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The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth protocols for the creation of nonpolar unlabeled gene deletions in Listeria (L.) monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe two different plasmid-based approaches, which facilitate the introduction of this spliced amplicon in place of the corresponding segment of chromosomal DNA: the pKSV7 system and the pORI280/pVE6007 system.

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth a protocol for the creation of nonpolar unlabelled gene deletions in Listeria monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe the pKSV7 plasmid-based approach, which facilitates the introduction of a spliced amplicon in place of the corresponding segment of chromosomal DNA.

Background. Specific bacterial species have been linked to several intestinal diseases, including colorectal cancer (CRC). In recent years, high abundances of an emerging pathogen, Fusobacterium nucleatum, have been identified in tumors and stool samples of CRC patients and it has been suggested that F. nucleatum contributes to CRC initiation and development. The possibility of suppressing the growth of F. nucleatum in the GI tract using antimicrobial-producing probiotic bacteria may reduce the overall risk of CRC development. Methods. Here, we screen a collection of faecal samples from healthy donors against F. nucleatum in an effort to discover an antimicrobial-producing isolate capable of selectively inhibiting this emerging human pathogen. Potential isolates with anti-Fusobacterial activity were then further analysed for the ability to inhibit the pathogen in cell culture and in a faecal fermentation system, which simulates the dynamic conditions of the human colon. Results. Culture-based screening of over 16,000 colonies of gastrointestinal origin resulted in the identification of one faecal isolate with probiotic potential displaying significant antagonistic activity against F. nucleatum initially in cell culture media and subsequently inhibition was confirmed in the simulated intestinal model. Conclusion. This study reveals that, a novel gut isolate demonstrates inhibition against the CRC-associated F. nucleatum in vitro and suppresses its growth in a model of the human distal colon. This is an important finding, suggesting the potential of a natural gut bacterium to supress the growth of a bacterial pathogen associated with CRC, which may contribute to reducing the overall risk of developing the disease.

The role of the gut microbiome in human health and disease is the focus of much attention. It has been widely agreed upon that our gut bacteria play a role in host immunity, nutrient absorption, digestion, metabolism, and other key drivers of health. Furthermore, certain microbial signatures and specific taxa have also been associated with the development of diseases, such as obesity; inflammatory bowel disease; and, indeed, colorectal cancer (CRC), which is the focus of this review. By extension, such taxa represent potential therapeutic targets. In particular, the emerging human pathogen Fusobacterium nucleatum represents an important agent in CRC development and its control within the gastrointestinal tract is desirable. This paper reviews the principal bacterial pathogens that have been associated with CRC to date and discusses the in vitro and human studies that have shown the potential use of biotherapeutic strains as a means of targeting CRC-associated bacteria.

Nisin P is a natural nisin variant, the genetic determinants for which were previously identified in the genomes of two Streptococcus species, albeit with no confirmed evidence of production. Here we describe Streptococcus agalactiae DPC7040, a human faecal isolate, which exhibits antimicrobial activity against a panel of gut and food isolates by virtue of producing nisin P. Nisin P was purified, and its predicted structure was confirmed by nanoLC-MS/MS, with both the fully modified peptide and a variant without rings B and E being identified. Additionally, we compared its spectrum of inhibition and minimum inhibitory concentration (MIC) with that of nisin A and its antimicrobial effect in a faecal fermentation in comparison with nisin A and H. We found that its antimicrobial activity was less potent than nisin A and H, and we propose a link between this reduced activity and the peptide structure.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.

There is accumulating evidence that physical fitness influences the gut microbiome and as a result, promotes health. Indeed, exercise-induced alterations in the gut microbiome can influence health parameters crucial to athletic performance, specifically, immune function, lower susceptibility to infection, inflammatory response and tissue repair. Consequently, maintenance of a healthy gut microbiome is essential for an athlete’s health, training and performance. This review explores the effect of exercise on the microbiome while also investigating the effect of probiotics on various potential consequences associated with over-training in athletes, as well as their associated health benefits.

Bacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius , a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius . While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.

The generation of the antimicrobial peptide caseicin A (IKHQGLPQE) from a casein substrate using proteolytic enzymes was assessed in silico. This bioinformatic analysis predicted that thermolysin (EC 3.4.24.27) and thermolysin-like enzymes (EC 3.4.24.28) are likely candidates to liberate the bioactive peptide from αS1-casein. Commercially available sources of the thermolysin enzyme from industrial suppliers were subsequently shown to liberate the 1049 Da caseicin A peptide, under various conditions of hydrolysis, at both lab and pilot scale. The antimicrobial ability of the hydrolysates to reduce pathogen numbers spiked in infant formula trials was subsequently confirmed. For example, numbers of Cronobacter sakazakii were reduced 1000 fold after 3 h of incubation at 37 °C. In conclusion, this study demonstrates the potential to improve the safety of infant milk formula using milk-derived bioactive peptides.

Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG . A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI . Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions—Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys—-and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin.

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 draft genome sequence of Staphylococcus saprophyticus DPC5671, isolated from cheddar cheese, was determined. S. saprophyticus is a common Gram-positive bacterium detected on the surface of smear-ripened cheese and other fermented foods.

This work was supported by Teagasc, Ireland and in part by a research grant from Science Foundation Ireland (SFI) under the Grant Number SFI/16/BBSRC/3389 and the BBSRC under the Grant Reference BB/P009875/1 (KNN and JRS) and by SFI Grant Number SFI/12/RC/2273 (JFC and APC Microbiome Institute). peer-reviewed We tested the hypothesis that dietary whey protein isolate (WPI) affects the intestinal mechanisms related to energy absorption and that the resulting energy deficit is compensated by changes in energy balance to support growth. C57BL/6 mice were provided a diet enriched with WPI with varied sucrose content, and the impact on energy balance-related parameters was investigated. As part of a high-sucrose diet, WPI reduced the hypothalamic expression of pro-opiomelanocortin gene expression and increased energy intake. The energy expenditure was unaffected, but epididymal weight was reduced, indicating an energy loss. Notably, there was a reduction in the ileum gene expression for amino acid transporter SLC6a19, glucose transporter 2, and fatty acid transporter 4. The composition of the gut microbiota also changed, where Firmicutes were reduced. The above changes indicated reduced energy absorption through the intestine. We propose that this mobilized energy in the adipose tissue and caused hypothalamic changes that increased energy intake, acting to counteract the energy deficit arising in the intestine. Lowering the sucrose content in the WPI diet increased energy expenditure. This further reduced epididymal weight and plasma leptin, whereupon hypothalamic ghrelin gene expression and the intestinal weight were both increased. These data suggest that when the intestine-adipose-hypothalamic pathway is subjected to an additional energy loss (now in the adipose tissue), compensatory changes attempt to assimilate more energy. Notably, WPI and sucrose content interact to enable the component mechanisms of this pathway. BBSRC Science Foundation Ireland

The polymorphism of 5 stress response genes (hrcA, ctsR, clpP, ftsH, dnaK) in 32 Lactobacillus plantarum strains was evaluated by multilocus restriction typing (MLRT) and by sequence analysis of ctsR, hrcA and clpP genes. Both these approaches allowed the discrimination of the subspecies L. plantarum ssp. plantarum and L. plantarum ssp. argentoratensis. HrcA sequence analysis also allowed discrimination at the species and subspecies level of several species of lactic acid bacteria, thus confirming that it can be used as a valuable taxonomic marker. No significant relationship was found between stress response gene polymorphism and resistance to heat treatments. The effect of temperature on growth kinetics and the protein expression were investigated for selected strains carrying different mutations in hrcA. L. plantarum ssp. argentoratensis NCIMB12120 and L. plantarum ssp. plantarum DPC2159, both of which had mutations in domains of HrcA which are important for the repressor functionality, had a reduced growth rate at all temperatures tested (25, 30, 37, 40, and 42 °C) compared to L. plantarum WCFS1. In L. plantarum DPC2159, protein expression upon temperature shifts from 25 to 40 °C or growth at 40 °C was altered compared to L. plantarum WCFS1, but further study is needed to unequivocally confirm the relationship with mutations in hrcA.

