Garvicin A, a novel class IId bacteriocin from Lactococcus garvieae that inhibits septum formation in L. garvieae strains

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Real-time PCR investigation on the expression of sboA and ituD genes in Bacillus spp

Aims: To investigate the expression of sboA and ituD genes among strains of Bacillus spp. at different pH and temperature. Methods and Results: Different Bacillus strains from the Amazon basin and Bacillus subtilis ATCC 19659 were investigated for the production of subtilosin A and iturin A by qRT-PCR, analysing sboA and ituD gene expression under different culture conditions. Amazonian strains presented a general gene expression level lower than B. subtilis ATCC 19659 for sboA. In contrast, when analysing the expression of ituD gene, the strains from the Amazon, particularly P40 and P45B, exhibited higher levels of expression. Changes in pH (6 and 8) and temperature (37 and 42 degrees C) caused a decrease in sboA expression, but increased ituD expression among strains from Amazonian environment. Conclusions: Temperature and pH have an important influence on the expression of genes sboA (subtilosin A) and ituD (iturin A) among Bacillus spp. The strains P40 and P45B can be useful for the production of antimicrobial peptide iturin A. Significance and Impact of the Study: Monitoring the expression of essential biosynthetic genes by qRT-PCR is a valuable tool for optimization of the production of antimicrobial peptides.

Nisin expression production from Lactococcus lactis in milk whey medium

BACKGROUND: Nisin is a commercially available bacteriocin produced by Lactococcus lactis ATCC 11454 and used as a natural agent in the biopreservation of food. In the current investigation, milk whey, a byproduct from dairy industries was used as a fermentation substrate for the production of nisin. Lactococcus lactis ATCC 11454 was developed in a rotary shaker (30 degrees C/36 h/100 rpm) using two different media with milk whey (i) without filtration, pH 6.8, adjusted with NaOH 2 mol L-1 and without pH adjustment, both autoclaved at 121 degrees C for 30 min, and (ii) filtrated (1.20 mu m and 0.22 mu m membrane filter). These cultures were transferred five times using 5 mL aliquots of broth culture for every new volume of the respective media. RESULTS: The results showed that culture media composed of milk whey without filtration supplied L. lactis its adaptation needs better than filtrated milk whey. Nisin titers, in milk whey without filtration (pH adjusted), was 11120.13 mg L-1 in the second transfer, and up to 1628-fold higher than the filtrated milk whey, 6.83 mg.L-1 obtained in the first(t) transfer. CONCLUSIONS: Biological processing of milk byproducts (milk whey) can be considered a profitable alternative, generating high-value bioproducts and contributing to decreasing river disposals by dairy industries. (C) 2008 Society of Chemical Industry.

Molecular identification of naturally occurring bacteriocinogenic and bacteriocinogenic-like lactic acid bacteria in raw milk and soft cheese

Lactic acid bacteria ( LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n = 2) and Lactococcus lactis ssp. lactis (n = 18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety. (C) 2008 Elsevier Ltd. All rights reserved.

Use of Carnobacterium maltaromaticum cultures and hydroalcoholic extract of Lippia sidoides Cham. against Listeria monocytogenes in fish model systems

Minimally processed refrigerated ready-to-eat fishes may offer health risk of severe infection to susceptible individuals due to contamination by the psychrotolerant bacterium L monocytogenes. In this work, inhibition of L monocytogenes by a plant extract and lactic acid bacteria (IAB) was studied in model fish systems kept at 5 degrees C for 35 days. For that, fillets of tropical fish ""surubim"" (Pseudoplatystoma sp.) and hydroalcoholic extract of the plant Lippia sidoides Cham. (""alecrim pimenta"") were used. Fish peptone broth (FPB), ""surubim"" broth and ""surubim"" homogenate were inoculated with combinations of L monocytogenes and bacteriocin-producing Carnobacterium maltaromaticum (C2 and A9b(+)) and non bacteriocin-producing C. maltaromaticum (A9b(-)), in the presence or absence of extract of ""alecrim pimenta"" (EAP). In all model systems, monocultures of L monocytogenes and carnobacteria reached final populations >= 10(8) CFU/ml after 35 days, except for L monocytogenes in ""surubim"" homogenate (10(4) CFU/ml). In FPB, EAP alone and combined with cultures of LAB inhibited L monocytogenes but carnobacteria without EAP were only weakly antilisterial. In ""surubim"" broth, EAP alone did not prevent L monocytogenes growth but cultures of carnobacteria combined or not with EAP inhibited L monocytogenes, with more pronounced effect being observed for C maltaromaticum C2, which produced bacteriocin. In ""surubim"" homogenate, EAP alone and combined with cultures of C. maltaromaticum A9b(-) and A9b(+) were strongly inhibitory to L monocytogenes, while C maltaromaticum C2 with EAP caused transient inhibition of L monocytogenes. No significant inhibition of L monocytogenes was observed for carnobacteria in ""surubim"" homogenate without EAP. In conclusion, it was observed that the use of EAP and cultures of carnobacteria have potential to inhibit L monocytogenes in fish systems and the applications should be carefully studied, considering the influence of food matrix. (c) 2011 Elsevier B.V. All rights reserved.

