing methods and greater discrimination was obtained between similar strains when the two methods were combined. Klebecin typing and serotyping revealed relationships between strains from five outbreaks of infection, and strains of the same serotype from different hospitals could frequently be distinguished by their klebecin typing patterns.