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A diet low in fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) is a successful therapeutic approach to alleviate symptoms of irritable bowel syndrome. However, wheat, as a fructan accumulating grain, is a major source of FODMAPs. Baker's yeast degrades fructans during fermentation, yet conventional whole wheat bread is often still high in FODMAPs. In this study, 96 yeast isolates from different environments were screened regarding their capability to metabolise FODMAPs. Two promising isolates were identified: Lachancea fermentati FST 5.1 and Cyberlindnera fabianii NTCyb, and their potential to produce low FODMAP whole wheat bread was compared to baker's yeast (Saccharomyces cerevisiae). A comprehensive characterisation of the carbohydrate metabolism by the different yeasts was achieved via HPAEC-PAD analysis of flour, doughs, and breads. L. fermentati FST 5.1 fermented fructans and excess fructose much more efficiently than baker's yeast and resulted in bread low in FODMAPs (below all cutoff levels known to induce symptoms). In contrast, C. fabianii NTCyb was unable to ferment FODMAPs in the wheat-dough-matrix. Furthermore, the yeasts' impact on the GC/MS-TOF profile of volatile aroma compounds, the sensory profile, the breads' ultrastructure, and the technological quality was examined. While C. fabianii NTCyb bread had poor technological and sensory attributes, the quality characteristics (volume, crumb structure, texture, sensory, aroma) of L. fermentati FST 5.1 bread were comparable to the baker's yeast bread. Ultimately, this study identified Lachancea fermentati FST 5.1 as an alternative to baker's yeast to produce low FODMAP whole wheat bread while maintaining optimal bread quality and consumer acceptance.

The search for natural biocontrol agents that allow the production of foods that are safe for human consumption and do not impact the taste, texture, and nutritional quality of the food, is a constant challenge for diverse food industries worldwide, particularly as the human population continues to rise globally, and multiple antibiotic resistance in pathogenic bacteria is increasingly prevalent. Bacteriophages (phages), the naturally occurring predators of bacteria, are harmless to humans and animals and are ubiquitous in the environment — and as such, have been recognised as promising antimicrobial agents to help control specific bacterial pathogens in food production. This short review details recent developments in relation to phage biocontrol in food, highlighting both their applicability for enhancing microbial safety and also the challenges within this area of food biotechnology. It also highlights developments in the use of phages for pathogen detection.

Campylobacter spp. is the leading cause of bacterial gastroenteritis worldwide and poultry are the primary reservoir. The aim of this study was to investigate the survival and/or growth of C. jejuni NCTC 11168 in broiler digestate prepared from commercial starter, grower and finisher feed formulations. Bolton broth and digestates were prepared, inoculated with C. jejuni NCTC 11168 (approximately 3 log10 CFU per ml) and incubated under microaerobic conditions at 42°C for 24 hours. Samples were taken at t = 0 (immediately after inoculation) and every 3 hours thereafter, serially diluted and plated onto mCCDA. C. jejuni grew as expected in Bolton broth (control) reaching the early stationary phase after approximately 15 hours. In contrast, although bacterial concentrations were maintained for at least 9 hours, none of the feed digestates supported the growth of C. jejuni, which were not detected after 15 hours. It is suggested that the nutrients available in the feed digestates are not enough to support C. jejuni growth and that additional factors may be at play in the avian gastrointestinal tract (GIT).

Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.

AimsCampylobacter fetus subsp fetus (CFF) can cause intestinal illness, particularly in immunocompromised humans, with the potential to cause severe systemic infections. CFF is a zoonotic pathogen with a broad host range among farm animals and humans, inducing abortion in sheep and cows. The current paper describes a strain of CFF isolated from a patient with prosthetic valve endocarditis in Mercy University Hospital, Cork, Ireland, during 2017. Only five cases of C. fetus as a cause of prosthetic valve endocarditis have been reported in the literature, with no reports of biofilm formation within the species.MethodsThe aetiological strain was speciated and subspeciated by the VITEK 2 NH card and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. CFF biofilm formation was analysed using a crystal violet staining method. C. jejuni National Collection of Type Cultures (NCTC) 11168 was used as a positive control organism. Strains were incubated statically in Mueller-Hinton broth and Mueller-Hinton broth supplemented with 0.025% sodium deoxycholate for 3 and 7 days at 37°C, microaerobically.ResultsThe CFF strain formed stronger attached biofilms on polystyrene plates on day 3 (72 hours) than the C. jejuni NCTC 11168 control strain, but were weaker than the control strain on day 7 in Mueller-Hinton broth. Monoculture of this C. fetus isolate was found to exist in three defined forms of biofilms (attached, air–liquid interface and floccules).ConclusionsThis clinically significant C. fetus isolate showed considerable biofilm-forming capability, which we suggest conferred a survivalist advantage, contributing to the genesis of infective prosthetic valve endocarditis.

Bacteriophages (phages) have traditionally been considered troublesome in food fermentations, as they are an important cause of starter-culture failure and trigger significant financial losses. In addition, from an evolutionary perspective, phages have contributed to the pathogenicity of many bacteria through transduction of virulence genes. In contrast, phages have played an important positive role in molecular biology. Moreover, these agents are increasingly being recognized as a potential solution to the detection and biocontrol of various undesirable bacteria, which cause either spoilage of food materials, decreased microbiological safety of foods, or infectious diseases in food animals and crops. The documented successful applications of phages and various phage-derived molecules are discussed in this review, as are many promising new uses that are currently under development.

This study investigates the effect of stocking density and population dynamics on broiler growth rates and productivity, while further validating the ability of the biosecurity cubes (BC) to protect birds from Campylobacter. In our methodology, six BC were constructed in a commercial broiler house containing approximately 28,500 birds. During three trials, the BC were stocked at densities of 12, 14, 16, 18, 20 and 22 birds/m2, with the main flock (20 birds/m2) considered the control. Periodically, 10 birds per density were weighed and examined. The Campylobacter status of the birds was monitored via faecal samples using the ISO 10272: 2017. The stocking density for maximum calculated yield was 20 (trials 1 and 2) or 22 birds/m2 (trial 3), followed by 18, 16, 14 and 12. At the stocking rate of 20 birds/m2, the birds in the pen grew faster than those at the same density in the main flock achieving 2 Kg 3–6 days faster. Birds in the BC were observed to be generally healthier, and in some cases, remained Campylobacter negative, even after the main flock was infected. Our results conclude that dividing the flock into sub-flocks of approximately 20 birds/m2 using BC could increase productivity up to 20%, while preventing Campylobacter.