Isolamento e caracterização de bactérias do ácido láctico, produtoras de bacteriocinas e sua aplicação no fabrico de queijo fresco

Dissertação de Mestrado em Tecnologia e Segurança Alimentar

Estudo de atividades antimicrobianas em bactérias do género Bacillus isoladas nos Açores : otimização das condições de produção, identificação e caracterização de bacteriocinas

Dissertação de Mestrado, Ciências Biomédicas, 18 de Novembro de 2015, Universidade dos Açores.

Formulação de um biofilme para controlo da listéria em queijos

Dissertação de Mestrado, Tecnologia e Segurança Alimentar, 12 Fevereiro de 2016, Universidade dos Açores.

Mutant prevention concentrations of fluoroquinolones for Enterobacteriaceae expressing the plasmid-carried quinolone resistance determinant qnrA1.

The influence of qnrA1 on the development of quinolone resistance in Enterobacteriaceae was evaluated by using the mutant prevention concentration parameter. The expression of qnrA1 considerably increased the mutant prevention concentration compared to strains without this gene. In the presence of qnrA1, mutations in gyrA and parC genes were easily selected to produce high levels of quinolone resistance.

Biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Lipopolysacharide (LPS) present on the outer leaflet of Gram-negative bacteria is important for the adaptation of the bacteria to the environment. Structurally, LPS can be divided into three parts: lipid A, core and O-polysaccharide (OPS). OPS is the outermost and also the most diverse moiety. When OPS is composed of identical sugar residues it is called homopolymeric and when it is composed of repeating units of oligosaccharides it is called heteropolymeric. Bacteria synthesize LPS at the inner membrane via two separate pathways, Lipid A-core via one and OPS via the other. These are ligated together in the periplasmic space and the completed LPS molecule is translocated to the surface of the bacteria. The genes directing the OPS biosynthesis are often clustered and the clusters directing the biosynthesis of heteropolymeric OPS often contain genes for i) the biosynthesis of required NDP-sugar precursors, ii) glycosyltransferases needed to build up the repeating unit, iii) translocation of the completed O-unit to the periplasmic side of the inner membrane (flippase) and iv) polymerization of the repeating units to complete OPS. The aim of this thesis was to characterize the biosynthesis of the outer core (OC) of Yersinia enterocolitica serotype O:3 (YeO3). Y. enterocolitica is a member of the Gram-negative Yersinia genus and it causes diarrhea followed sometimes by reactive arthritis. The chemical structure of the OC and the nucleotide sequence of the gene cluster directing its biosynthesis were already known; however, no experimental evidence had been provided for the predicted functions of the gene products. The hypothesis was that the OC biosynthesis would follow the pathway described for heteropolymeric OPS, i.e. a Wzy-dependent pathway. In this work the biochemical activities of two enzymes involved in the NDP-sugar biosynthesis was established. Gne was determined to be a UDP-N-acetylglucosamine-4-epimerase catalyzing the conversion of UDP-GlcNAc to UDP-GalNAc and WbcP was shown to be a UDP-GlcNAc- 4,6-dehydratase catalyzing the reaction that converts UDP-GlcNAc to a rare UDP-2-acetamido- 2,6-dideoxy-d-xylo-hex-4-ulopyranose (UDP-Sugp). In this work, the linkage specificities and the order in which the different glycosyltransferases build up the OC onto the lipid carrier were also investigated. In addition, by using a site-directed mutagenesis approach the catalytically important amino acids of Gne and two of the characterized glycosyltranferases were identified. Also evidence to show the enzymes involved in the ligations of OC and OPS to the lipid A inner core was provided. The importance of the OC to the physiology of Y. enterocolitica O:3 was defined by determining the minimum requirements for the OC to be recognized by a bacteriophage, bacteriocin and monoclonal antibody. The biological importance of the rare keto sugar (Sugp) was also shown. As a conclusion this work provides an extensive overview of the biosynthesis of YeO3 OC as it provides a substantial amount of information of the stepwise and coordinated synthesis of the Ye O:3 OC hexasaccharide and detailed information of its properties as a receptor.