peer-reviewed The human gut microbiota comprises approximately 100 trillion microbial cells and has a significant effect on many aspects of human physiology including metabolism, nutrient absorption and immune function. Disruption of this population has been implicated in many conditions and diseases, including examples such as obesity, inflammatory bowel disease and colorectal cancer that are highlighted in this review. A logical extension of these observations suggests that the manipulation of the gut microbiota can be employed to prevent or treat these conditions. Thus, here we highlight a variety of options, including the use of changes in diet (including the use of prebiotics), antimicrobial-based intervention, probiotics and faecal microbiota transplantation, and discuss their relative merits with respect to modulating the intestinal community in a beneficial way. C.J.W, C.M.G. and P.D.C are supported by a SFI PI award “Obesibiotics” (11/PI/1137)

The composition of the microbiota associated with the human ileum and colon in the early weeks of life of two preterm infants was examined, with particular emphasis on the Lactobacillus and Bifidobacterium members. Culturing work showed that bifidobacteria and lactobacilli in the ileostomy changed over time, compared with the colostomy effluent where there was far less variation. The colostomy infant was dominated by two phyla, Actinobacteria and Firmicutes, while in the ileostomy samples, Proteobacteria emerged at the expense of Actinobacteria. Bacteroidetes were only detected following the reversal of the ileostomy in the final fecal sample and were not detected in any colonic fluid samples. Clostridia levels were unstable in the colostomy fluid, suggesting that the ileostomy/colostomy itself influenced the gut microbiota, in particular the strict anaerobes. Pyrosequencing analysis of microbiota composition indicated that bifidobacteria and lactobacilli are among the dominant genera in both the ileal and colonic fluids. Bifidobacteria and lactobacilli levels were unstable in the ileostomy fluid, with large reductions in numbers and relative proportions of both observed. These decreases were characterized by an increase in proportions of Streptococcus and Enterobacteriaceae. Clostridium was detected only in the colonic effluent, with large changes in the relative proportions over time.

The diverse and dynamic microbiota of the gastrointestinal tract represents a vast source of bioactive substances. These include bacteriocins, which are antimicrobial peptides with the potential to modulate gut populations to impact positively on human health. Although several gut-derived bacteriocins have been isolated, there remain only a few exceptional studies in which their influence on microbial populations within the gut has been investigated. To facilitate such investigations, in vitro faecal fermentation systems can be used to simulate the anaerobic environment of the colon. In this instance, such a system was employed to explore the impact of bactofencin A, a novel broad spectrum class IId bacteriocin produced by gut isolates of Lactobacillus salivarius, on intestinal populations and overall microbial diversity. The study reveals that, although bactofencin A is a broad spectrum bacteriocin, it has a relatively subtle influence on intestinal communities, with a potentially positive impact on anaerobic populations such as Bacteroides, Clostridium and Bifidibacterium spp. The strategy taken is an important first step in investigating the merits of using bactofencin A to manipulate the gut microbiota in a beneficial way for health.