Phenotypic antimicrobial resistance testing of 160 Campylobacter jejuni isolates from broiler caeca and neck skin samples collected at key stages during processing from 32 batches was conducted to: (i) determine the prevalence of antimicrobial resistance in C. jejuni isolates (ii) investigate if C. jejuni strains with particular resistance profiles were more frequently recovered at the end of processing, (iii) to establish if multiple strains with different resistance profiles were present within individual batches of broilers and (iv) to determine the virulence profiles of isolates. C. jejuni isolates were tested for resistance to erythromycin (ERY), ciprofloxacin (CIP), tetracycline (TET), gentamicin (GEN), nalidixic acid (NAL) and streptomycin (STR) using a broth dilution phenotypic method. Resistance to TET was most prevalent (46%), followed by resistance to CIP and NAL (29%). All isolates were susceptible to ERY and GEN, while resistance to STR was low (2%). Strains with different resistance profiles were found within 8 individual batches of broilers. Both resistant and susceptible strains were present within 4 individual batches of broilers. Whole genome sequencing of 132 C. jejuni isolates was conducted to: (i) determine relationship between resistant phenotypes and genotypes, (ii) examine genomic diversity of strains present in caeca and in neck skin samples collected at different processing stages and (iii) gain insights into the genomic profiles, including the resistome and virulome of C. jejuni isolates. A high level of correlation between resistant phenotypes and genotypes was observed (99.2%). Resistance determinants identified in sequenced isolates using ResFinder included the tet(O) gene, tet(O/32/O) gene, gyrA p.T86I mutation and blaOXA-61 like genes. A high correlation between sequence type (ST) and antimicrobial resistance was observed. The 132 sequenced C. jejuni isolates were assigned to 10 sequence types (STs), which belonged to 7 clonal complexes, based on multi-locus sequence typing (MLST). ST-257 was prevalent with 58 isolates assigned to it, followed by ST-51 with 25 isolates, ST-10089 with 16 isolates, ST-48 with 13 isolates and ST-50 with 12 isolates. Four isolates were assigned to ST-354, while 1 isolate was assigned to STs 464, 42, 1489 and 6209. All sequenced isolates were assigned to 17 cgSTs. Virulence genes which might have a role in Campylobacter survival and environmental adaptation during key processing stages and a number of virulence genes associated with human infection and colonization of broilers were detected. This study is one of the first to provide data on genomic diversity of C. jejuni isolates from broilers collected during processing in Ireland.

To date, Certrevirus is one of two genera of bacteriophage (phage), with phages infecting Pectobacterium atrosepticum, an economically important phytopathogen that causes potato blackleg and soft rot disease. This study provides a detailed description of Pectobacterium phage CB7 (vB_PatM_CB7), which specifically infects P. atrosepticum. Host range, morphology, latent period, burst size and stability at different conditions of temperature and pH were examined. Analysis of its genome (142.8 kbp) shows that the phage forms a new species of Certrevirus, sharing sequence similarity with other members, highlighting conservation within the genus. Conserved elements include a putative early promoter like that of the Escherichia coli sigma70 promoter, which was found to be shared with other genus members. A number of dissimilarities were observed, relating to DNA methylation and nucleotide metabolism. Some members do not have homologues of a cytosine methylase and anaerobic nucleotide reductase subunits NrdD and NrdG, respectively. Furthermore, the genome of CB7 contains one of the largest numbers of homing endonucleases described in a single phage genome in the literature to date, with a total of 23 belonging to the HNH and LAGLIDADG families. Analysis by RT-PCR of the HNH homing endonuclease residing within introns of genes for the large terminase, DNA polymerase, ribonucleotide reductase subunits NrdA and NrdB show that they are splicing competent. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was also performed on the virion of CB7, allowing the identification of 26 structural proteins&mdash, 20 of which were found to be shared with the type phages of the genera of Vequintavirus and Seunavirus. The results of this study provide greater insights into the phages of the Certrevirus genus as well as the subfamily Vequintavirinae.

Campylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element.

Background The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called “substrate”; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. Results L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290‐2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. Conclusion These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross‐contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry.

Background: Most publicly available genomes of Salmonella enterica are from human disease in the US and the UK, or from domesticated animals in the US. Methods: Here we describe a historical collection of 10,000 strains isolated between 1891-2010 in 73 different countries. They encompass a broad range of sources, ranging from rivers through reptiles to the diversity of all S. enterica isolated on the island of Ireland between 2000 and 2005. Genomic DNA was isolated, and sequenced by Illumina short read sequencing. Results: The short reads are publicly available in the Short Reads Archive. They were also uploaded to EnteroBase, which assembled and annotated draft genomes. 9769 draft genomes which passed quality control were genotyped with multiple levels of multilocus sequence typing, and used to predict serovars. Genomes were assigned to hierarchical clusters on the basis of numbers of pair-wise allelic differences in core genes, which were mapped to genetic Lineages within phylogenetic trees. Conclusions: The University of Warwick/University College Cork (UoWUCC) project greatly extends the geographic sources, dates and core genomic diversity of publicly available S. enterica genomes. We illustrate these features by an overview of core genomic Lineages within 33,000 publicly available Salmonella genomes whose strains were isolated before 2011. We also present detailed examinations of HC400, HC900 and HC2000 hierarchical clusters within exemplar Lineages, including serovars Typhimurium, Enteritidis and Mbandaka. These analyses confirm the polyphyletic nature of multiple serovars while showing that discrete clusters with geographical specificity can be reliably recognized by hierarchical clustering approaches. The results also demonstrate that the genomes sequenced here provide an important counterbalance to the sampling bias which is so dominant in current genomic sequencing.

