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Bacteriophage (phage) are bacterial viruses and are considered to be the most widely distributed and diverse natural biological entities. Soon after their discovery, bacteriophage were found to have antimicrobial properties that were exploited in many early anti-infection trials. However, the subsequent discovery of antibiotics led to a decline in the popularity of bacteriophage in much of the Western world, although work continued in the former Soviet Union and Eastern Europe. As a result of the emergence of antibiotic resistance in a number of bacterial pathogens, focus has been redirected back to bacteriophage and bacteriophage lysins as a means of pathogen control. Although bacteriophage have certain limitations, significant progress has been made toward their applications in food and has resulted in the U.S. Food and Drug Administration approving the use of a bacteriophage-based additive for the control of Listeria monocytogenes contamination. Furthermore, a number of animal studies have revealed the potential of bacteriophage for the control of various foodborne pathogens within the animal gastrointestinal tract and to subsequently decrease the likelihood of foodborne outbreaks. From a biopreservative perspective, phage have a number of key properties, including relative stability during storage, an ability to self-replicate, and a nontoxic nature. The purpose of this review is to highlight the recent developments in the use of phages and their lysins for biocontrol and to address their potential future applications.

There is a lack of fundamental knowledge about the influence of bacteriophage on probiotic bacteria and other commensals in the gut. Here, we present the isolation and morphological and genetic characterization of a virulent narrow-host-range bacteriophage, phiLb338-1. This phage was isolated from fresh sewage and was shown to infect the probiotic cheese strain Lactobacillus paracasei NFBC 338. Electron microscopy studies revealed that phiLb338-1 is a member of the Myoviridae family, with an isometric head, a medium-sized contractile tail, and a complex base plate. Genome sequencing revealed a 142-kb genome with 199 open reading frames. Putative functions could be assigned to 22% of the open reading frames; these had significant homology to genes found in the broad-host-range SPO1-like group of phages which includes the Enterococcus faecalis phage phiEF24C, Listeria phage A511, and Lactobacillus plantarum phage LP65. Interestingly, no significant genomic similarity was observed between the phage and the probiotic host strain. Future studies will determine if the presence of bacteriophage phiLb338-1 or others in the human or animal gut plays an antagonistic role against the probiotic effect of beneficial bacteria.

Bacteriophage infection of Streptococcus thermophilus is becoming increasingly problematic in many industry fermentations such as yoghurt and mozzarella manufacture. This study describes the development of an efficient and rapid 3-step approach for the generation of bacteriophage insensitive mutants (BIMs) of these starter strains. The method initially involves infection of a culture in solid media at a multiplicity of infection (M.O.I.) of 10 which is then incubated in milk overnight. BIMs are then isolated following successive rounds (20-25) of growth in 10% reconstituted skimmed milk (RSM) in the presence of high phage titres. The method selects for BIMs which can grow efficiently in milk. Using this approach BIMs of two industrial strains were generated, whose starter performance was comparable to the parent starters in terms of performance in milk. Genomic fingerprinting used to validate the identity of each BIM, revealed a number of restriction fragment length polymorphisms (RFLPs) in two of the resultant BIMs. This method provides a simple and reliable method for generation of BIMs of industrial starters which does not require any specialised equipment and should be widely applicable.

A series of styrylquinolines and quinolineamides based on the 8-hydroxyquinoline moiety were investigated as potential antimycobacterial agents. The lipophilicity of the compounds was measured using RP-HPLC and the tests of their activity against Mycobacterium kansasii, the M. avium complex, M. smegmatis, M. abscessus, M. tuberculosis and M. avium paratuberculosis was performed. Several of the compounds that were obtained appeared to be more effective than isoniazid and ciprofloxacin. The 5,7-dinitro-8-hydroxyquinoline derivative possessed the highest potency against M. abscessus and M. Smegmatis, which was about twice as effective as ciprofloxacin, while 2-(2-hydroxystyryl)-8-hydroxyquinoline-7-carboxylic acid appeared to be comparable with the standard drugs that are against the M. avium complex. The structure activity relationships are discussed.

The physical characteristics of bacteriophages establish them as viable candidates for downstream development of pathogen detection assays and biocontrol measures. To utilize phages for such purposes, a detailed knowledge of their host interaction mechanisms is a prerequisite. There is currently a wealth of knowledge available concerning Gram-negative phage-host interaction, but little by comparison for Gram-positive phages and Listeria phages in particular. In this research, the lytic spectrum of two recently isolated Listeria monocytogenes phages (vB_LmoS_188 and vB_LmoS_293) was determined, and the genomic basis for their observed serotype 4b/4e host-specificity was investigated using comparative genomics. The late tail genes of these phages were identified to be highly conserved when compared to other serovar 4-specific Listeria phages. Spontaneous mutants of each of these phages with broadened host specificities were generated. Their late tail gene sequences were compared with their wild-type counterparts resulting in the putative identification of the products of ORF 19 of vB_LmoS_188 and ORF 20 of vB_LmoS_293 as the receptor binding proteins of these phages. The research findings also indicate that conserved baseplate architectures and host interaction mechanisms exist for Listeria siphoviruses with differing host-specificities, and further contribute to the current knowledge of phage-host interactions with regard to Listeria phages.

This study was undertaken to assess the antifungal performance of three different Lactobacillus species.Experiments were conducted in vitro and in situ to extend the shelf life of wheat bread. Standard sourdough analyses were performed characterising acidity and carbohydrate levels. Overall, the strains showed good inhibition in vitro against the indicator mould Fusarium culmorum TMW4.2043. Sourdough bread fermented with Lactobacillus amylovorus DSM19280 performed best in the in situ shelf life experiment. An average shelf life extension of six more mould-free days was reached when compared to the non-acidified control bread. A range of antifungal-active acids like 3-phenyllactic acid, 4-hydroxyphenyllactic acid and 2-hydroxyisocaproic acid in quantities between 0.1 and 360 mg/kg were present in the freeze-dried sourdoughs. Their concentration differed greatly amongst the species.However, a higher concentration of these compounds could not completely justify the growth inhibition of environmental moulds. In particular, although Lb. reuteri R29 produced the highest total concentration of these active compounds in the sourdough, its addition to bread did not result in a longest shelf life. Nevertheless, when the artificial compounds were spiked into a chemically acidified dough, it succeeded in a longer shelf life (+25 %) than achieved only by acidifying the dough. This provides evidence of their contribution to the antifungal activity and their synergy in concentration levels far below their single minimal inhibition concentrations under acidic conditions.

