Impact of bacteriocin-producing strains on bacterial community composition in a simplified human intestinal microbiota.

Bacteriocins are antimicrobial peptides that have been studied for decades as food bio-preservatives or as alternatives to antibiotics. They also have potential as modulators of the gut microbiome, which has been linked to human health. However, it is difficult to predict a priori how bacteriocins will impact complex microbial communities through direct and indirect effects. Here we assess the effect of different bacteriocin-producing strains on a Simplified Human Intestinal Microbiota (SIHUMI) model, using a set of bacteriocin-producing strains (Bac+) and otherwise isogenic non-producers (Bac-). Bacteriocins from different classes and with different activity spectra were selected, including lantibiotics such as lacticin 3147 and nisin A, and pediocin-like bacteriocins such as pediocin PA-1 among other peptides. SIHUMI is a bacterial consortium of seven diverse human gut species that assembles to a predictable final composition in a particular growth medium. Each member can be individually tracked by qPCR. Bac+ and Bac- strains were superimposed on the SIHUMI system, and samples were taken at intervals up to 48 h. The genome copy number of each SIHUMI member was evaluated using specific primers. We establish that the composition of the community changes in response to the presence of either broad- or narrow-spectrum bacteriocin producers and confirm that there are significant off-target effects. These effects were analyzed considering antagonistic inter-species interactions within the SIHUMI community, providing a comprehensive insight into the possible mechanisms by which complex communities can be shaped by bacteriocins.

A Bioengineered Nisin Derivative To Control Streptococcus uberis Biofilms.

Antimicrobial peptides are evolving as novel therapeutic options against the increasing problem of multidrug-resistant microorganisms, and nisin is one such avenue. However, some bacteria possess a specific nisin resistance system (NSR), which cleaves the peptide reducing its bactericidal efficacy. NSR-based resistance was identified in strains of Streptococcus uberis, a ubiquitous pathogen that causes mastitis in dairy cattle. Previous studies have demonstrated that a nisin A derivative termed nisin PV, featuring S29P and I30V, exhibits enhanced resistance to proteolytic cleavage by NSR. Our objective was to investigate the ability of this nisin derivative to eradicate and inhibit biofilms of S. uberis DPC 5344 and S. uberis ATCC 700407 (nsr+) using crystal violet (biomass), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (viability) assays, and confocal microscopy (viability and architecture). When preestablished biofilms were assessed, both peptides reduced biofilm biomass by over 60% compared to that of the untreated controls. However, a 42% higher reduction in viability was observed following treatment with nisin PV compared to that of nisin A. Accordingly, confocal microscopy analysis revealed significantly more dead cells on the biofilm upper surface and a reduced thickness following treatment with nisin PV. When biofilm inhibition was assessed, nisin PV inhibited biofilm formation and decreased viability up to 56% and 85% more than nisin A, respectively. Confocal microscopy analysis revealed a lack of biofilm for S. uberis ATCC 700407 and only dead cells for S. uberis DPC 5344. These results suggest that nisin PV is a promising alternative to effectively reduce the biofilm formation of S. uberis strains carrying NSR. IMPORTANCE One of the four most prevalent species of bovine mastitis-causing pathogens is S. uberis. Its ability to form biofilms confers on the bacteria greater resistance to antibiotics, requiring higher doses to be more effective. In a bid to limit antibiotic resistance development, the need for alternative antimicrobials is paramount. Bacteriocins such as nisin represent one such alternative that could alleviate the impact of mastitis caused by S. uberis. However, many strains of S. uberis have been shown to possess nisin resistance determinants, such as the nisin resistance protein (NSR). In this study, we demonstrate the ability of nisin and a nisin derivative termed PV that is insensitive to NSR to prevent and remove biofilms of NSR-producing S. uberis strains. These findings will add new information to the antimicrobial bacteriocins and control of S. uberis research fields specifically in relation to biofilms and nsr+ mastitis-associated strains.

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk.

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments.

Strategies for Pathogen Biocontrol Using Lactic Acid Bacteria and Their Metabolites: A Focus on Meat Ecosystems and Industrial Environments.

The globalization of trade and lifestyle ensure that the factors responsible for the emergence of diseases are more present than ever. Despite biotechnology advancements, meat-based foods are still under scrutiny because of the presence of pathogens, which causes a loss of consumer confidence and consequently a fall in demand. In this context, Lactic Acid Bacteria (LAB) as GRAS organisms offer an alternative for developing pathogen-free foods, particularly avoiding Listeria monocytogenes, with minimal processing and fewer additives while maintaining the foods' sensorial characteristics. The use of LAB strains, enabling us to produce antimicrobial peptides (bacteriocins) in addition to lactic acid, with an impact on quality and safety during fermentation, processing, and/or storage of meat and ready-to-eat (RTE) meat products, constitutes a promising tool. A number of bacteriocin-based strategies including the use of bioprotective cultures, purified and/or semi-purified bacteriocins as well as their inclusion in varied packaging materials under different storage conditions, have been investigated. The application of bacteriocins as part of hurdle technology using non-thermal technologies was explored for the preservation of RTE meat products. Likewise, considering that food contamination with L. monocytogenes is a consequence of the post-processing manipulation of RTE foods, the role of bacteriocinogenic LAB in the control of biofilms formed on industrial surfaces is also discussed.