Exopolysaccharides produced by lactic acid bacteria are typically high molecular weight polymers that display physiochemical properties similar to commercial hydrocolloids or gums i.e. ability to form a network and bind water. Such properties, coupled with their production in-situ, mean these exopolysaccharides represent a natural alternative to commercial hydrocolloids for the enhancement of both gluten-containing and gluten-free cereal products. Research in the past decade has shown the potential of these lactic acid bacteria exopolysaccharides, in combination with sourdough technology, to enhance the quality of gluten-containing (primarily wheat) products, particularly with respect to loaf volume, shelf-life and staling rate. Such techno-functional properties can be exploited in the gluten-free area and shows promise in helping to overcome the challenge of producing gluten-free products of the same quality and acceptability as their gluten-containing counterparts. The aim of this review is to present an overview of our current knowledge of exopolysaccharides produced by lactic acid bacteria, and how they can be exploited in the area of cereal science and gluten-free.

Campylobacter phage vB_CjeM_Los1 was recently isolated from a slaughterhouse in the Republic of Ireland using the host Campylobacter jejuni subsp. jejuni PT14, and full-genome sequencing and annotation were performed. The genome was found to be 134,073 bp in length and to contain 169 predicted open reading frames. Transmission electron microscopy images of vB_CjeM_Los1 revealed that it belongs to the family Myoviridae, with tail fibres observed in both extended and folded conformations, as seen in T4. The genome size and morphology of vB_CjeM_Los1 suggest that it belongs to the genus Cp8virus, and seven other Campylobacter phages with similar size characteristics have also been fully sequenced. In this work, comparative studies were performed in relation to genomic rearrangements and conservation within each of the eight genomes. None of the eight genomes were found to have undergone internal rearrangements, and their sequences retained more than 98% identity with one another despite the widespread geographical distribution of each phage. Whole-genome phylogenetics were also performed, and clades were shown to be representative of the differing number of tRNAs present in each phage. This may be an indication of lineages within the genus, despite their striking homology.

peer-reviewed As the new technology of whole genome sequencing (WGS) has been shown to have greater discriminatory power in differentiating strains than the much-used pulsed-field gel electrophoresis (PFGE), there is currently a transition from using PFGE to WGS for disease outbreak investigation. Therefore, there is a need for comparison of bacterial isolates using both PFGE and WGS. In this study, two pairs of L. monocytogenes strains with geographically diverse sources of isolation but which had indistinguishable or closely related PFGE profiles, were subjected to WGS analysis. Comparative analysis of their genomes showed that one pair of strains which had closely related PFGE profiles in fact differed significantly from one another in terms of their antibiotic and heavy metal stress resistance determinants, and mobile genetic elements. Therefore, this research demonstrated the ability of WGS analysis to differentiate very closely related strains and that WGS analysis represents the most effective tool available for subtyping L. monocytogenes isolates.

The species Leuconostoc citreum is often isolated from grain and vegetable fermentations such as sourdough, sauerkraut and kimchi. Lc. citreum has seen an increase in its use as a starter culture for various fermentations and food applications. The strain Lc. citreum TR116 has been applied previously in this laboratory aimed at sugar depletion through metabolism resulting in the reduction of fructose to mannitol, a polyol considered as a sweet carbohydrate. Besides reducing sugar, TR116 showed flavour modulating characteristics and contributes to the extension of microbial shelf life. In order to obtain a better understanding of this strain and to fully use its set of abilities, the genome of Lc. citreum TR116 was sequenced using the Illumina MiSeq, assembly with SPAdes and annotated by the Prokaryotic Genome Annotation Pipeline. Metabolic reconstruction was employed to elucidate carbohydrate, organic acid and amino acid metabolism in the strain. Of particular interest was the gene expression analysis ascertained the influence of fructose on the genes mdh and manX involved in the uptake of fructose and its conversion to mannitol. This investigation, the first in Lc. citreum, illustrates the metabolic processes involved in fermentation used by this strain and demonstrates that in the presence of fructose, expression of the genes mdh and manX is increased. The resulting transparency of the skill set of TR116 contributes highly to future functionalisation of food systems and food ingredients.

Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus–host infection networks have been used to characterize the “predator–prey” interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage–bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

Supplementary data to this article can be found online at: http://dx. doi.org/10.1016/j.ijfoodmicro.2017.04.009., In recent years, the microbiological safety of powdered infant formula has gained increasing attention due to the identification of contaminating C. sakazakii and its epidemiological link with life-threatening neonatal infections. Current intervention strategies have fallen short of ensuring the production of infant formula that is free from C. sakazakii. In this study, we describe the isolation and characterisation of three bacteriophages (phages) and their application as a phage cocktail to inhibit the growth of C. sakazakii in different brands of infant formula, while also assessing the phages ability to prevent biofilm formation. All three phages, isolated from slurry, possess a relatively broad host range, verified by their ability to infect across genera and species. When all three phages were combined and used as part of a phage cocktail, 73% coverage was obtained across all Cronobacter strains tested. Optimum thermo-tolerance and pH stability were determined between 4 °C37 °C, and pH 68, respectively, well within the normal range of application of infant formula. Genome sequencing and analysis revealed all the phages to be free from lysogenic properties, a trait which renders each favourable for phage therapy applications. As such, the combined-phage preparation (3 × 108 pfu/mL) was found to possess a strong bactericidal effect on C. sakazakii/C. sakazakii LUX cells ( 104 cfu/mL), resulting in a significant reduction in cell numbers, to below the limit of detection (, This work was funded by Technological Sector Research Strand III ref. CRS/07/CR03. Angela Back from MRI Kiel is acknowledged for technical assistance in preparations for electron microscopy. Hugo Oliveira and Rob Lavigne contributed to the genome sequencing analysis, supported by the KULeuven GOA (GOA/15/006) Grant Phagebiosystems., info:eu-repo/semantics/publishedVersion

The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34 pg and 10 4 CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 10 3 CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 10 3 CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 10 2 CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and with its unique melting curve profile adds increased specificity to MAP diagnostic tests.