Listeria monocytogenes is a foodborne pathogen and is the causative agent of listeriosis among humans and animals. The draft genome sequence of L. monocytogenes DPC6895, a serotype 1/2b strain isolated from the raw milk of a cow with subclinical bovine mastitis, is reported.

Illness caused by the consumption of contaminated food products continues to represent one of the main challenges facing food manufacturers worldwide. Even with current intervention technologies and increased hygiene measures, foodborne illness remains a significant threat to public health. This coupled with the increasing emergence of multidrug resistant pathogens has increased the need for the development of novel technologies for pathogen control. Bacterial derived peptidoglycan hydrolases represent a vast and highly diverse group of enzymes with potential for biocontrol of a range of Gram-positive and Gram-negative foodborne pathogens. In this study, we describe the identification, cloning, expression and purification of a peptidoglycan hydrolase (LysCs4) derived from Cronobacter sakazakii for biocontrol of the aforementioned infant formula pathogen itself. In silico analysis of LysCs4 revealed the gene to display greatest sequence similarity to a putative lysozyme encoded by the lytic Cronobacter phage ES2. Conserved domain analysis of LysCs4 revealed the presence of a single catalytic domain predicted to display O-Glycosyl hydrolase activity and to be a member of the GH24 family. The ability of this enzyme to hydrolyse the peptidoglycan of 25 Gram-negative strains, across 4 different genera, highlights its potential as a novel candidate for biocontrol of C. sakazakii and other Gram-negative food related pathogens.

A series of fifteen new N-alkoxyphenylanilides of 3-hydroxynaphthalene-2-carboxylic acid was prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Staphylococcus aureus, three methicillin-resistant S. aureus strains, Mycobacterium tuberculosis H37Ra and M. avium subsp. paratuberculosis. Some of the tested compounds showed antibacterial and antimycobacterial activity against the tested strains comparable with or higher than that of the standards ampicillin or rifampicin. 3-Hydroxy-N-(2-propoxyphenyl)naphthalene-2-carboxamide and N-[2-(but-2-yloxy)-phenyl]-3-hydroxynaphthalene-2-carboxamide had MIC = 12 µM against all methicillin-resistant S. aureus strains, thus their activity is 4-fold higher than that of ampicillin. The second mentioned compound as well as 3-hydroxy-N-[3-(prop-2-yloxy)phenyl]-naphthalene-2-carboxamide had MICs = 23 µM and 24 µM against M. tuberculosis respectively. N-[2-(But-2-yloxy)phenyl]-3-hydroxynaphthalene-2-carboxamide demonstrated higher activity against M. avium subsp. paratuberculosis than rifampicin. Screening of the cytotoxicity of the most effective antimycobacterial compounds was performed using THP-1 cells, and no significant lethal effect was observed for the most potent compounds. The compounds were additionally tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (IC50 = 4.5 µM) was the most active PET inhibitor. The structure-activity relationships are discussed.

In this study, a series of twenty-two ring-substituted 8-hydroxyquinoline-2-carboxanilides was prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium tuberculosis H37Ra, Mycobacterium avium complex and M. avium subsp. paratuberculosis. Some of the tested compounds showed the antimycobacterial activity against M. avium subsp. paratuberculosis comparable with or higher than that of rifampicin. 8-Hydroxy-N-[3-(trifluoromethyl)phenyl]- and 8-hydroxy-N-[4-(trifluoromethyl)phenyl]quinoline-2-carboxamide showed MIC=24 μM against all tested mycobacterial strains. 3-Methoxyphenyl- and 3-methylphenyl derivatives expressed MIC=27 or 29 μM also against all the tested strains. Their activity against M. avium subsp. paratuberculosis was 4-fold higher than that of rifampicin. 2-Bromophenyl- and 2-(trifluoromethyl)phenyl derivatives had MIC=23 or 24 μM against M. tuberculosis. A significant decrease of mycobacterial cell metabolism (viability of M. tuberculosis H37Ra) was observed using MTT assay. Screening of cytotoxicity of the compounds was performed using the THP-1 cells, and no significant lethal effect was observed up to tested concentration 30 μM. The structure-activity relationships are discussed.

Listeria monocytogenes is responsible for the rare disease listeriosis, which is associated with the consumption of contaminated food products. We report here the complete genome sequences of vB_LmoS_188 and vB_LmoS_293, phages isolated from environmental sources and that have host specificity for L. monocytogenes strains of the 4b and 4e serotypes.

Staphylococcus epidermidis (S. epidermidis), one of the leading etiological agents of nosocomial infections poses a significant economic burden globally. Introduced in 2000, linezolid (LZD) has become an important antibiotic, used in nearly seventy countries worldwide to treat infections caused by Grampositive pathogens such as meticillin-resistant Staphylococcus and Streptococcus species along with vancomycin-resistant enterococci. Resistance to LZD in clinical settings remains rare. Here, we report the emergence of meticillin resistant S. epidermidis (MRSE) clinical isolates from two voluntary general acute hospitals exhibiting higher than typically reported levels of LZD resistance (MIC>256 mg/ml). The MRSE ST-2 clone isolated from eight patients (2010e2011) not only possessed resistance-conferring mutations such as G2576T in domain V of 23S rRNA gene (as determined by HRMPCR analysis) and R172C substitution in the ribosomal protein L3, but also carried the cfr gene (the only known transmissible mechanism of LZD resistance). All isolates possessed several key biofilm-associated genes (such as icaA, icaD, aap and atlE) and resistance to multiple clinically significant antibiotics was recorded. This study reports the earliest incidence (2010) of clinical MRSE in the Republic of Ireland demonstrating multiple LZD resistance mechanisms both mutational and potentially transmissible, and characterises this emerging resistance from a molecular perspective. © 2015, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases.