The objective of the study was to isolate potentialBacillus cereussensu lato (B.cereus s.l.)from a range of farm environments. Samples of tap water, milking equipment rinse water, milk sediment filter, grass, soil and bulk tank milk were collected from 63 farms. In addition, milk liners were swabbed at the start and the end of milking, and swabs were taken from cows’ teats prior to milking. The samples were plated on mannitol egg yolk polymyxin agar (MYP) and presumptiveB. cereus s.l. colonies were isolated and stored in nutrient broth with 20% glycerol and frozen at -80 °C. These isolates were then plated on chromogenic medium (BACARA) and colonies identified as presumptiveB. cereus s.l. on this medium were subjected to 16S ribosomal RNA (rRNA) sequencing. Of the 507 isolates presumed to beB. cereus s.l. on the basis of growth on MYP, only 177 showed growth typical ofB. cereus s.l. on BACARA agar. The use of 16S rRNA sequencing to identify isolates that grew on BACARA confirmed that the majority of isolates belonged toB. cereus s.l. A total of 81 of the 98 isolates sequenced were tentatively identified as presumptiveB. cereus s.l. Pulsed-field gel electrophoresis was carried out on milk and soil isolates from seven farms that were identified as having presumptiveB. cereus s.l. No pulsotype was shared by isolates from soil and milk on the same farm. PresumptiveB. cereus s.l. was widely distributed within the dairy farm environment.

The field of probiotic research has grown considerably in recent years. Despite in many cases a long history of use, putatively probiotic strains are now subject to extensive clinical and experimental analysis to establish efficacy and safety. There has recently been an ever-increasing reliance on genomic data from such strains to deepen our understanding of their beneficial attributes in addition to extracting important information with regard to their safe use. This chapter reviews the sequencing technologies currently available for providing these data and how this information can be harnessed while also highlighting challenges associated with sequencing annotation and the downstream analysis of data.

The infant gut microbiota undergoes dramatic changes during the first 2 years of life. The acquisition and development of this population can be influenced by numerous factors, and antibiotic treatment has been suggested as one of the most significant. Despite this, however, there have been relatively few studies which have investigated the short-term recovery of the infant gut microbiota following antibiotic treatment. The aim of this study was to use high-throughput sequencing (employing both 16S rRNA and rpoB -specific primers) and quantitative PCR to compare the gut microbiota of nine infants who underwent parenteral antibiotic treatment with ampicillin and gentamicin (within 48 h of birth), 4 and 8 weeks after the conclusion of treatment, relative to that of nine matched healthy controls. The investigation revealed that the gut microbiota of the antibiotic-treated infants had significantly higher proportions of Proteobacteria ( P = 0.0049) and significantly lower proportions of Actinobacteria ( P = 0.00001) (and the associated genus Bifidobacterium [ P = 0.0132]) as well as the genus Lactobacillus ( P = 0.0182) than the untreated controls 4 weeks after the cessation of treatment. By week 8, the Proteobacteria levels remained significantly higher in the treated infants ( P = 0.0049), but the Actinobacteria , Bifidobacterium , and Lactobacillus levels had recovered and were similar to those in the control samples. Despite this recovery of total Bifidobacterium numbers, rpoB -targeted pyrosequencing revealed that the number of different Bifidobacterium species present in the antibiotic-treated infants was reduced. It is thus apparent that the combined use of ampicillin and gentamicin in early life can have significant effects on the evolution of the infant gut microbiota, the long-term health implications of which remain unknown.

Aims: The aim of this study was to identify Bacillus isolates capable of degrading sodium caseinate and subsequently to generate bioactive peptides with antimicrobial activity. Methods and results: Sodium caseinate (2·5% w/v) was inoculated separately with 16 Bacillus isolates and allowed to ferment overnight. Protein breakdown in the fermentates was analysed using gel permeation-HPLC (GP-HPLC) and screened for peptides (