Characterization of Xanthomonas spp. strains by bacteriocins

Twenty-five strains of Xanthomonas axonopodis pv. citri and 14 strains of Xanthomonas spp. were tested for bacteriocin production. X. axonopodis pv. passiflorae strains were sensitive to the bacteriocins produced by the 25 X. axonopodis pv. citri strains evaluated in this study while strains of X. axonopodis pv. manihotis and X. campestris pv. campestris showed variable sensitivity. Only five of the 25 X. axonopodis pv. citri strains were not inhibited by the bacteriocins produced by the two X. axonopodis pv. passiflorae strains. The bacteriocins produced by the Xanthomonas axonopodis pv. citri (FDC-806) and X. axonopodis pv. passiflorae (Mar-2850 A) strains were thermolabile, resistant to lysozyme and sensitive to DNAse. The bacteriocin produced by X. axonopodis pv. passiflorae was resistant to the action of proteinase K, trypsin and RNAse while the bacteriocin produced by X. axonopodis pv. citri was sensitive to these enzymes. The bacteriocins produced by X. axonopodis pv. passiflorae and X. axonopodis pv. citri were called passifloricin and citricin, respectively.

Secretion of Streptomyces tendae antifungal protein 1 by Lactococcus lactis

Lactococcus lactis, the model lactic acid bacterium, is a good candidate for heterologous protein production in both foodstuffs and the digestive tract. We attempted to produce Streptomyces tendae antifungal protein 1 (Afp1) in L. lactis with the objective of constructing a strain able to limit fungal growth. Since Afp1 activity requires disulfide bond (DSB) formation and since intracellular redox conditions are reportedly unfavorable for DSB formation in prokaryotes, Afp1 was produced as a secreted form. An inducible expression-secretion system was used to drive Afp1 secretion by L. lactis; Afp1 was fused or not with LEISSTCDA, a synthetic propeptide (LEISS) that has been described to be a secretion enhancer. Production of Afp1 alone was not achieved, but production of LEISS-Afp1 was confirmed by Western blot and immunodetection with anti-Afp1 antibodies. This protein (molecular mass: 9.8 kDa) is the smallest non-bacteriocin heterologous protein ever reported to be secreted in L. lactis via the Sec-dependent pathway. However, no anti-fungal activity was detected, even in concentrated samples of induced supernatant. This could be due to a too low secretion yield of Afp1 in L. lactis, to the absence of DSB formation, or to an improper DSB formation involving the additional cysteine residue included in LEISS propeptide. This raises questions about size limits, conformation problems, and protein secretion yields in L. lactis.

Antimicrobial properties of lactic acid bacteria isolated from uruguayan artisan cheese

Uruguayan artisan cheese is elaborated with raw milk and non-commercial starters. The associated native microbiota may include lactic acid bacteria and also potentially pathogenic bacteria. Lactic acid bacteria were isolated from artisan cheese, raw milk, and non-commercial starter cultures, and their potential bacteriocin production was assessed. A culture collection of 509 isolates was obtained, and five isolates were bacteriocin-producers and were identified as Enterococcus durans,Lactobacillus casei, and Lactococcus lactis. No evidence of potential virulence factors were found in E. durans strains. These are promising results in terms of using these native strains for cheese manufacture and to obtain safe products.