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1.  A review of the systematic review process and its applicability for use in evaluating evidence for health claims on probiotic foods in the European Union 
Nutrition Journal  2015;14:16.
This paper addresses the use of systematic review and meta-analysis to evaluate the strength of evidence for health benefits of probiotic foods, especially relating to health claim substantiation in the European Union. A systematic review is a protocol-driven, transparent and replicable approach, widely accepted in a number of scientific fields, and used by many policy-setting organizations to evaluate the strength of evidence to answer a focused research question. Many systematic reviews have been published on the broad category of probiotics for many different outcomes. Some of these reviews have been criticized for including poor quality studies, pooling heterogeneous study results, and not considering publication bias. Well-designed and -conducted systematic reviews should address such issues. Systematic reviews of probiotics have an additional challenge – rarely addressed in published reviews - in that there must be a scientifically sound basis for combining evidence on different strains, species or genera. The European Food Safety Authority (EFSA) is increasingly adopting the systematic review methodology. It remains to be seen how health claims supported by systematic reviews are evaluated within the EFSA approval process. The EFSA Panel on Dietetic Products, Nutrition and Allergies deems randomized trials to be the best approach to generating evidence about the effects of foods on health outcomes. They also acknowledge that systematic reviews (with or without meta-analyses) are the best approach to assess the totality of the evidence. It is reasonable to use these well-established methods to assess objectively the strength of evidence for a probiotic health claim. Use of the methods to combine results on more than a single strain or defined blend of strains will require a rationale that the different probiotics are substantively similar, either in identity or in their mode of action.
doi:10.1186/s12937-015-0004-5
PMCID: PMC4334596
Systematic reviews; Meta-analysis; Probiotics; EFSA; Regulatory; Health claims
2.  Heterologous Expression of Thuricin CD Immunity Genes in Listeria monocytogenes 
Bacteriocins are ribosomally synthesized peptides that can have a narrow or broad spectrum of antimicrobial activity. Bacteriocin producers typically possess dedicated immunity systems that often consist of an ATP-binding cassette (ABC) transporter system and/or a dedicated immunity protein. Here we investigated the genes responsible for immunity to thuricin CD, a narrow-spectrum two-peptide sactibiotic produced by Bacillus thuringiensis DPC6431. Heterologous expression of putative thuricin CD immunity determinants allowed us to identify and investigate the relative importance of the individual genes and gene products that contribute to thuricin CD immunity. We established that TrnF and TrnG are the individual components of an ABC transporter system that provides immunity to thuricin CD. We also identified a hitherto overlooked open reading frame located upstream of trnF predicted to encode a 79-amino-acid transmembrane protein. We designated this newly discovered gene trnI and established that TrnI alone can provide protection against thuricin CD.
doi:10.1128/AAC.00090-14
PMCID: PMC4068476  PMID: 24709257
3.  Divergent Evolution of the Activity and Regulation of the Glutamate Decarboxylase Systems in Listeria monocytogenes EGD-e and 10403S: Roles in Virulence and Acid Tolerance 
PLoS ONE  2014;9(11):e112649.
The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains.
doi:10.1371/journal.pone.0112649
PMCID: PMC4227838  PMID: 25386947
4.  Metagenomic Identification of a Novel Salt Tolerance Gene from the Human Gut Microbiome Which Encodes a Membrane Protein with Homology to a brp/blh-Family β-Carotene 15,15′-Monooxygenase 
PLoS ONE  2014;9(7):e103318.
The human gut microbiome consists of at least 3 million non-redundant genes, 150 times that of the core human genome. Herein, we report the identification and characterisation of a novel stress tolerance gene from the human gut metagenome. The locus, assigned brpA, encodes a membrane protein with homology to a brp/blh-family β-carotene monooxygenase. Cloning and heterologous expression of brpA in Escherichia coli confers a significant salt tolerance phenotype. Furthermore, when cultured in the presence of exogenous β-carotene, cell pellets adopt a red/orange pigmentation indicating the incorporation of carotenoids in the cell membrane.
doi:10.1371/journal.pone.0103318
PMCID: PMC4110020  PMID: 25058308
5.  Rethinking “probiotics” 
Gut Microbes  2013;4(4):269-270.