The aim of this study was to investigate the efficacy of IS_MAP04 as a potential new diagnostic quantitative PCR (qPCR) target for the detection of Mycobacterium avium subspecies paratuberculosis from bovine faeces. IS_MAP04 primers were designed and tested negative against non-MAP strains. The detection limit of IS_MAP04 qPCR was evaluated on different MAP K-10 DNA concentrations and on faecal samples spiked with different MAP K-10 cell dilutions. A collection of 106 faecal samples was analysed and the efficacy of IS_MAP04 was statistically compared with IS900 and IS_MAP02. The detection limits observed for IS_MAP04 and IS900 on MAP DNA was 34 fg and 3.4 fg respectively. The detection limit of MAP from inoculated faecal samples was 102 CFU/g for both IS_MAP04 and IS900 targets and a detection limit of 102 CFU/g was also achieved with a TaqMan qPCR targeting IS_MAP04. The efficacy of IS_MAP04 to detect positive MAP faecal samples was 83.0% compared to 85.8% and 83.9% for IS900 and IS_MAP02 respectively. Strong kappa agreements were observed between IS_MAP04 and IS900 (κ=0.892) and between IS_MAP04 and IS_MAP02 (κ=0.897). As a new molecular target, IS_MAP04 showed that the detection limit was comparable to IS900 to detect MAP from inoculated faecal material. The MAP detection efficacy of IS_MAP04 from naturally infected faecal samples proved to be relatively comparable to IS_MAP02, but yielded efficacy results slightly less than IS900. Moreover, IS_MAP04 could be of significant value when used in duplex or multiplex qPCR assays.

Aims This study was designed to investigate the ability of naturally occurring bacteria isolated from mushroom substrate to prevent biofilm formation by L. monocytogenes or to remove existing biofilms in mushroom production facilities. Methods and Results It is generally recognised that Listeria monocytogenes forms biofilms that can facilitate its survival in food processing environments. Eleven bacteriocin-producing isolates were identified and the bacteriocins characterised based on heat and enzyme inactivation studies. Further characterisation was undertaken by MALDI-TOF mass spectrometry, PCR and sequencing. Production of Nisin Z (by Lactococcus lactis isolates), subtilomycin (by Bacillus subtilis isolates) and lichenicidin (by Bacillus licheniformis and Bacillus sonorensis isolates) was detected. In co-culture with L. monocytogenes, the bacteriocin-producing strains could prevent biofilm formation and reduce pre-formed biofilms. Conclusions Mushroom substrate can be source of bacteriocin-producing bacteria that can antagonize L. monocytogenes. Significance and Impact of the Study The results highlight the potential of bacteriocin-producing strains from mushroom substrate to reduce L. monocytogenes biofilm in food production environments, contributing to a reduction in the risk of food contamination from the environment. This article is protected by copyright. All rights reserved.

The true prevalence of tet(A), which codes for a tetracycline efflux pump, in thermophilic Camplyobacter spp. requires clarification after reports emerged in Iran (2014) and Kenya (2016) of the novel detection of tet(A) in Campylobacter. During our investigation of antibiotic resistance mechanisms in a sample of Irish thermophilic Campylobacter broiler isolates, it was determined that 100% of tetracycline-resistant isolates (n = 119) harboured tet(O). Accessory tetracycline-resistance mechanisms were considered as tetracycline minimum inhibitory concentrations ranged from 4 to ≥ 64 mg/L. Primers previously reported for the detection of tet(A) in Campylobacter failed to produce an amplicon using a positive control strain (Escherichia coli K12 SK1592 containing the pBR322 plasmid) and a selection of Campylobacter isolates. Accordingly, we designed new tet(A)-targeting primers on SnapGene2.3.2 that successfully generated a 407 bp product from the positive control strain only. Further in silico analysis using BLASTn and SnapGene2.3.2 revealed that previously reported Campylobacter tet(A) sequences deposited on GenBank shared 100% homology with Campylobacter tet(O). We postulate that this gave rise to the erroneous report of a high tet(A) prevalence among a pool of Kenyan broiler Campylobacter isolates that were tested using primers designed based on these apparent tet(A) sequences. In conclusion, further work would be required to determine whether the homology between tet(A) potentially present in Campylobacter and known tet(A) genes would be sufficient to allow amplification using the primers designed in our study. Finally, the existence of tet(A) in thermophilic Campylobacter spp. remains to be demonstrated.

© The Author(s)., Listeria monocytogenes is a ubiquitous Gram-positive bacterium that is a major concern for food business operators because of its pathogenicity and ability to form biofilms in food production environments. Bacteriophages (phages) have been evaluated as biocontrol agents for L. monocytogenes in a number of studies and, indeed, certain phages have been approved for use as anti-listerial agents in food processing environments (ListShield and PhageGuard Listex). Endolysins are proteins produced by phages in the host cell. They cleave the peptidoglycan cell wall, thus allowing release of progeny phage into the environment. In this study, the amidase domain of the phage vB_LmoS_293 endolysin (293-amidase) was cloned and expressed in Escherichia. coli (E. coli). Muralytic activity at different concentrations, pH and temperature values, lytic spectrum and activity against biofilms was determined for the purified 293-amidase protein. The results showed activity on autoclaved cells at three different temperatures (20 °C, 37 °C and 50 °C), with a wider specificity (L. monocytogenes 473 and 3099, a serotype 4b and serogroup 1/2b-3b-7, respectively) compared to the phage itself, which targets only L. monocytogenes serotypes 4b and 4e. The protein also inhibits biofilm formation on abiotic surfaces. These results show the potential of using recombinant antimicrobial proteins against pathogens in the food production environment., This research was funded by the Food Institutional Research Measure under project 14/F/881, grant BFU2014-53425-P from the Spanish Ministry of Science, Innovation and Universities and the Teagasc Walsh Fellowship Short-Term Overseas Training Awards.