Lactic acid bacteria (LAB) are of interest to cereal and beverage industry due to their contribution to the microbial safety and quality of fermented food/beverages. The main objectives of this study were to optimize the production of an antifungal cell-free-supernatant (cfs), based on lactic acid bacteria, using a wort-base substrate; and the subsequent identification of its active acid-base antifungal compounds. Two antifungal strains ( Lactobacillus amylovorus DSM19280 and Lactobacillus reuteri R29) and a negative control strain ( L. amylovorus DSM20552) were used. Barley based malt extract (wort) was produced and used as a fermentation substrate. Worts fermented with LAB were characterized in detail (total cell counts, pH, and total titratable acids (TTA) over time (0–120 h). A microtiter plate and an overlay agar based assay were used to evaluate the antifungal activity of the LAB cfs. Sugars, organic acids of the fermented and non-fermented wort (control) were analysed by HPLC. LAB antifungal metabolites were quantified using a QuEChERS method and an HPLC-UV/PDA system. Results show that wort produced from barley malt is a suitable substrate for LAB and dependent on the species and the fermentation time. The type of antifungal compounds can vary significantly. L. amylovorus DSM19280 cfs inhibited Fusarium culmorum spores at levels of 10 4 spores.mL −1 , for 7 days, whereas L. reuteri R29 cfs inhibited up to 10 5 spores.mL −1 for the same time. Thirteen acid-base antifungal compounds were identified in L. reuteri R29 cfs and twelve in L. amylovorus DSM19280. Among them, phenyllactic, OH-phenylactic and benzoic acids were present at a significant concentration. This study demonstrates that the LAB wort-base cfs exhibits potent antifungal activity and is the ideal substrate for applications in a wide range of cereal products.

Lactococci are one of the most exploited microorganisms used in the manufacture of food. These intensively used cultures are generally characterized by having a rich plasmid complement. It could be argued that it is the plasmid complement of commercially utilized cultures that gives them their technical superiority and individuality. Consequently, it is timely to reflect on the desirable characteristics encoded on lactococcal plasmids. It is argued that plasmids play a key role in the evolution of modern starter strains and are a lot more than just selfish replicosomes but more essential necessities of intensively used commercial starters. Moreover, the study of plasmid biology provides a genetic blueprint that has proved essential for the generation of molecular tools for the genetic improvement of Lactococcus lactis.

Analysis of the complete nucleotide sequence of the lactococcal phage 4268, which is lytic for the cheese starter Lactococcus lactis DPC4268, is presented. Phage 4268 has a linear genome of 36,596 bp, which is modularly organised and encompasses 49 open reading frames. Putative functions were assigned to approximately 45% of the predicted products of these open reading frames based on sequence similarity with known proteins, N-terminal sequence analysis and identification of conserved domains. Significantly, a segment of the genome has homology to the recently sequenced lysogenic module in lactococcal phage ϕ31 that contains a lytic switch but no phage integrase or attachment site. This suggests that it is derived from a prophage. A phage 4268-encoded and a host-encoded methylase were found to be highly similar, having only two nucleotide mismatches, suggesting that the phage acquired the methylase gene to protect it from a host endonuclease. Comparative genomic analysis revealed significant homology between phage 4268 and the lactococcal phage BK5-T. The comparative analysis also supported the classification of phage 4268 and other BK5-T-related phage as separate from the proposed P335 species of lactococcal phage.

Insertional inactivation of the plasmid-encoded determinants for Mg 2+ and Co 2+ transport, orf18 / corA , provides a tool for screening recombinant clones in Lactococcus , based on the observation that overexpression of orf18 / corA results in cell growth inhibition on certain concentrations of CoCl 2 . The lacticin 3147 immunity gene, ltnI , was used to insertionally inactivate orf18 / corA . The resulting clones were capable of growth on concentrations of CoCl 2 that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA , the system has potential as a screen for selecting recombinant lactococcal clones.

The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant Staphylococcus aureus strains from hospitals and other species of Staphylococcus isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant S. aureus (MRSA) strains, S. aureus with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti- Staphylococcus activity from the cream on plates and in broth.

Phage K is a polyvalent phage of the Myoviridae family which is active against a wide range of staphylococci. Phage genome sequencing revealed a linear DNA genome of 127,395 bp, which carries 118 putative open reading frames. The genome is organized in a modular form, encoding modules for lysis, structural proteins, DNA replication, and transcription. Interestingly, the structural module shows high homology to the structural module from Listeria phage A511, suggesting intergenus horizontal transfer. In addition, phage K exhibits the potential to encode proteins necessary for its own replisome, including DNA ligase, primase, helicase, polymerase, RNase H, and DNA binding proteins. Phage K has a complete absence of GATC sites, making it insensitive to restriction enzymes which cleave this sequence. Three introns ( lys -I1, pol -I2, and pol -I3) encoding putative endonucleases were located in the genome. Two of these ( pol -I2 and pol -I3) were found to interrupt the DNA polymerase gene, while the other ( lys -I1) interrupts the lysin gene. Two of the introns encode putative proteins with homology to HNH endonucleases, whereas the other encodes a 270-amino-acid protein which contains two zinc fingers (CX 2 CX 22 CX 2 C and CX 2 CX 23 CX 2 C). The availability of the genome of this highly virulent phage, which is active against infective staphylococci, should provide new insights into the biology and evolution of large broad-spectrum polyvalent phages.

We describe a novel HRM-PCR (high-resolution melting) assay capable of the accurate identification of the G2576T point mutation in domain V of the 23 S rRNA genes attributed to linezolid resistance in Staphylococcus epidermidis. This rapid method demonstrated 100% correlation with the previously established NheI restriction digest assay.