doi:10.4161/gmic.25143
PMCID: PMC3744511  PMID: 23778363
probiotic; meta-analysis; regulatory; EFSA; health effects
6.  Combined metagenomic and phenomic approaches identify a novel salt tolerance gene from the human gut microbiome 
In the current study, a number of salt-tolerant clones previously isolated from a human gut metagenomic library were screened using Phenotype MicroArray (PM) technology to assess their functional capacity. PM's can be used to study gene function, pathogenicity, metabolic capacity and identify drug targets using a series of specialized microtitre plate assays, where each well of the microtitre plate contains a different set of conditions and tests a different phenotype. Cellular respiration is monitored colorimetrically by the reduction of a tetrazolium dye. One clone, SMG 9, was found to be positive for utilization/transport of L-carnitine (a well-characterized osmoprotectant) in the presence of 6% w/v sodium chloride (NaCl). Subsequent experiments revealed a significant growth advantage in minimal media containing NaCl and L-carnitine. Fosmid sequencing revealed putative candidate genes responsible for the phenotype. Subsequent cloning of two genes did not replicate the L-carnitine-associated phenotype, although one of the genes, a σ54-dependent transcriptional regulator, did confer salt tolerance to Escherichia coli when expressed in isolation. The original clone, SMG 9, was subsequently found to have lost the original observed phenotype upon further investigation. Nevertheless, this study demonstrates the usefulness of a phenomic approach to assign a functional role to metagenome-derived clones.
doi:10.3389/fmicb.2014.00189
PMCID: PMC4010731  PMID: 24808895
metagenomics; functional metagenomics; gut microbiome; microbiota; salt tolerance; BIOLOG; phenotype microarray; transcriptional regulator
7.  Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature 
Natural product reports  2013;30(1):108-160.
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
doi:10.1039/c2np20085f
PMCID: PMC3954855  PMID: 23165928
8.  Atypical Listeria innocua strains possess an intact LIPI-3 
BMC Microbiology  2014;14:58.
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.
doi:10.1186/1471-2180-14-58
PMCID: PMC3974016  PMID: 24606727
9.  Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207) 
Applied and Environmental Microbiology  2013;79(17):5179-5185.
Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.
doi:10.1128/AEM.01394-13
PMCID: PMC3753947  PMID: 23793637
10.  Streptolysin S-like virulence factors: the continuing sagA 
Nature reviews. Microbiology  2011;9(9):670-681.
Streptolysin S (SLS) is a potent cytolytic toxin and virulence factor produced by nearly all Streptococcus pyogenes strains. Despite a 100-year history of research on this toxin, it has only recently been established that SLS represents the archetypal example of an extended family of post-translationally modified virulence factors also produced by some other streptococci and Gram-positive pathogens, such as Listeria monocytogenes and Clostridium botulinum. In this Review we describe the identification, genetics, biochemistry and various functions of SLS. We also discuss the shared features of the virulence-associated SLS-like peptides, as well as their place within the rapidly expanding family of thiazole/oxazole-modified microcins (TOMMs).
doi:10.1038/nrmicro2624
PMCID: PMC3928602  PMID: 21822292
11.  Effects of Combined Radiation and Burn injury on the Renin-Angiotensin System 
The renin angiotensin system (RAS) plays an important role in wound repair; however, little is known pertaining to RAS expression in response to thermal and the combination of radiation plus burn injury (CRBI). The purpose of this study was to test the hypothesis that thermal injury modifies expression of RAS components and CRBI delayed this up-regulation of RAS. Skin from uninjured mice was compared to mice receiving local thermal injury or CRBI (injury site). Skin was analyzed for gene and protein expression of RAS components. There was an initial increase in the expression of various components of RAS following thermal injury. However, in the higher CRBI group there is an initial decrease in AT1b (vasoconstriction, pro-proliferative) AT2 (vasodilation, differentiation) and Mas (vasodilation, anti-inflammatory) gene expression. This corresponded with a delay and decrease in AT1, AT2 and MAS protein expression in fibroblasts and keratinocytes. The reduction in RAS receptor positive fibroblasts and keratinocytes correlated with a reduction in collagen deposition and keratinocyte infiltration into the wounded area resulting in a delay of re-epithelialization following CRBI. These data support the hypothesis that delayed wound healing observed in subjects following radiation exposure may be in part due to decreased expression of RAS.
doi:10.1111/j.1524-475X.2012.00867.x
PMCID: PMC3540151  PMID: 23231670
angiotensin; combined radiation and burn injury; wound healing
12.  Movers and shakers 
Gut Microbes  2013;4(1):4-16.