Summary Staphylococci adapt specifically to various animal hosts by genetically determined mechanisms that are not well understood. One such adaptation involves the ability to coagulate host plasma, by which strains isolated from ruminants or horses can be differentiated from closely related human strains. Here, we report first that this differential coagulation activity is due to animal-specific alleles of the von Willebrand factor-binding protein (vWbp) gene, vwb, and second that these vwb alleles are carried by highly mobile pathogenicity islands, SaPIs. Although all Staphylococcus aureus possess chromosomal vwb as well as coagulase (coa) genes, neither confers species-specific coagulation activity; however, the SaPI-coded vWbps possess a unique N-terminal region specific for the activation of ruminant and equine prothrombin. vWbp-encoding SaPIs are widely distributed among S. aureus strains infecting ruminant or equine hosts, and we have identified and characterized four of these, SaPIbov4, SaPIbov5, SaPIeq1 and SaPIov2, which encode vWbpSbo4, vWbpSbo5, vWbpSeq1 and vWbpSov2 respectively. Moreover, the SaPI-carried vwb genes are regulated differently from the chromosomal vwb genes of the same strains. We suggest that the SaPI-encoded vWbps may represent an important host adaptation mechanism for S. aureus pathogenicity, and therefore that acquisition of vWbp-encoding SaPIs may be determinative for animal specificity.

A common clone (ET3) of Staphylococcus aureus is responsible for a large proportion of cases of bovine mastitis and occasionally causes zoonotic infections of humans. In the present study, we report the identification of a virulent clonal subtype (ST151) of ET3, which resulted in increased tissue damage and mortality in a mouse model of mastitis. ST151 has undergone extensive diversification in virulence and regulatory‐gene content, including the acquisition of genetic elements encoding toxins not made by other ET3 strains. Furthermore, ST151 had elevated levels of RNAIII and cytolytic toxin–gene expression, consistent with the enhanced virulence observed during experimental infection. Previously, the ST151 clone was shown to be hypersusceptible to the acquisition of vancomycin‐resistance genes from Enterococcus spp. Taken together, these data indicate the emergence of a virulent subtype of the common ET3 clone, which could present an enhanced risk to public health. A common clone (ET3) of Staphylococcus aureus is responsible for a large proportion of cases of bovine mastitis and occasionally causes zoonotic infections of humans. In the present study, we report the identification of a virulent clonal subtype (ST151) of ET3, which resulted in increased tissue damage and mortality in a mouse model of mastitis. ST151 has undergone extensive diversification in virulence and regulatory‐gene content, including the acquisition of genetic elements encoding toxins not made by other ET3 strains. Furthermore, ST151 had elevated levels of RNAIII and cytolytic toxin–gene expression, consistent with the enhanced virulence observed during experimental infection. Previously, the ST151 clone was shown to be hypersusceptible to the acquisition of vancomycin‐resistance genes from Enterococcus spp. Taken together, these data indicate the emergence of a virulent subtype of the common ET3 clone, which could present an enhanced risk to public health. Teagasc

While the potential emergence of food spoilage and pathogenic bacteria with resistance to lantibiotics is a concern, the creation of derivatives of starter cultures and adjuncts that can grow in the presence of these antimicrobials may have applications in food fermentations. Here a bank of Lactococcus lactis IL1403 mutants was created and screened, and a number of novel genetic loci involved in lantibiotic resistance were identified.

peer-reviewed Background The human gut microbiota comprises approximately 100 trillion microbial cells which significantly impact many aspects of human physiology - including metabolism, nutrient absorption and immune function. Disturbances in this population have been implicated in many conditions and diseases, including obesity, type-2 diabetes and inflammatory bowel disease. This suggests that targeted manipulation or shaping of the gut microbiota, by bacteriocins and other antimicrobials, has potential as a therapeutic tool for the prevention or treatment of these conditions. With this in mind, several studies have used traditional culture-dependent approaches to successfully identify bacteriocin-producers from the mammalian gut. In silico-based approaches to identify novel gene clusters are now also being utilised to take advantage of the vast amount of data currently being generated by next generation sequencing technologies. In this study, we employed an in silico screening approach to mine potential bacteriocin clusters in genome-sequenced isolates from the gastrointestinal tract (GIT). More specifically, the bacteriocin genome-mining tool BAGEL3 was used to identify potential bacteriocin producers in the genomes of the GIT subset of the Human Microbiome Project’s reference genome database. Each of the identified gene clusters were manually annotated and potential bacteriocin-associated genes were evaluated. Results We identified 74 clusters of note from 59 unique members of the Firmicutes, Bacteroidetes, Actinobacteria, Fusobacteria and Synergistetes. The most commonly identified class of bacteriocin was the >10 kDa class, formerly known as bacteriolysins, followed by lantibiotics and sactipeptides. Conclusions Multiple bacteriocin gene clusters were identified in a dataset representative of the human gut microbiota. Interestingly, many of these were associated with species and genera which are not typically associated with bacteriocin production. CJW, CMG and PDC are supported by a SFI PI award to PDC “Obesibiotics” (11/PI/1137).