In this study, we describe a novel HRM-PCR assay that clearly differentiates the two main types of Mycobacterium avium subsp. paratuberculosis (cattle and sheep) based on the polymorphic variation of a previously described tandem repeat. This modern genotyping technique has several advantages over alternative methods, including cost, ease of use and rapidity.

Aims: Use of lacticin 481 to facilitate the conjugal transfer of the bacteriophage resistance plasmid pCBG104 to various starter cultures. Methods and Results: A raw milk isolate of Lactococcus was found to harbour determinants for lacticin 481 production and immunity and phage resistance on a plasmid designated pCBG104. The lacticin 481 was successfully used to mobilize the phage resistance determinant to a variety of cheese starters enabling the formation of highly phage resistant starters. In addition, it facilitated the stacking of a number of phage resistance genes, namely a type I restriction modification system, a phage abortive infection system and a phage adsorption blocking system in a single Lactococcus strain without the use of recombinant techniques. The transconjugants were all shown to produce lacticin 481 and to contain the entire 481 operon. Subsequently one transconjugant was selected and successfully used for large-scale cheddar cheese manufacture. Conclusions: Lacticin 481 could be used as a food-grade selectable marker to facilitate the introduction of advantageous traits to starter cultures for industrial food fermentations. Significance and Impact of the Study: Food-grade selectable markers greatly facilitate the introduction of various advantageous traits to starter cultures for industrial food fermentation. Indeed self-cloning which is becoming increasingly important for strain improvement has a requirement for the identification and demonstration of the utility of tools such as lacticin 481.

Novel 1-(2-{3-/4-[(alkoxycarbonyl)amino]phenyl}-2-hydroxyethyl)-4-(2-fluorophenyl)-piperazin-1-ium chlorides (alkoxy = methoxy to butoxy; 8a–h) have been designed and synthesized through multistep reactions as a part of on-going research programme focused on finding new antimycobacterials. Lipophilic properties of these compounds were estimated by RP-HPLC using methanol/water mobile phases with a various volume fraction of the organic modifier. The log kw values, which were extrapolated from intercepts of a linear relationship between the logarithm of a retention factor k (log k) and volume fraction of a mobile phase modifier (ϕM), varied from 2.113 (compound 8e) to 2.930 (8h) and indicated relatively high lipophilicity of these salts. Electronic properties of the molecules 8a–h were investigated by evaluation of their UV/Vis spectra. In a next phase of the research, the compounds 8a–h were in vitro screened against M. tuberculosis CNCTC My 331/88 (identical with H37Rv and ATCC 2794), M. kansasii CNCTC My 235/80 (identical with ATCC 12478), a M. kansasii 6 509/96 clinical isolate, M. avium CNCTC My 330/80 (identical with ATCC 25291) and M. avium intracellulare ATCC 13950, respectively, as well as against M. kansasii CIT11/06, M. avium subsp. paratuberculosis CIT03 and M. avium hominissuis CIT10/08 clinical isolates using isoniazid, ethambutol, ofloxacin, ciprofloxacin or pyrazinamide as reference drugs. The tested compounds 8a–h were found to be the most promising against M. tuberculosis; a MIC = 8 μM was observed for the most effective 1-(2-{4-[(butoxycarbonyl)amino]phenyl}-2-hydroxyethyl)-4-(2-fluorophenyl)piperazin-1-ium chloride (8h). In addition, all of them showed low (insignificant) in vitro toxicity against a human monocytic leukemia THP-1 cell line, as observed LD50 values > 30 μM indicated. The structure–antimycobacterial activity relationships of the analyzed 8a–h series are also discussed.

Starter inhibition by bacteriophage infection in dairy fermentations can limit the usage of specific bacterial strains used in the manufacture of Cheddar, Mozzarella and other cheeses and can result in substantial economic losses. A variety of practical measures to alleviate the problem of phage infection have been adopted over the years but has invariably resulted in a very limited number of strains which can withstand intensive usage in industry. The application of genetic techniques to improve the phage-resistance of starter cultures for dairy fermentations has been intensively studied for the last 20 years to a point where this approach now has significant potential to alleviate the problem. This paper highlights the recent findings and developments that have been described in the literature that will have an impact on improvement of the phage-resistance of starter cultures.

Weissella is a genus of lactic acid bacteria (LAB) consisting of species formerly included in the Leuconostoc paramesenteroides group. Similar to other LAB, they are commonly found in fermented foods but have also been isolated from environmental and human samples. Currently there are 20 recognized species. Herein, three Weissella cibaria genomes were sequenced using Illumia Mi-Seq and Roche 454 technologies. Annotation was performed using the Prokka and JGI IMG pipelines. A thorough analysis of the genomics of the W. cibaria strains was performed, in addition to brief comparative analyses of the genus Weissella as a whole. Genomic sequence data from the newly sequenced W. cibaria strains and data available in GenBank for other Weissella strains was used (n = 10; four Weissella cibaria, one Weissella ceti, one Weissella confusa, one Weissella halotolerans, two Weissella koreensis and one Weissella paramesenteroides). The genomes had sizes varying from 1.3 to 2.4 Mb. DNA G+C contents ranged from 35 to 45 mol%. The core- and pan-proteome at genus and species levels were determined. The genus pan-proteome was found to comprise 4712 proteins. Analysis of the four W. cibaria genomes indicated that the core-proteome, consisting of 729 proteins, constitutes 69 % of the species pan-proteome. This large core-set may explain the divergent niches in which this species has been found. In W. cibaria, in addition to a number of phosphotransferase systems conferring the ability to assimilate plant-associated polysaccharides, an extensive proteolytic system was identified.

Bacteriophage vB_EcoM_112 (formerly e11/2) is an Escherichia coli phage with specificity for the O157:H7 serotype. The vB_EcoM_112 genome sequence shares high degrees of similarity with the phage T4 genome sequence.

It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.