Proteomic and genetics insights on the response of the bacteriocinogenic Lactobacillus sakei CRL1862 during biofilm formation on stainless steel surface at 10°C.

Some lactic acid bacteria have the ability to form biofilms on food-industry surfaces and this property could be used to control food pathogens colonization. Lactobacillus sakei CR1862 was selected considering its bacteriocinogenic nature and ability to adhere to abiotic surfaces at low temperatures. In this study, the proteome of L. sakei CRL1862 grown either under biofilm on stainless steel surface and planktonic modes of growth at 10°C, was investigated. Using two-dimensional gel electrophoresis, 29 out of 43 statistically significant spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ten proteins resulted up-regulated whereas 16 were down-regulated during biofilm formation. Differentially expressed proteins were found to belong to carbohydrate, nucleotide, aminoacid and lipid metabolisms as well as translation, peptide hydrolysis, cell envelope/cell wall biosynthesis, adaption to atypical conditions and protein secretion. Some proteins related to carbohydrate and nucleotide metabolisms, translation and peptide degradation were overexpressed whereas those associated to stress conditions were synthesized in lower amounts. It seems that conditions for biofilm development would not imply a stressful environment for L. sakei CRL1862 cells, directing its growth strategy towards glycolytic flux regulation and reinforcing protein synthesis. In addition, L. sakei CRL1862 showed to harbor nine out of ten assayed genes involved in biofilm formation and protein anchoring. By applying qRT-PCR analysis, four of these genes showed to be up regulated, srtA2 being the most remarkable. The results of this study contribute to the knowledge of the physiology of L. sakei CRL1862 growing in biofilm on a characteristic food contact surface. The use of this strain as green biocide preventing L. monocytogenes post-processing contamination on industrial surfaces may be considered.

Control of Listeria monocytogenes biofilms on industrial surfaces by the bacteriocin-producing Lactobacillus sakei CRL1862.

The effect of the bacteriocin-producing Lactobacillus sakei CRL1862 and its bacteriocin in the control of Listeria biofilm formation on industrial surfaces at 10°C was investigated. A screening among different Listeria species was performed allowing selecting L. monocytogenes FBUNT for its use as a biofilm producer on stainless steel (SS) and polytetrafluoroe-thylene (PTFE) surfaces. Three conditions were simulated to evaluate the ability of the bacteriocinogenic strain to displace, exclude and compete pathogen biofilm formation. Lactobacillus sakei CRL1862 effectively inhibited biofilm formation by L. monocytogenes FBUNT through the three assayed mechanisms, pathogen inhibition being more efficient on PTFE than on SS surface. Moreover, co-culture of L. monocytogenes FBUNT with the bacteriocin-producer displayed the highest efficacy reducing the pathogen by 5.54 ± 0.12 and 4.52 ± 0.01 on PTFE and SS, respectively. Industrially, the pre-treatment with L. sakei CRL1862 or its bacteriocin (exclusion) constitutes the most realistic way to prevent pathogen biofilm settlement. The use of bacteriocins and/or the bacteriocin-producer strain represents a safe and environmentally-friendly sanitation method to mitigate post-processing food contamination.

Evaluation of anti-Listeria meat borne Lactobacillus for biofilm formation on selected abiotic surfaces.

The ability of meat borne anti-Listeria Lactobacillus to form biofilms under different in vitro conditions and on abiotic surfaces was investigated. Biofilm formation by the adhesion to polystyrene microtiter plates was determined, this being higher for Lactobacillus curvatus CRL1532 and CRL705 and Lactobacillus sakei CRL1862. The physicochemical properties of the cell surface were relatively hydrophilic and acidic in character; L. sakei CRL1862 exhibiting the strongest autoaggregation. The adhesion of lactobacilli to stainless steel (SS) and polytetrafluoroethylene (PTFE) supports at 10°C was found to be maximal for L. sakei CRL1862 on SS after 6 days. When biofilm architecture was characterized by epifluorescence and SEM, L. sakei CRL1862 homogeneously covered the SS surface while cell clusters were observed on PTFE; the extracellular polymeric substance matrix adapted to the topography and hydrophilic/hydrophobic characteristics of each material. The feasibility of L. sakei CRL1862 to form biofilm on materials used in meat processing highlights its potential as a control strategy for Listeria monocytogenes biofilms.