The human intestinal microbiota is one of the most densely populated ecosystems on Earth, containing up to 1013 bacteria/g and in some respects can be considered an organ itself given its role in human health. Bacteriophages (phages) are the most abundant replicating entities on the planet and thrive wherever their bacterial hosts exist. They undoubtedly influence the dominant microbial populations in many ecosystems including the human intestine. Within this setting, lysogeny appears to be the preferred life cycle, presumably due to nutrient limitations and lack of suitable hosts protected in biofilms, hence the predator/prey dynamic observed in many ecosystems is absent. On the other hand, free virulent phages in the gut are more common among sufferers of intestinal diseases and have been shown to increase with antibiotic usage. Many of these phages evolve from prophages of intestinal bacteria and emerge under conditions where their bacterial hosts encounter stress suggesting that prophages can significantly alter the microbial community composition. Based on these observations, we propose the “community shuffling” model which hypothesizes that prophage induction contributes to intestinal dysbiosis by altering the ratio of symbionts to pathobionts, enabling pathobiont niche reoccupation. The consequences of the increased phage load on the mammalian immune system are also addressed. While this is an area of intestinal biology which has received little attention, this review assembles evidence from the literature which supports the role of phages as one of the biological drivers behind the composition of the gut microbiota.
doi:10.4161/gmic.22371
PMCID: PMC3555884  PMID: 23022738
gut; microbiota; bacteriophages; phages; prophages; induction; community shuffling
13.  Antimicrobials 
Gut Microbes  2013;4(1):48-53.
Obesity is associated with a number of serious health consequences, including type 2 diabetes, cardiovascular disease and a variety of cancers among others and has been repeatedly shown to be associated with a higher risk of mortality. The relatively recent discovery that the composition and metabolic activity of the gut microbiota may affect the risk of developing obesity and related disorders has led to an explosion of interest in this distinct research field. A corollary of these findings would suggest that modulation of gut microbial populations can have beneficial effects with respect to controlling obesity. In this addendum, we summarize our recent data, showing that therapeutic manipulation of the microbiota using different antimicrobial strategies may be a useful approach for the management of obesity and metabolic conditions. In addition, we will explore some of the mechanisms that may contribute to microbiota-induced susceptibility to obesity and metabolic diseases.
doi:10.4161/gmic.22328
PMCID: PMC3555886  PMID: 23018760
obesity; antimicrobials; gut microbiota; firmicutes; metabolic disease
14.  In Vivo Assessment of Growth and Virulence Gene Expression during Commensal and Pathogenic Lifestyles of luxABCDE-Tagged Enterococcus faecalis Strains in Murine Gastrointestinal and Intravenous Infection Models 
Applied and Environmental Microbiology  2013;79(13):3986-3997.
Cytolysin and gelatinase are prominent pathogenicity determinants associated with highly virulent Enterococcus faecalis strains. In an effort to explore the expression profiles of these virulence traits in vivo, we have employed E. faecalis variants expressing the luxABCDE cassette under the control of either the P16S, cytolysin, or gelatinase promoter for infections of Galleria mellonella caterpillars and mice. Systemic infection of G. mellonella with bioluminescence-tagged E. faecalis MMH594 revealed temporal regulation of both gelatinase and cytolysin promoters and demonstrated that these traits were induced in response to the host environment. Gavage of mice pretreated perorally with antibiotics resulted in efficient colonization of the murine gastrointestinal tract (GIT) in a strain-dependent manner, where the commensal baby isolate EF62 was more persistent than the nosocomial isolate MMH594. A highly significant correlation (R2 > 0.94) was found between bioluminescence and the CFU counts in mouse fecal samples. Both strains showed similar preferences for growth and persistence in the ileum, cecum, and colon. Cytolysin expression was uniform in these compartments of the intestinal lumen. In spite of high numbers (109 CFU/g of intestinal matter) in the ileum, cecum, and colon, no evidence of translocation or systemic infection could be observed. In the murine intravenous infection model, cytolysin expression was readily detected in the liver, kidneys, and bladder. At 72 h postinfection, the highest bacterial loads were found in the liver, kidneys, and spleen, with organ-specific expression levels of cytolysin ∼400- and ∼900-fold higher in the spleen and heart, respectively, than in the liver and kidneys. Taken together, this system based on the bioluminescence imaging technology is established as a new, powerful method to monitor the differential regulation of E. faecalis virulence determinants and to study the spatiotemporal course of infection in living animals in real time.