The present study investigated the antifungal activity of Lactobacillus amylovorus DSM19280 as a starter culture for gluten-free quinoa sourdough bread under pilot-plant conditions to extend the microbial shelf life. Challenge tests against environmental moulds were conducted and a negative control with non-antifungal strain, L. amylovorus DSM20531(T), as well as a chemically acidified and a non-acidified control were included. Organic acid production, antifungal metabolites, carbohydrates changes during fermentation and bread quality were compared to wheat counterparts. The application of quinoa sourdough fermented with the antifungal L. amylovorus DSM19280 extended the mould free shelf life by 4 days compared to the non-acidified control. No significant difference in lactic acid production was found between the lactobacilli strains. HPLC-UV/DAD was used to quantify antifungal compounds. The concentration of 4-hydroxyphenyllactic acid, phloretic acid, 3-phenyllactic acid and hydroferulic acid were significantly higher (P

Despite advances in modern technologies, the food industry is continuously challenged with the threat of microbial contamination. The overuse of antibiotics has further escalated this problem, resulting in the increasing emergence of antibiotic-resistant foodborne pathogens. Efforts to develop new methods for controlling microbial contamination in food and the food processing environment are extremely important. Accordingly, bacteriophages (phages) and their derivatives have emerged as novel, viable, and safe options for the prevention, treatment, and/or eradication of these contaminants in a range of foods and food processing environments. Whole phages, modified phages, and their derivatives are discussed in terms of current uses and future potential as antimicrobials in the traditional farm-to-fork context, encompassing areas such as primary production, postharvest processing, biosanitation, and biodetection. The review also presents some safety concerns to ensure safe and effective exploitation of bacteriophages in the future.

peer reviewed Lactococci isolated from non-dairy sources have been found to possess enhanced metabolic activity when compared to dairy strains. These capabilities may be harnessed through the use of these strains as starter or adjunct cultures to produce more diverse flavor profiles in cheese and other dairy products. To understand the interactions between these organisms and the phages that infect them, a number of phages were isolated against lactococcal strains of non-dairy origin. One such phage, ΦL47, was isolated from a sewage sample using the grass isolate L. lactis ssp. cremoris DPC6860 as a host. Visualization of phage virions by transmission electron microscopy established that this phage belongs to the family Siphoviridae and possesses a long tail fiber, previously unseen in dairy lactococcal phages. Determination of the lytic spectrum revealed a broader than expected host range, with ΦL47 capable of infecting 4 industrial dairy strains, including ML8, HP and 310, and 3 additional non-dairy isolates. Whole genome sequencing of ΦL47 revealed a dsDNA genome of 128, 546 bp, making it the largest sequenced lactococcal phage to date. In total, 190 open reading frames (ORFs) were identified, and comparative analysis revealed that the predicted products of 117 of these ORFs shared greater than 50% amino acid identity with those of L. lactis phage Φ949, a phage isolated from cheese whey. Despite their different ecological niches, the genomic content and organization of ΦL47 and Φ949 are quite similar, with both containing 4 gene clusters oriented in different transcriptional directions. Other features that distinguish ΦL47 from Φ949 and other lactococcal phages, in addition to the presence of the tail fiber and the genome length, include a low GC content (32.5%) and a high number of predicted tRNA genes (8). Comparative genome analysis supports the conclusion that ΦL47 is a new member of the 949 lactococcal phage group which currently includes the dairy Φ949.

[Background] Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge., [Methods] The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants., [Results] The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity., [Conclusions] The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity., The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X grant agreement no. 283570 and was sponsored by grant BFU2011-24843 (MJvR) from the Spanish Ministry of Economy and Competitiveness, a Masters fellowship (MSG) and an FPU Ph.D. fellowship (CGD) from the Spanish Ministry of Education, Culture and Sports. We also acknowledge financial support from TSR-StrandIII:CRS/07/CR03 and FIRM:08RDCIT600 of the Irish Department of Agriculture.

Lactobacillus amylovorus DSM 19280 is an antifungal strain that is inhibitory to a range of fungi including Penicillium expansum, Penicillium roqueforti, Aspergillus niger, Aspergillus fumigatus and Fusarium culmorum. In this study, the strain was used as an adjunct culture in a Cheddar cheese model system. During the ripening period, P. expansum spores were applied to the cheese surface to mimic fungal contamination. The presence of the antifungal L. amylovorus adjunct resulted in a four-day delay in appearance of Penicillium growth on the cheese in comparison to the adjunct-free control. When cheeses were exposed to natural airborne fungi, the presence of the adjunct resulted in a six-day delay in the appearance of mycelia on the cheese surface. Significantly, its presence had no detectable negative impact on cheese quality. The results indicate that the strain could have an application for extending the shelf-life of cheeses which are prone to fungal spoilage.

Aims: To facilitate the horizontal transfer and selection of phage-resistance plasmids in industrial lactococci. Methods and Results: Cadmium-resistance properties similar to those previously identified in Lactococcus were linked to the well-known phage-resistance plasmid pNP40. This finding was exploited to facilitate delivery of the plasmid to an industrial cheese starter Lactococcus lactis DPC4268. Additionally, 25 different cadmium-sensitive cheese starter lactococci were also identified as potential recipients for the phage-resistance plasmid pNP40, and also the plasmids pAH90/pAH82 which also encode cadmium resistance. All three plasmids were successfully conjugated to strain DPC4268. Cheddar cheese was manufactured in industry with the pNP40 phage-resistant transconjugant. Significance and Impact of the Study: Food-grade enhancement of phage resistance in industrial starter strains has been made simpler by the use of this selection, especially since the majority of potential recipient starter strains analysed were cadmium sensitive.