The demand for sugar reduction in products across the food and beverage industries has evoked the development of novel processes including the application of fermentation with lactic acid bacteria. Heterofermentative lactic acid bacteria (LAB) are diverse in their ability to utilise fermentable sugars and can also convert fructose into the sweet tasting polyol, mannitol. The sourdough microbiota has long been recognised as an ecological niche for a range of homofermentative and heterofermentative lactic acid bacteria. A leading determinant in the biodiversity of sourdough microbial populations is the type of flour used. Ten non-wheat flours were used and back-slopped for 7 days resulting in the isolation of 52 mannitol producing isolates which spanned six heterofermentative species of the genera Lactobacillus, Leuconostoc and Weissella. Assessment of mannitol productivity in fructose concentrations up to 100 g/L found Leuconostoc citreum TR116, to have the best mannitol producing characteristics, consuming 95% of available fructose and yielding 0.68 g of mannitol per gram of fructose consumed which equates to the maximal theoretical yield. Investigation of the effects of initial pH on mannitol production and other fermentation parameters in the isolates found pH 7 to be best for isolates Lactobacillus brevis TR052, Leuconostoc fallax TR111, Leuconostoc citreum TR116, Leuconostoc mesenteroides TR154 and Weissella paramesenteroides TR212, while pH 6 was optimal for Leuconostoc pseudomesenteroides TR080. The fermentation of apple juice with each isolate resulted in sugar reduction ranging from 30.3–74.0 g/L (34–72%). When apple juice fermentation with Leuconostoc citreum TR116 was scaled up to 1 L bioreactor a reduction in sugar of 98.6 g/L (83%) was achieved along with the production of 61.6 g/L mannitol. This demonstrates a fermentative process for sugar reduction in fruit juice with concomitant production of the sweet metabolite mannitol to create a fermentate that is suitable for further development as a low sugar fruit juice alternative.

Bacteriophages are the principal cause of starter culture failure in many food fermentations carried out by lactic acid bacteria (LAB). The understanding and correct exploitation of phage resistance mechanisms are vital for the development of robust LAB starter cultures that can withstand phage infection and consistently produce desirable products. Plasmid-encoded mechanisms can be particularly functional if the plasmid can be mobilized to other strains via food-grade methods. Chromosomally encoded phage resistance mechanisms can be categorized as phage adsorption inhibition, inhibition of phage deoxyribonucleic acid injection, restriction/modification, abortive infection, and the clustered regularly interspaced short palindromic repeat-Cas system. The increased availability of dairy streptococcal phage genome sequences has permitted the development of a multiplex polymerase chain reaction system that encompasses all known phage groups for the bacterial species. Lactobacillus is a highly diverse bacterial genus with in excess of 200 species recognized.

Campylobacter fetus is a Gram-negative, zoonotic pathogen and a member of the class Epsilonproteobacteria . We report the draft genome sequence of C. fetus subsp. fetus CITCf01, isolated from a patient with subacute bacterial endocarditis.

The world is facing a big problem of non-communicable diseases, such as obesity, cardiovascular disease and diabetes. An excessive sugar consumption is considered as a main factor, which triggers these diseases. The two main sources of sugar in processed products on the market are sugar-sweetened beverages and sweet bakery products. Sugar reduction is challenging, especially in baked goods, since it interacts significantly with all ingredients. These interactions cause an increase in gelatinization temperature, a delay in gluten network development, an increase or decrease in yeast activity depending on the sugar concentration, as well as an enhancement of emulsification. Reflecting the molecular interactions on the product quality characteristics of different types of baked goods, sugar also contributes to browning reactions and extension of microbial shelf life. During cake preparation, sugar supports the batter aeration which results in the typical soft cake crumb. Furthermore, it contributes to the spreading process of biscuits during baking and enhances surface cracking due to recrystallization. Sugar reduction requires the development of different strategies; Two well-known strategies are the replacement of added sugar by the combination of bulking agents and high-intensive sweeteners, or by sweet bulking ingredients, such as polyols. The in-situ production of polyols to enhance sweetness, and exopolysaccharides to improve texture, in a sourdough system shows high potential as sugar replacement. Lactobacillus sanfranciscensis, Leuconostoc mesenteroides and Leuconostoc citreum are high mannitol producing lactic acid bacteria (LAB) strains with yields of 70-98% and Leuconostoc oenos was found to produce erythritol. Furthermore, the yeast strain Candida milleri isolated from sourdough produces xylitol in the presence of xylose. Exopolysaccharides produced by LAB and/or yeasts are known to improve the texture and structure of bakery products and, thus, have high potential as natural functional ingredients to compensate quality loss in sweet bakery goods.

This study examined the impact of key processing stages and flock variables on the prevalence of Campylobacter on broiler carcasses. Overall, the prevalence of Campylobacter was 62% in caeca, and 68%, 65% and 62% in neck skin samples collected after evisceration, final wash and carcass chilling, respectively. Campylobacter were found in 32% of caeca, and 52%, 40% and 32% of neck skin samples collected after evisceration, final wash and carcass chilling, respectively from first thin broiler batches. Final thin broiler batches were more frequently contaminated with prevalences of 83% found in caeca, 80% in neck skin samples collected after evisceration and 83% found in neck skin samples collected after both final wash and carcass chilling stages (p < 0.05). Thinning status had a significant effect on Campylobacter counts with significantly higher counts observed in samples from final thin batches (p < 0.05). Highest Campylobacter concentrations in neck skin samples were observed at the evisceration stage in both first and final thin samples, with counts ranging from 2.0 to 3.8 log10 CFU/g and 2.3 to 4.8 log10 CFU/g in first and final thin batches, respectively. All first thin samples had counts below the European Union (EU) Process Hygiene Criterion threshold level of 3 log10 CFU/g after chilling while 52% of final thin batches had counts above this limit.

Endolysins (lysins) are bacteriophage-encoded enzymes that have evolved to degrade specific bonds within the bacterial cell wall. These enzymes represent a novel class of antibacterial agents against infectious pathogens, especially in light of multidrug-resistant bacteria, which have made antibiotic therapy increasingly redundant. Lysins have been used successfully to eliminate/control bacterial pathogens in various anatomical locations in mouse and other animal models. Engineering tactics have also been successfully applied to improve lysin function. This review discusses the structure and function of lysins. It highlights protein-engineering tactics utilised to improve lysin activity. It also reviews the applications of lysins towards food biopreservation, therapeutics, bioifilm elimination and diagnostics.