doi:10.1128/AEM.00831-13
PMCID: PMC3697570  PMID: 23603680
15.  Listeria monocytogenes: survival and adaptation in the gastrointestinal tract 
The foodborne pathogen Listeria monocytogenes has the capacity to survive and grow in a diverse range of natural environments. The transition from a food environment to the gastrointestinal tract begins a process of adaptation that may culminate in invasive systemic disease. Here we describe recent advances in our understanding of how L. monocytogenes adapts to the gastrointestinal environment prior to initiating systemic infection. We will discuss mechanisms used by the pathogen to survive encounters with acidic environments (which include the glutamate decarboxylase and arginine deiminase systems), and those which enable the organism to cope with bile acids (including bile salt hydrolase) and competition with the resident microbiota. An increased understanding of how the pathogen survives in this environment is likely to inform the future design of novel prophylactic approaches that exploit specific pharmabiotics; including probiotics, prebiotics, or phages.
doi:10.3389/fcimb.2014.00009
PMCID: PMC3913888  PMID: 24551601
Listeria; stress; acid; bile; gastrointestinal; virulence; pathogenesis; infection
16.  Functional Environmental Screening of a Metagenomic Library Identifies stlA; A Unique Salt Tolerance Locus from the Human Gut Microbiome  
PLoS ONE  2013;8(12):e82985.
Functional environmental screening of metagenomic libraries is a powerful means to identify and assign function to novel genes and their encoded proteins without any prior sequence knowledge. In the current study we describe the identification and subsequent analysis of a salt-tolerant clone from a human gut metagenomic library. Following transposon mutagenesis we identified an unknown gene (stlA, for “salt tolerance locus A”) with no current known homologues in the databases. Subsequent cloning and expression in Escherichia coli MKH13 revealed that stlA confers a salt tolerance phenotype in its surrogate host. Furthermore, a detailed in silico analysis was also conducted to gain additional information on the properties of the encoded StlA protein. The stlA gene is rare when searched against human metagenome datasets such as MetaHit and the Human Microbiome Project and represents a novel and unique salt tolerance determinant which appears to be found exclusively in the human gut environment.
doi:10.1371/journal.pone.0082985
PMCID: PMC3861447  PMID: 24349412
17.  A method for extracting high-quality RNA from diverse plants for next-generation sequencing and gene expression analyses1 
Applications in Plant Sciences  2013;1(12):apps.1300070.
• Premise of the study: To study gene expression in plants, high-quality RNA must be extracted in quantities sufficient for subsequent cDNA library construction. Field-based collections are often limited in quantity and quality of tissue and are typically preserved in RNAlater. Obtaining sufficient and high-quality yield from variously preserved samples is essential to studies of comparative biology. We present a protocol for the extraction of high-quality RNA from even the most recalcitrant plant tissues.
• Methods and Results: Tissues from mosses, cycads, and angiosperm floral organs and leaves were preserved in RNAlater or frozen fresh at −80°C. Extractions were performed and quality was measured for yield and purity.
• Conclusions: This protocol results in the extraction of high-quality RNA from a variety of plant tissues representing vascular and nonvascular plants. RNA was used for cDNA synthesis to generate libraries for next-generation sequencing and for expression studies using quantitative PCR (qPCR) and semiquantitative reverse transcription PCR (RT-PCR).
doi:10.3732/apps.1300070
PMCID: PMC4103122  PMID: 25202509
cDNA library; gene expression; Illumina; RNA-Seq; RNA extraction; transcriptome
18.  Analysis of Anti-Clostridium difficile Activity of Thuricin CD, Vancomycin, Metronidazole, Ramoplanin, and Actagardine, both Singly and in Paired Combinations 
Due to the ongoing problem of recurrence of Clostridium difficile-associated diarrhea following antibiotic treatment, there is an urgent need for alternative treatment options. We assessed the MICs of five antimicrobials singly and in combinations against a range of C. difficile clinical isolates. Ramoplanin-actagardine combinations were particularly effective, with partial synergistic/additive effects observed against 61.5% of C. difficile strains tested.
doi:10.1128/AAC.00261-13
PMCID: PMC3716125  PMID: 23571539
19.  Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives 
PLoS ONE  2013;8(11):e79563.
Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.
doi:10.1371/journal.pone.0079563
PMCID: PMC3823697  PMID: 24244524
20.  Bactofencin A, a New Type of Cationic Bacteriocin with Unusual Immunity 
mBio  2013;4(6):e00498-13.