Exopolysaccharide-synthesizing Lactobacillus mucosae DPC 6426 is a heterofermentative strain, which has demonstrated cholesterol-lowering properties in an animal model of lipid-driven atherosclerosis. The genome revealed a plethora of homologues linked to carbohydrate metabolism and mucin binding.

The capacity of microbial pathogens to alter their host-tropism leading to epidemics in distinct\ud host-species populations is a global public and veterinary health concern. In order to investigate\ud the molecular basis of a bacterial host-switching event in a tractable host-species, we traced the\ud evolutionary trajectory of the common rabbit clone of Staphylococcus aureus. We report that it\ud evolved through a likely human-to-rabbit host jump over 40 years ago, and that only a single\ud natural nucleotide mutation was required and sufficient to convert a human-specific S. aureus strain into one which could infect rabbits. Related mutations were identified at the same locus in\ud other rabbit strains of distinct clonal origin, consistent with convergent evolution. This first report\ud of a single mutation that was sufficient to alter the host-tropism of a micro-organism during its\ud evolution highlights the capacity of some pathogens to readily expand into novel host-species\ud populations.

Seven open reading frames, annotated as potential penicillin-binding-protein-encoding genes (lmo0441, lmo0540, lmo1438, lmo1892, lmo2039, lmo2229, and lmo2754), were targeted for insertional mutagenesis in Listeria monocytogenes EGDe. These genes were found to contribute in various degrees to β-lactam resistance, cell morphology, or the virulence potential of this organism.

This review will discuss the potential for certain lactococcal bacteriocins to be used as tools for influencing food safety and quality. The focus will be on the potential use of a broad-spectrum bacteriocin, lacticin 3147, as a tool to control nonstarter lactic acid bacteria (NSLAB) in cheese, to inhibit pathogens in fermented and nonfermented foods and to extend the shelf life of certain products. Different strategies used to incorporate this bacteriocin into foods will also be described, as will possible problems that may arise when using a bacteriocin in foods. The narrow spectrum bacteriocins, lactococcin ABM (which actually corresponds to three bacteriocins, lactococcins A, B and M produced in a single strain) and the medium-spectrum bacteriocin lacticin 481, also have certain potential applications (albeit limited) in the food industry. In particular, these bacteriocins exhibit a bacteriolytic effect on target cells and so can be used for inducing lysis on sensitive cells during cheese manufacture which concomitantly can lead to accelerated flavour development.

The ability to manipulate chromosomally encoded genes is a fundamental biological tool for the analysis of gene function. Here, we provide in greater depth protocols for the creation of nonpolar unlabeled gene deletions in Listeria (L.) monocytogenes that are facilitated by the splicing overlap extension PCR technique. For mutagenesis in L. monocytogenes, we describe two different plasmid-based approaches, which facilitate the introduction of this spliced amplicon in place of the corresponding segment of chromosomal DNA: the pKSV7 system and the pORI280/pVE6007 system.