Exopolysaccharides (EPS)-producing lactic acid bacteria (LAB) are industrially important microorganisms in the development of functional food products and are used as starter cultures or coadjutants to develop fermented foods. There is large variability in EPS production by LAB in terms of chemical composition, quantity, molecular size, charge, presence of side chains, and rigidity of the molecules. The main body of the review will cover practical aspects concerning the structural diversity structure of EPS, and their concrete application in food industries is reported in details. To strengthen the food application and process feasibility of LAB EPS at industrial level, a future academic research should be combined with industrial input to understand the technical shortfalls that EPS can address.

Pectobacterium atrosepticum is a phytopathogen of economic importance as it is the causative agent of potato blackleg and soft rot. Here we describe the Pectobacterium phage vB_PatP_CB5 (abbreviated as CB5), which specifically infects the bacterium. The bacteriophage is characterized in detail and TEM micrographs indicate that it belongs to the Podoviridae family. CB5 shares significant pairwise nucleotide identity (&ge, 80%) with P. atrosepticum phages &phi, M1, Peat1, and PP90 and also shares common genome organization. Phylograms constructed using conserved proteins and whole-genome comparison-based amino acid sequences show that these phages form a distinct clade within the Autographivirinae. They also possess conserved RNA polymerase recognition and specificity loop sequences. Their lysis cassette resembles that of KP34virus, containing in sequential order a U-spanin, a holin, and a signal&ndash, arrest&ndash, release (SAR) endolysin. However, they share low pairwise nucleotide identity with the type phage of the KP34virus genus, Klebsiella phage KP34. In addition, phage KP34 does not possess several conserved proteins associated with these P. atrosepticum phages. As such, we propose the allocation of phages CB5, Peat1, &phi, M1, and PP90 to a separate new genus designated Phimunavirus.

The interest for plant-based dairy substitutes is expanding rapidly and consumers are opting for nutritious and healthy dairy alternatives. The reduction of sugar using different exogenous enzymes in combination with lactic acid fermentation in a quinoa-based milk substitute was explored in this study. Different amylolytic enzymes were used to release sugar from the raw material, which were further metabolised to mannitol, due to fermentation with two heterofermentative lactic acid bacteria. Using these two biotechnological techniques enables the reduction of sugar, while also preserving some of the sweetness. Leuconostoc citreum TR116, and Lactobacillus brevis TR055 were isolated from sourdough. Both strains showed high viable cell counts with L. citreum TR116 > 8.4 and L. brevis TR055 > 9.3 log cfu/mL, and a reduction in pH to 3.7 and 3.5 respectively. When fructose was available, mannitol was produced in conjunction with acetic acid in addition to lactic acid. Due to these processes, the original glucose value was reduced from 50 mmol/100 g to approximately 30 mmol/100 g, which equates to a glucose reduction of 40%. In respect to mannitol production, both strains performed well: L. citreum TR116 showed a conversion factor of 1:1 from fructose to mannitol, while L. brevis TR055 showed a lower yield, with a conversion factor of 1:0.8. Glycaemic load was reduced by more than a third, bringing it down to the low range with a value of about 10. Overall, enzymatic modification in conjunction with mannitol-producing lactic acid bacteria shows great potential for further possible application in the development of nutritious and sugar reduced plant-based milk substitutes.

peer-reviewed The increase in antibiotic resistance in pathogenic bacteria is a public health danger requiring alternative treatment options, and this has led to renewed interest in phage therapy. In this respect, we describe the distinct host ranges of Staphylococcus phage K, and two other K-like phages against 23 isolates, including 21 methicillin-resistant S. aureus (MRSA) representative sequence types representing the Irish National MRSA Reference Laboratory collection. The two K-like phages were isolated from the Fersisi therapeutic phage mix from the Tbilisi Eliava Institute, and were designated B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1). The sequence relatedness of B1 and JA1 to phage K was observed to be 95% and 94% respectively. In terms of host range on the 23 Staphylococcus isolates, B1 and JA1 infected 73.9% and 78.2% respectively, whereas K infected only 43.5%. Eleven open reading frames (ORFs) present in both phages B1 and JA1 but absent in phage K were identified by comparative genomic analysis. These ORFs were also found to be present in the genomes of phages (Team 1, vB_SauM-fRuSau02, Sb_1 and ISP) that are components of several commercial phage mixtures with reported wide host ranges. This is the first comparative study of therapeutic staphylococcal phages within the recently described genus Kayvirus.

The challenge remains for the baking industry to reduce salt levels in yeasted bread as directed by governments, retailers and consumers around the world. The two main problems associated with the reduction of salt are a lack of salty taste and the reduction in shelf-life. Both of these issues are addressed in the presented work. A range of breads containing different levels of salt (0.0%, 0.3% and 1.2% of NaCl) in combination with various levels of sourdough (0%, 6%, 12%, 18%, 24%) was produced. The different doughs were analysed for their rheological behaviour. The bread quality characteristics such as loaf volume, crumb structure, staling rate and microbial shelf life were also determined. The sourdoughs were analysed for their different metabolites: organic acids, sugars, exopolysaccharides (EPS), and antifungal compounds. A trained sensory panel was used to perform descriptive analysis of the bread samples. The object of this paper is to use functional sourdoughs, containing Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1 to compensate for the quality problems that occur when salt is reduced in yeasted bread. The application of functional sourdoughs containing exopolysaccharides and/or antifungal substances in salt reduced breads significantly improved the quality. The application of functional sourdoughs allows the reduction of salt to a level of 0.3%.