ABSTRACT
Bacteriocin production is an important probiotic trait of intestinal bacteria. In this study, we identify a new type of bacteriocin, bactofencin A, produced by a porcine intestinal isolate Lactobacillus salivarius DPC6502, and assess its potency against pathogenic species including Staphylococcus aureus and Listeria monocytogenes. Genome sequencing of the bacteriocin producer revealed bfnA, which encodes the mature and highly basic (pI 10.59), 22-amino-acid defensin-like peptide. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectral analysis determined that bactofencin A has a molecular mass of 2,782 Da and contains two cysteine residues that form an intramolecular disulfide bond. Although an ABC transporter and transport accessory protein were also present within the bacteriocin gene cluster, a classical bacteriocin immunity gene was not detected. Interestingly, a dltB homologue was identified downstream of bfnA. DltB is usually encoded within the dlt operon of many Gram-positive bacteria. It is responsible for d-alanylation of teichoic acids in the cell wall and has previously been associated with bacterial resistance to cationic antimicrobial peptides. Heterologous expression of this gene conferred bactofencin A-specific immunity on sensitive strains of L. salivarius and S. aureus (although not L. monocytogenes), establishing its role in bacteriocin immunity. An analysis of the distribution of bfnA revealed that it was present in four additional isolates derived from porcine origin and absent from five human isolates, suggesting that its distribution is host specific. Given its novelty, we anticipate that bactofencin A represents the prototype of a new class of bacteriocins characterized as being cationic, with a DltB homologue providing a cognate immunity function.
IMPORTANCE
This study describes the identification, purification, and characterization of bactofencin A, a novel type of bacteriocin produced by L. salivarius DPC6502. Interestingly, bactofencin A is not similar to any other known bacteriocin but instead shares similarity with eukaryotic cationic antimicrobial peptides, and here, we demonstrate that it inhibits two medically significant pathogens. Genome sequence analysis of the producing strain also revealed the presence of an atypical dltB homologue in the bacteriocin gene cluster, which was lacking a classical bacteriocin immunity gene. Furthermore, cloning this gene rendered sensitive strains resistant to the bacteriocin, thereby establishing its role in providing cognate bacteriocin immunity. Four additional L. salivarius isolates, also of porcine origin, were found to contain the bacteriocin biosynthesis genes and successfully produced bactofencin A, while these genes were absent from five human-derived strains investigated.
doi:10.1128/mBio.00498-13
PMCID: PMC3809560  PMID: 24169573
21.  The Effect of Dietary Supplementation with Spent Cider Yeast on the Swine Distal Gut Microbiome 
PLoS ONE  2013;8(10):e75714.
Background
There is an increasing need for alternatives to antibiotics for promoting animal health, given the increasing problems associated with antibiotic resistance. In this regard, we evaluated spent cider yeast as a potential probiotic for modifying the gut microbiota in weanling pigs using pyrosequencing of 16S rRNA gene libraries.
Methodology and Principal Findings
Piglets aged 24–26 days were assigned to one of two study groups; control (n = 12) and treatment (n = 12). The control animals were fed with a basal diet and the treatment animals were fed with basal diet in combination with cider yeast supplement (500 ml cider yeast containing ∼7.6 log CFU/ml) for 21 days. Faecal samples were collected for 16s rRNA gene compositional analysis. 16S rRNA compositional sequencing analysis of the faecal samples collected from day 0 and day 21 revealed marked differences in microbial diversity at both the phylum and genus levels between the control and treatment groups. This analysis confirmed that levels of Salmonella and Escherichia were significantly decreased in the treatment group, compared with the control (P<0.001). This data suggest a positive influence of dietary supplementation with live cider yeast on the microbial diversity of the pig distal gut.
Conclusions/Significance
The effect of dietary cider yeast on porcine gut microbial communities was characterized for the first time using 16S rRNA gene compositional sequencing. Dietary cider yeast can potentially alter the gut microbiota, however such changes depend on their endogenous microbiota that causes a divergence in relative response to that given diet.
doi:10.1371/journal.pone.0075714
PMCID: PMC3794030  PMID: 24130736
22.  Functional metagenomics reveals novel salt tolerance loci from the human gut microbiome 
The ISME Journal  2012;6(10):1916-1925.