M.M. MAHER, K.N. JORDAN, M.E. UPTON AND A. COFFEY. 2001. Aims: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. Methods and Results: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1·52 ± 0·03 log cfu ml–1 in the milk (34 ± 2 cfu ml–1), the numbers increased to 3·4 ± 0·05 log cfu g–1 in the cheese at day 1. During ripening, the numbers decreased to

A significant amount of Cheddar cheese manufactured world-wide relies on bulk starter cultures instead of direct vat set (DVS) cultures. While the inclusion of S. thermophilus is some- times used to counteract failure due to lactococcal phage in the latter system, it is considered difficult to implement in bulk starter systems and is normally avoided. This stems from the problem in con- trolling the ratio of S. thermophilusto lactococci during the bulk starter preparation such that suitable acidification rates can be achieved. The current study demonstrates how S. thermophilusnumbers can be controlled during growth in the bulk starter medium prior to inoculation of a culture, based on three lactococcal strains and S. thermophilusDPC1842, into the cheese vat. The concentration of inor- ganic phosphate necessary to inhibit the growth of strain DPC1842 in a whey-based bulk starter medium was found to be 0.18 mol.L -1 . Since higher levels of phosphate exist in different commer- cial bulk starter media used for mesophilic cultures, a number of these media can be used for prop- agation of this starter blend without domination of strain DPC1842 over its lactococcal counter- parts. Strain DPC1842 is highly phage resistant and is particularly acid-fast in the cheese milk and reduces the pH efficiently at very low inocula. Cheddar cheese was manufactured in a commercial plant with this system and the resulting cheese had good flavour characteristics. This study demonstrates the effectiveness of the S. thermophilus DPC1842 component in rescuing the fermentation in the event of severe lactococcal phage attack.

Developments in the molecular genetics of food bacteria have now made possible the construction of designer cultures with the potential to improve important characteristics of cheese including production efficiency, safety and health properties. Such advances are set to expand the range and understanding of both starter and probiotic cultures, especially at a time when the total genomes of many strains are being determined and will become available. To illustrate the types of improvements which such technologies can bring, examples are given such as development of bacteriophage resistant starters and bacteriocin-producing starters with the potential to control adventitious cheese flora.

A microtiter alamarBlue assay was adapted and optimized for Mycobacterium avium subsp. paratuberculosis . Using cell concentrations ranging from 10 4 to 10 8 CFU/ml, a minimum incubation time to indicate viability was obtained after 24 h. Rifampin (rifampicin) was used to demonstrate that this method has applications for high-throughput screening against M. avium subsp. paratuberculosis .

Lactococcus lactis DPC4268 is widely used for Cheddar cheese manufacture in Ireland where it is recognised for its reliable fast acid producing ability in dairy environments. A transconjugant of this strain, L. lactis DPC4275, was generated which produces the broad spectrum, two-component bacteriocin lacticin 3147, which is inhibitory to a variety of undesirable gram-positive bacteria including Clostridium, Bacillus, Enterococcus, Listeria, Staphylococcus and Streptococcus. Both DPC4268 and DPC4275 were used as single strain starters for manufacture of salamis. These products were compared with a salami manufactured with a conventional starter (Lactobacillus sake and Staphylococcus carnosus) in terms of pH development, a(w)-value, weight loss, colour development and sensory characteristics. Salamis produced with either lactococcal culture exhibited pH values below 5.1 and a(w)-values below 0.90 which is favourable for preservation and hygienic stability. In addition, these salamis had good sensory and colorimetric qualities. A minimum lacticin 3147 concentration of 640 AU/g was detected in the salamis which were produced with L. lactis DPC4275.

This study was designed to evaluate the effects of algal and yeast β-glucans on the porcine gastrointestinal microbiota, specifically the community of Lactobacillus, Bifidobacterium and coliforms. A total of 48 pigs were fed four diets over a 28-day period to determine the effect that each had on these communities. The control diet consisted of wheat and soya bean meal. The remaining three diets contained wheat and soya bean meal supplemented with β-glucan at 250 g/tonne from Laminaria digitata, Laminaria hyperborea or Saccharomyces cerevisiae. Faecal samples were collected from animals before feeding each diet and after the feeding period. The animals were slaughtered the following day and samples were collected from the stomach, ileum, caecum, proximal colon and distal colon. Alterations in Lactobacillus in the gastrointestinal tract (GIT) were analysed using denaturing gradient gel electrophoresis (DGGE) profiles generated by group-specific 16S rRNA gene PCR amplicons. Plate count analysis was also performed to quantify total coliforms. DGGE profiles indicated that all β-glucan diets provoked the emergence of a richer community of Lactobacillus. The richest community of lactobacilli emerged after feeding L. digitata (LD β-glucan). Plate count analysis revealed that the L. hyperborea (LH β-glucan) diet had a statistically significant effect on the coliform counts in the proximal colon in comparison with the control diet. β-glucan from L. digitata and S. cerevisiae also generally reduced coliforms but to a lesser extent. Nevertheless, the β-glucan diets did not significantly reduce levels of Lactobacillus or Bifidobacterium. DGGE analysis of GIT samples indicated that the three β-glucan diets generally promoted the establishment of a more varied range of Lactobacillus species in the caecum, proximal and distal colon. The LH β-glucan had the most profound reducing effect on coliform counts when compared with the control diet and diets supplemented with L. digitata and S. cerevisiae β-glucans.

Aims: The growing interest of governments and industry in developing healthy and natural alternative foods and beverages that will fulfil the consumer drive towards a healthy lifestyle and clean-label, natural diet has led to an increase in traditional lactic acid bacteria fermentation research. In particular, this research aims to address the organoleptic modulation of beverages using in situ-produced bacterial polysaccharides. Methods and Results: Weissella cibaria MG1 is capable of producing exopolysaccharides (dextran) and oligosaccharides (glucooligosaccharides) during sucrose-supplemented barley-malt-derived wort fermentation. Up to 36 4g l 1 of dextran was produced in an optimized system, which improved the rheological profile of the resulting fermentate. Additionally, small amounts of organic acids were formed, and ethanol remained below 05% (v/v), the threshold volume for a potential health claim designation. Conclusions: The results suggest that the cereal fermentate produced by W. cibaria MG1 could be potentially used for the production of a range of novel, nutritious and functional beverages. Significance and Impact of the Study: Using conventional raw materials and traditional processes, novel LAB-fermented beverages can be produced representing an innovative mechanism towards fulfilling the aim to decrease government and personal costs as well as potentially ameliorating consumer lifestyle regarding dietary-related disease.