Background Listeria monocytogenes is a food-borne pathogen which is the causative agent of listeriosis and can be divided into three evolutionary lineages I, II and III. While all strains possess the well established virulence factors associated with the Listeria pathogenicity island I (LIPI-1), lineage I strains also possess an additional pathogenicity island designated LIPI-3 which encodes listeriolysin S (LLS), a post-translationally modified cytolytic peptide. Up until now, this pathogenicity island has been identified exclusively in a subset of lineage I isolates of the pathogen Listeria monocytogenes. Results In total 64 L. innocua strains were screened for the presence of LIPI-3. Here we report the identification of an intact LIPI-3 in 11 isolates of L. innocua and the remnants of the cluster in several others. Significantly, we can reveal that placing the L. innocua lls genes under the control of a constitutive promoter results in a haemolytic phenotype, confirming that the cluster is capable of encoding a functional haemolysin. Conclusions Although the presence of the LIPI-3 gene cluster is confined to lineage I isolates of L. monocytogenes, a corresponding gene cluster or its remnants have been identified in many L. innocua strains.

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.

peer-reviewed Controlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains. This work was supported by a grant from the Austrian Science Fund (FWF, http://www.fwf.ac.at/) to SSE (grant no. P22703‐B17), by the European Union funded integrated project BIOTRACER (contract no. 036272) under the 6th RTD framework and by the EU grant FP7‐KBBE‐2010‐4 (FOODSEG).

The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health., IMPORTANCE There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

Probiotics are “live microorganisms which, when consumed in adequate amounts, confer a health benefit to the host”. A number of attributes are highly sought after among these microorganisms, including immunomodulation, epithelial barrier maintenance, competitive exclusion, production of short-chain fatty acids, and bile salt metabolism. Bacteriocin production is also generally regarded as a probiotic trait, but it can be argued that, in contrast to other traits, it is often considered a feature that is desirable, rather than a key probiotic trait. As such, the true potential of these antimicrobials has yet to be realised.

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.

The Listeria monocytogenes LiaSR two-component system (2CS) encoded by lmo1021 and lmo1022 plays an important role in resistance to the food preservative nisin. A nonpolar deletion in the histidine kinase-encoding component (Δ liaS ) resulted in a 4-fold increase in nisin resistance. In contrast, the Δ liaS strain exhibited increased sensitivity to a number of cephalosporin antibiotics (and was also altered with respect to its response to a variety of other antimicrobials, including the active agents of a number of disinfectants). This pattern of increased nisin resistance and reduced cephalosporin resistance in L. monocytogenes has previously been associated with mutation of a second histidine kinase, LisK, which is a predicted regulator of liaS and a penicillin binding protein encoded by lmo2229 . We noted that lmo2229 transcription is increased in the Δ liaS mutant and in a Δ liaS Δ lisK double mutant and that disruption of lmo2229 in the Δ liaS Δ lisK mutant resulted in a dramatic sensitization to nisin but had a relatively minor impact on cephalosporin resistance. We anticipate that further efforts to unravel the complex mechanisms by which LiaSR impacts on the antimicrobial resistance of L. monocytogenes could facilitate the development of strategies to increase the susceptibility of the pathogen to these agents.

The draft genome of Bifidobacterium breve DPC 6330, isolated from an elderly patient, was determined. B. breve DPC 6330 was previously identified to synthesize the beneficial metabolite conjugated linoleic acid from free linoleic acid. The sequence will allow identification and characterization of the genetic determinants of its putative beneficial properties.

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone., Author Summary Staphylococcus aureus is a global pathogen, responsible for an array of different illnesses in humans and animals. In particular, community-associated methicillin-resistant S. aureus (CA-MRSA) strains of the pandemic USA300 clone have the capacity to cause lethal human necrotizing pneumonia, but the molecular basis for the enhanced virulence remains unclear. Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in severe systemic illnesses such as toxic shock syndrome (TSS). However, all S. aureus SAgs identified to date are encoded by mobile genetic elements found only in a proportion of clinical isolates. Here, we report the discovery of a unique core genome-encoded SAg (SElX) which was acquired by an ancestor of the S. aureus species and which has undergone genetic and functional diversification in pathogenic clones infecting humans and animals. Importantly, we report that SElX made by pandemic USA300 contributes to lethality in a rabbit model of human necrotizing pneumonia revealing a novel virulence determinant of severe CA-MRSA infection.