Metagenomics is a powerful tool that allows for the culture-independent analysis of complex microbial communities. One of the most complex and dense microbial ecosystems known is that of the human distal colon, with cell densities reaching up to 1012 per gram of faeces. With the majority of species as yet uncultured, there are an enormous number of novel genes awaiting discovery. In the current study, we conducted a functional screen of a metagenomic library of the human gut microbiota for potential salt-tolerant clones. Using transposon mutagenesis, three genes were identified from a single clone exhibiting high levels of identity to a species from the genus Collinsella (closest relative being Collinsella aerofaciens) (COLAER_01955, COLAER_01957 and COLAER_01981), a high G+C, Gram-positive member of the Actinobacteria commonly found in the human gut. The encoded proteins exhibit a strong similarity to GalE, MurB and MazG. Furthermore, pyrosequencing and bioinformatic analysis of two additional fosmid clones revealed the presence of an additional galE and mazG gene, with the highest level of genetic identity to Akkermansia muciniphila and Eggerthella sp. YY7918, respectively. Cloning and heterologous expression of the genes in the osmosensitive strain, Escherichia coli MKH13, resulted in increased salt tolerance of the transformed cells. It is hoped that the identification of atypical salt tolerance genes will help to further elucidate novel salt tolerance mechanisms, and will assist our increased understanding how resident bacteria cope with the osmolarity of the gastrointestinal tract.
doi:10.1038/ismej.2012.38
PMCID: PMC3446828  PMID: 22534607
Akkermansia ;  Collinsella ;  Eggerthella ; human gut microbiome; metagenomics; salt tolerance
23.  The two peptide lantibiotic lacticin 3147 acts synergistically with polymyxin to inhibit Gram negative bacteria 
BMC Microbiology  2013;13:212.
Background
The emergence of bacterial drug resistance encourages the re-evaluation of the potential of existing antimicrobials. Lantibiotics are post-translationally modified, ribosomally synthesised antimicrobial peptides with a broad spectrum antimicrobial activity. Here, we focussed on expanding the potential of lacticin 3147, one of the most studied lantibiotics and one which possesses potent activity against a wide range of Gram positive species including many nosocomial pathogens. More specifically, our aim was to investigate if lacticin 3147 activity could be enhanced when combined with a range of different clinical antibiotics.
Results
Initial screening revealed that polymyxin B and polymyxin E (colistin) exhibited synergistic activity with lacticin 3147. Checkerboard assays were performed against a number of strains, including both Gram positive and Gram negative species. The resultant fractional inhibitory concentration (FIC) index values established that, while partial synergy was detected against Gram positive targets, synergy was obvious against Gram negative species, including Cronobacter and E. coli.
Conclusions
Combining lacticin 3147 with low levels of a polymyxin could provide a means of broadening target specificity of the lantibiotic, while also reducing polymyxin use due to the lower concentrations required as a result of synergy.
doi:10.1186/1471-2180-13-212
PMCID: PMC3849175  PMID: 24069959
Antimicrobial; Synergy; Lantibiotic; Bacteriocin; Lacticin 3147; Polymyxin
24.  Subspecies diversity in bacteriocin production by intestinal Lactobacillus salivarius strains 
Gut Microbes  2012;3(5):468-473.
A recent comparative genomic hybridization study in our laboratory revealed considerable plasticity within the bacteriocin locus of gastrointestinal strains of Lactobacillus salivarius. Most notably, these analyses led to the identification of two novel unmodified bacteriocins, salivaricin L and salivaricin T, produced by the neonatal isolate L. salivarius DPC6488 with immunity, regulatory and export systems analogous to those of abp118, a two-component bacteriocin produced by the well characterized reference strain L. salivarius UCC118. In this addendum we discuss the intraspecific diversity of our seven bacteriocin-producing L. salivarius isolates on a genome-wide level, and more specifically, with respect to their salivaricin loci.
doi:10.4161/gmic.21417
PMCID: PMC3466502  PMID: 22892690
Lactobacillus salivarius; bacteriocin; comparative genomic hybridization; probiotic; salivaricin
25.  Selection for Loss of RpoS in Cronobacter sakazakii by Growth in the Presence of Acetate as a Carbon Source 
We demonstrate that growth of Cronobacter sakazakii in the presence of acetate as a carbon source promotes loss of RpoS, with a consequent reduction in stress tolerance. This suggests that C. sakazakii is capable of regulating cell fitness through mutation of the rpoS gene.
doi:10.1128/AEM.03302-12
PMCID: PMC3592228  PMID: 23335773

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