Recently, aTyphi isolate producing CTX-M-15 extended spectrum β-lactamase (ESBL) and with decreased ciprofloxacin susceptibility was isolated in the Democratic Republic of the Congo. We have selected bacteriophages that show strong lytic activity against this isolate and have potential for phage-based treatment of. Typhi, andin general. ispartof: Viruses vol:10 issue:4 ispartof: location:Switzerland status: published

Aims The aims of this study were to characterize the genetic diversity of Listeria monocytogenes isolates obtained from commercial mushroom production, to establish the persistence, recontamination and the risk of cross-contamination from the working environment to the final products, creating awareness about the presence of L. monocytogenes thus helping to prevent the possibility of cross-contamination. Methods and results From an extensive analysis of commercial mushroom production, analysed with BS EN ISO 11290-1:1996/Amd 1:2004 and BS EN ISO 11290-2:1998/Amd 1:2004, 279 L. monocytogenes isolates were obtained. All of the isolates were characterized by pulsed-field gel electrophoresis, species PCR and serogroup PCR. All the isolates were confirmed as L. monocytogenes; 30·1% were serogroup 1/2b-3b-7, 40·8% were serogroup 1/2a-3a and 29·1% were serogroup 4b-4d-4e. There were 77 pulsotypes from the 279 isolates, 40 of the pulsotypes had only one strain and 37 had two or more strains, indicating great diversity in the isolates. Conclusions The high genetic diversity is indicative of the fact that current hygiene practices are successful at removing L. monocytogenes but that recontamination of the production environment is frequent. Significance and impact of the study The results obtained are very valuable in creating awareness of L. monocytogenes in mushroom production and for the improvement of hygiene practices.

Pectobacterium atrosepticum is an economically important phytopathogen that is responsible for potato blackleg and soft rot, and for which current control strategies are limited. In this study, stem samples of potato crops exhibiting blackleg were taken from three farms in Co. Cork, Ireland, and they were found to be infected with P. atrosepticum. Three closely related bacteriophages (phages) that are specific to this phytopathogen were isolated and characterized, namely vB_PatP_CB1, vB_PatP_CB3, and vB_PatP_CB4 (abbreviated as CB1, CB3, and CB4). Both CB1 and CB3 were determined to infect 12 strains and CB4 10 strains of the 19 strains of P. atrosepticum tested. Morphology, latent periods, burst sizes, and their stability at various temperatures and pHs were also examined. Genome sequencing of the three phages revealed that they shared a minimum nucleotide identity of 93% with each other. Their genomes exhibited an Enquartavirinae genome organization, possessing several conserved proteins that were associated with phages of this group, like the type species Escherichia virus N4. Tandem electrospray ionization-mass spectrometry (ESI-MS/MS) allowed for the identification of ten structural proteins that form the virion of CB1, six that are conserved in phage N4. Biocontrol experiments demonstrated that the phages suppress soft rot formation upon co-inoculation with P. atrosepticum on whole tubers. The results of this study indicate that CB1 related phages could be good candidates for phage-based control. ispartof: PHARMACEUTICALS vol:11 issue:2 ispartof: location:Switzerland status: published

Exopolysaccharides produced by lactic acid bacteria are a diverse group of polysaccharides produced by many species. They vary widely in their molecular, compositional, and structural characteristics, including mechanisms of synthesis. The physiochemical properties of these polymers mean that they can be exploited for the sensorial and textural enhancement of a variety of food and beverage products. Traditionally, lactic acid bacteria exopolysaccharides have an important role in fermented dairy products and more recently are being applied for the improvement of bakery products. The health benefits that are continually being associated with these polysaccharides enable the development of dual function, added-value, and clean-label products. To fully exploit and understand the functionality of these exopolysaccharides, their isolation, purification, and thorough characterization are of great importance. This review considers each of the above factors and presents the current knowledge on the importance of lactic acid bacteria exopolysaccharides in the food and beverage industry.

To date, a small number of jumbo myoviruses have been reported to possess atypical whisker-like structures along the surface of their contractile tails. Erwinia amylovora phage vB_EamM_Y3 is another example. It possesses a genome of 261,365 kbp with 333 predicted ORFs. Using a combination of BLASTP, Interproscan and HHpred, about 21% of its putative proteins could be assigned functions involved in nucleotide metabolism , DNA replication , virion structure and cell wall degradation. The phage was found to have a signal-arrest-release (SAR) endolysin (Y3_301) possessing a soluble lytic transglycosylase domain. Like other SAR endolysins, inducible expression of Y3_301 caused Escherichia coli lysis, which is dependent on the presence of an N-terminal signal sequence. Phylogenetic analysis showed that its closest relatives are other jumbo phages including Pseudomonas aeruginosa phage PaBG and P. putida phage Lu11, sharing 105 and 87 homologous proteins respectively. Like these phages, Y3 also shares a distant relationship to Ralstonia solanacearum phage ΦRSL1 (sharing 55 homologous proteins). As these phages are unrelated to the Rak2-like group of hairy phages, Y3 along with Lu11 represent a second lineage of hairy myoviruses.

Lactic acid bacteria (LAB) with antifungal activity can be applied to the malting process in order to improve the microbial stability and safety of malt. The main objectives of this project was to evaluate the influence of the antifungal activity of LAB cell-free-supernatant (cfs) in the malting process and to investigate antifungal effects of selected LAB towards the mould growth and mycotoxin production of Fusarium culmorum. The impact of substrate concentration on the production of LAB antifungal compounds and the changes in the malt quality attributes was also investigated. Barley grains infected with F. culmorum were used in the malting trials. Lactobacillus amylovorus and Lactobacillus reuteri cfs were generated in a wort based substrate and applied in the early stages of malting. F. culmorum growth was quantified and the mycotoxin deoxynivalenol (DON) was detected in the malted grains. The various supernatants were characterized based on their sugar and organic acid composition. Antifungal metabolites were quantified using a QuEChERS and HPLC-UV/PDA method. Standard EBC methods were used to evaluate the malt quality attributes. Results show that L. reuteri R29 cfs produced in 3 °P substrate successfully inhibited Fusarium growth by 23% and mycotoxin DON by 83%. Using a 3 °P wort substrate concentration, 68% of the phenyllactic acid (PLA) was produced, when compared to the 12 °P substrate. PLA plays an essential role in the supernatant antifungal activity. Malt quality attributes resulted in highly modified grains, lower pH, higher colouration, and higher extract yield.