Aims: The study set out to sample €10 banknotes for the presence ofcoagulase-positive staphylococci (CPS) such as Staphylococcus aureus(S. aureus) and coagulase-negative staphylococci (CoNS) in Southern Ireland,to assess the levels of antibiotic resistance among those isolated, and determinethe persistence of S. aureus on €10 banknotes and €2 coins.Methods and Results: We report that 97% of €10 banknotes screened(n = 155) harboured multiple species of staphylococci. From the generated bankof strains, a total of 150 representative staphylococci isolates were used forfurther study, 71 were CPS and 79 were CoNS. Of these, we found that 62% ofthe staphylococci demonstrated resistance to at least one of the first-lineantibiotics (52 !11% of CPS isolates and 76 71% of the CoNS isolates). Resistanceto multiple antibiotics was seen in 31!18% of the resistant isolates. In relation topersistence studies, S. aureus was shown to remain viable on euro banknotes andcoins for significant periods (on average, 19!33 days on €10 banknotes and16!67 days on €2 coins) as determined using bioluminescence.Conclusions: We advocate the expansion of antibiotic surveillance programs,with a view to tracking/monitoring antibiotic resistance disseminationamong environmental contaminants. Additionally, we propose that ‘cashlesstransactions’ should be encouraged in high-risk environments such as hospitalsand healthcare settings, as well as stricter infection controls.Significance and Impact of the Study: Although it is accepted that circulatingcurrency has the potential to harbour disease-causing pathogens, studiesinvestigating prevalence and persistence of such pathogens on euro currencyare virtually nonexistent. In an attempt to rectify this, we examined theprevalence of staphylococci on €10 banknotes in Ireland and reported relativelyhigh levels of antibiotic resistance among the isolates. Furthermore, we haveestablished the persistence of S. aureus on euro currency for the first time.IntroductionMoney has historically been suspected of being a meansfor disease transmission. Even during the ‘Black Death’or bubonic and pneumonic plague pandemics in Eng-land, historical reports show that money was stronglybelieved to carry fatal infections. According to records,affected villages would leave money in water troughsfilled with vinegar, or under running well water inattempts to decontaminate the money in exchange forfood and other supplies from the surrounding uninfectedvillages (Tolba et al. 2007).Contamination from the skin, wounds, anal region,nasal secretions and aerosols generated by sneezing andcoughing are all potential sources of bacteria on currency(Tagoe et al. 2010). A study in the U.S. reported that amere 6% of banknotes examined were found to be freefrom bacterial contamination (Aidoo 2011). Various

A series of twenty substituted 2-hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(alkoxycarbonyl)amino]benzoates were prepared and characterized. As similar compounds have been described as potential antimycobacterials, primaryin vitroscreening of the synthesized carbamates was also performed against two mycobacterial species. 2-Hydroxy-3-[2-(2,6-dimethoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, 2-hydroxy-3-[2-(4-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, and 2-hydroxy-3-[2-(2-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride showed higher activity againstM. aviumsubsp.paratuberculosisandM. intracellularethan the standards ciprofloxacin, isoniazid, or pyrazinamide. Cytotoxicity assay of effective compounds was performed using the human monocytic leukaemia THP-1 cell line. Compounds with predicted amphiphilic properties were also tested for their effects on the rate of photosynthetic electron transport (PET) in spinach (Spinacia oleraceaL.) chloroplasts. All butyl derivatives significantly stimulated the rate of PET, indicating that the compounds can induce conformational changes in thylakoid membranes resulting in an increase of their permeability and so causing uncoupling of phosphorylation from electron transport.

Bacteriophages (phages) are viruses that infect bacterial hosts, and since their discovery over a century ago they have been primarily exploited to control bacterial populations and to serve as tools in molecular biology. In this commentary, we highlight recent diverse advances in the field of phage research, going beyond bacterial control using whole phage, to areas including biocontrol using phage-derived enzybiotics, diagnostics, drug discovery, novel drug delivery systems and bionanotechnology.

The human intestinal microbiota is one of the most densely populated ecosystems on Earth, containing up to 10 ( 13) bacteria/g and in some respects can be considered an organ itself given its role in human health. Bacteriophages (phages) are the most abundant replicating entities on the planet and thrive wherever their bacterial hosts exist. They undoubtedly influence the dominant microbial populations in many ecosystems including the human intestine. Within this setting, lysogeny appears to be the preferred life cycle, presumably due to nutrient limitations and lack of suitable hosts protected in biofilms, hence the predator/prey dynamic observed in many ecosystems is absent. On the other hand, free virulent phages in the gut are more common among sufferers of intestinal diseases and have been shown to increase with antibiotic usage. Many of these phages evolve from prophages of intestinal bacteria and emerge under conditions where their bacterial hosts encounter stress suggesting that prophages can significantly alter the microbial community composition. Based on these observations, we propose the "community shuffling" model which hypothesizes that prophage induction contributes to intestinal dysbiosis by altering the ratio of symbionts to pathobionts, enabling pathobiont niche reoccupation. The consequences of the increased phage load on the mammalian immune system are also addressed. While this is an area of intestinal biology which has received little attention, this review assembles evidence from the literature which supports the role of phages as one of the biological drivers behind the composition of the gut microbiota.