In this study, a series of twenty-two ring-substituted 6-hydroxynaphthalene-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. Derivatives substituted by trifluoromethyl, bromo, methyl and methoxy moieties in C′ (3) and C′ (4) positions of the anilide ring showed 2-fold higher activity against M. tuberculosis than isoniazid and 4.5-fold higher activity against M. avium subsp. paratuberculosis than rifampicin. 6-Hydroxy- N -(2-methylphenyl)naphthalene-2-carboxamide had MIC = 29 μM against M. avium complex. A significant decrease of mycobacterial cell metabolism (viability of M. tuberculosis H37Ra) was observed using MTT assay. Screening of the cytotoxicity of the most effective antimycobacterial compounds was performed using the THP-1 cells, and no significant lethal effect was observed. The structure–activity relationships are discussed.

Bacteriophages and their derivatives are continuously gaining impetus as viable alternative therapeutic agents to control harmful multidrug-resistant bacterial pathogens, particularly in the food industry. The reduced efficacy of conventional antibiotics has resulted in a quest to find novel alternatives in the war against infectious disease. This study describes the full-genome sequence of Cronobacter phage vB_CsaP_Ss1, with subsequent cloning and expression of its endolysin, capable of hydrolysing Gram-negative peptidoglycan. Cronobacter phage vB_CsaP_Ss1 is composed of 42 205 bp of dsDNA with a G+C content of 46.1 mol%. A total of 57 ORFs were identified of which 18 could be assigned a putative function based on similarity to characterized proteins. The genome of Cronobacter phage vB_CsaP_Ss1 showed little similarity to any other bacteriophage genomes available in the database and thus was considered unique. In addition, functional analysis of the predicted endolysin (LysSs1) was also investigated. Zymographic experiments demonstrated the hydrolytic activity of LysSs1 against Gram-negative peptidoglycan, and this endolysin thus represents a novel candidate with potential for use against Gram-negative pathogens.

The emergence of antibiotic-resistant bacteria and decrease in the discovery rate of novel antibiotics takes mankind back to the ‘pre-antibiotic era' and search for alternative treatments. Bacteriophages have been one of promising alternative agents which can be utilised for medicinal and biological control purposes in agriculture and related fields. The idea to treat bacterial infections with phages came out of the pioneering work of Félix d‘Hérelle but this was overshadowed by the success of antibiotics. Recent renewed interest in phage therapy is dictated by its advantages most importantly by their specificity against the bacterial targets. This prevents complications such as antibiotic-induced dysbiosis and secondary infections. This article is compiled by the participants of the Expert Round Table conference ‘Bacteriophages as tools for therapy, prophylaxis and diagnostics' (19–21 October 2015) at the Eliava Institute of Bacteriophage, Microbiology and Virology, Tbilisi, Georgia. The first paper from the Round Table was published in the Biotechnology Journal1. This In Focus article expands from this paper and includes recent developments reported since then by the Expert Round Table participants, including the implementation of the Nagoya Protocol for the applications of bacteriophages.

Mycobacterial interspersed repetitive unit and variable number tandem repeat (MIRU-VNTR) has been developed as a simple, rapid and cost efficient molecular typing method to differentiate Mycobacterium avium subspecies paratuberculosis (MAP) isolates. The aim of this study was to determine the genomic diversity of MAP across the Republic of Ireland by utilising the MIRU-VNTR typing method on a large collection of MAP isolates. A total of 114 MAP isolates originated from 53 herds across 19 counties in the Republic of Ireland were genotyped based on eight established MIRU-VNTR loci. Four INMV groups were observed during this study. INMV 1 was found in 67 MAP isolates (58.8%) and INMV 2 was observed in 45 isolates (39.4%). INMV 3 and INMV 116 recorded only one isolate each (0.9%). The unique INMV 116 group has never been reported among herds thus far and the molecular pattern of the MAP isolate classified in INMV 116 showed a difference at the MIRU-VNTR X3 locus compared to the other three INMV groups observed. INMV 1, INMV 2 and INMV 3 are observed frequently in Europe and comprised 99.1% of the total MAP isolates characterised in this study, indicating that MAP exhibited low level of genetic diversity across the Republic of Ireland using the MIRU-VNTR method. By the implementation of SNP analysis or MLSSR as an additional typing method, MAP genetic diversity would increase. INMV 3 is unique to Ireland and whereas INMV 116 has never been previously reported among herds by MIRU-VNTR typing.

Losses in crop yields due to disease need to be reduced in order to meet increasing global food demands associated with growth in the human population. There is a well recognised need to develop new environmentally-friendly control strategies to combat bacterial crop disease. Current control measures involving the use of traditional chemicals or antibiotics are losing their efficacy due to the natural development of bacterial resistance to these agents. In addition, there is an increasing awareness that their use is environmentally unfriendly. Bacteriophages, the viruses of bacteria, have received increased research interest in recent years as a realistic environmentally friendly means of controlling bacterial diseases. Their use presents a viable control measure for a number of destructive bacterial crop diseases, with some phage-based products already becoming available on the market. Phage biocontrol possesses advantages over chemical controls in that tailor-made phage cocktails can be adapted to target specific disease-causing bacteria. Unlike chemical control measures, phage mixtures can be easily adapted for bacterial resistance which may develop over time. In this review, we will examine the progress and challenges for phage-based disease biocontrol in food crops.

Enterobacteria phage vB_PcaM_CBB is a jumbo phage belonging to the family Myoviridae. It possesses highly atypical whisker-like structures along the length of its contractile tail. It has a broad host range with the capability of infecting species of the genera Erwinia, Pectobacterium, and Cronobacter. With a genome of 355,922 bp, excluding a predicted terminal repeat of 22,456 bp, phage CBB is the third largest phage sequenced to date. Its genome was predicted to encode 554 ORFs with 33 tRNAs. Based on prediction and proteome analysis of the virions, 29% of its predicted ORFs could be functionally assigned. Protein comparison shows that CBB shares between 3338% of its proteins with Cronobacter phage GAP32, coliphages PBECO4 and 121Q as well as Klebsiella phage vB_KleM_Rak2. This work presents a detailed and comparative analysis of vB_PcaM_CBB of a highly atypical jumbo myoviridae phage, contributing to a better understanding of phage diversity and biology., Funding was provided by Cork Institute of Technology as a PhD fellowship to CB.