Fungal food spoilage plays a pivotal role in the deterioration of food and feed systems and some of them are also able to produce toxic compounds for humans and animals. The mycotoxins produced by fungi can cause serious health hazards, including cancerogenic, immunotoxic, teratogenic, neurotoxic, nephrotoxic and hepatotoxic effects, and Kashin-Beck disease. In addition to this, fungal spoilage/pathogens are causing losses of marketable quality and hygiene of foodstuffs, resulting in major economic problem throughout the world. Nowadays, food spoilage can be prevented using physical and chemical methods, but no efficient strategy has been proposed so far to reduce the microbial growth ensuring public health. Therefore, lactic acid bacteria (LAB) can play an important role as natural preservatives. The protection of food products using LAB is mainly due to the production of antifungal compounds such as carboxylic acids, fatty acids, ethanol, carbon dioxide, hydrogen peroxide, and bacteriocins. In addition to this, LAB can also positively contribute to the flavor, texture, and nutritional value of food products. This review mainly focuses on the use of LAB for food preservation given their extensive industrial application in a wide range of foods and feeds. The attention points out the several industrial patents concerning the use of antifungal LAB as biocontrol agent against spoilage organisms in different fermented foods and feeds.

Pseudomonas aeruginosa is a common cause of infection in the lungs of patients with cystic fibrosis (CF). In addition, biofilm formation and antibiotic resistance of Pseudomonas are major problems that can complicate antibiotic therapy. We evaluated the efficacy of using bacteriophages to kill the pathogen in both biofilms and in the murine lung. We isolated and characterized two phages from a local wastewater treatment plant, a myovirus (ϕNH-4) and a podovirus (ϕMR299-2). Both phages were active against clinical isolates of P. aeruginosa . Together, the two phages killed all 9 clinical isolate strains tested, including both mucoid and nonmucoid strains. An equal mixture of the two phages was effective in killing P. aeruginosa NH57388A (mucoid) and P. aeruginosa MR299 (nonmucoid) strains when growing as a biofilm on a cystic fibrosis bronchial epithelial CFBE41o- cell line. Phage titers increased almost 100-fold over a 24-h period, confirming replication of the phage. Furthermore, the phage mix was also effective in killing the pathogen in murine lungs containing 1 × 10 7 to 2 × 10 7 P. aeruginosa . Pseudomonas was effectively cleared (reduced by a magnitude of at least 3 to 4 log units) from murine lungs in 6 h. Our study demonstrates the efficacy of these two phages in killing clinical Pseudomonas isolates in the murine lung or as a biofilm on a pulmonary cell line and supports the growing interest in using phage therapy for the control and treatment of multidrug-resistant Pseudomonas lung infections in CF patients. IMPORTANCE Given the rise in antibiotic resistance, nonantibiotic therapies are required for the treatment of infection. This is particularly true for the treatment of Pseudomonas infection in patients with cystic fibrosis. We have identified two bacterial viruses (bacteriophages) that can kill Pseudomonas growing on human lung cells and in an animal model of lung infection. The use of bacteriophages is particularly appropriate because the killing agent can replicate on the target cell, generating fresh copies of the bacteriophage. Thus, in the presence of a target, the killing agent multiplies. By using two bacteriophages we can reduce the risk of resistant colonies developing at the site of infection. Bacteriophage therapy is an exciting field, and this study represents an important demonstration of efficacy in validated infection models.

A total of 220 lactic acid bacteria isolates were screened for antifungal activity using Aspergillus fumigatus and Aspergillus niger as the target strains. Four Lactobacillus strains exhibited strong inhibitory activity on agar surfaces. All four were also identified as having strong inhibitory activity against the human pathogenic fungi Microsporum canis, Microsporum gypseum and Epidermophyton floccosum. One of the four lactobacilli, namely Lb. reuteri ee1p exhibited the most inhibition against dermatophytes. Cell-free culture supernatants of Lb. reuteri ee1p and of the non-antifungal Lb. reuteri M13 were freeze-dried and used to access and compare antifungal activity in agar plate assays and microtiter plate assays. Addition of the Lb. reuteri ee1p freeze-dried cell-free supernatant powder into the agar medium at concentrations greater than 2% inhibited all fungal colony growth. Addition of the powder at 5% to liquid cultures caused complete inhibition of fungal growth on the basis of turbidity. Freeze-dried supernatant of the non-antifungal Lb. reuteri M13 at the same concentrations had a much lesser effect. As Lb. reuteri M13 is very similar to the antifungal strain ee1p in terms of growth rate and final pH in liquid culture, and as it has little antifungal activity, it is clear that other antifungal compounds must be specifically produced (or produced at higher levels) by the anti-dermatophyte strain Lb. reuteri ee1p. Reuterin was undetectable in all four antifungal strains. The cell free supernatant of Lb. reuteri ee1p was analyzed by LC-FTMS using an Accela LC coupled to an LTQ Orbitrap XL mass spectrometer. The high mass accuracy spectrum produced by compounds in the Lb. reuteri ee1p strain was compared with both a multianalyte chromatogram and individual spectra of standard anti-fungal compounds, which are known to be produced by lactic acid bacteria. Ten antifungal metabolites were detected.

Fungal food spoilage plays a pivotal role in the deterioration of food and feed systems and some of them are also able to produce toxic compounds for humans and animals. The mycotoxins produced by fungi can cause serious health hazards, including cancerogenic, immunotoxic, teratogenic, neurotoxic, nephrotoxic and hepatotoxic effects, and Kashin-Beck disease. In addition to this, fungal spoilage/pathogens are causing losses of marketable quality and hygiene of foodstuffs, resulting in major economic problem throughout the world. Nowadays, food spoilage can be prevented using physical and chemical methods, but no efficient strategy has been proposed so far to reduce the microbial growth ensuring public health. Therefore, lactic acid bacteria (LAB) can play an important role as natural preservatives. The protection of food products using LAB is mainly due to the production of antifungal compounds such as carboxylic acids, fatty acids, ethanol, carbon dioxide, hydrogen peroxide, and bacteriocins. In addition to this, LAB can also positively contribute to the flavor, texture, and nutritional value of food products. This review mainly focuses on the use of LAB for food preservation given their extensive industrial application in a wide range of foods and feeds. The attention points out the several industrial patents concerning the use of antifungal LAB as biocontrol agent against spoilage organisms in different fermented foods and feeds.