Sarah O'Flaherty

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples.

Regulation of induced colonic inflammation by Lactobacillus acidophilus deficient in lipoteichoic acid

Imbalance in the regulatory immune mechanisms that control intestinal cellular and bacterial homeostasis may lead to induction of the detrimental inflammatory signals characterized in humans as inflammatory bowel disease. Induction of proinflammatory cytokines (i.e., IL-12) induced by dendritic cells (DCs) expressing pattern recognition receptors may skew naive T cells to T helper 1 polarization, which is strongly implicated in mucosal autoimmunity. Recent studies show the ability of probiotic microbes to treat and prevent numerous intestinal disorders, including Clostridium difficile-induced colitis. To study the molecular mechanisms involved in the induction and repression of intestinal inflammation, the phosphoglycerol transferase gene that plays a key role in lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus NCFM (NCK56) was deleted. The data show that the L. acidophilus LTA-negative in LTA (NCK2025) not only down-regulated IL-12 and TNFα but also significantly enhanced IL-10 in DCs and controlled the regulation of costimulatory DC functions, resulting in their inability to induce CD4+ T-cell activation. Moreover, treatment of mice with NCK2025 compared with NCK56 significantly mitigated dextran sulfate sodium and CD4+CD45RBhighT cell-induced colitis and effectively ameliorated dextran sulfate sodium-established colitis through a mechanism that involves IL-10 and CD4+FoxP3+ T regulatory cells to dampen exaggerated mucosal inflammation. Directed alteration of cell surface components of L. acidophilus NCFM establishes a potential strategy for the treatment of inflammatory intestinal disorders.

Bacteriophage and their lysins for elimination of infectious bacteria

When phages were originally identified, the possibility of using them as antibacterial agents against pathogens was immediately recognized and put into practise based on the knowledge available at the time. However, with the advent of antibiotics a decline in the use of phage as therapeutics followed. Phages did, however, become more useful in the study of fundamental aspects of molecular biology and in the diagnostic laboratory for the identification of pathogenic bacteria. More recently, the original application of phage as therapeutics to treat human and animal infections has been rekindled, particularly in an era where antibiotic resistance has become so problematic/commonplace. Phage lysins have also been studied and utilized in their own right as potential therapeutics for the treatment of bacterial infections. Indeed the past decade has seen a considerable amount of research worldwide focused on the engineering of phages as antibacterial agents in a wide range of applications. Furthermore, the US Food and Drug Administration and/or the US Department of Agriculture have recently approved commercial phage preparations to prevent bacterial contamination of livestock, food crops, meat and other foods. Such developments have prompted this review into the status of phage research as it pertains to the control of infectious bacteria.

Potential of the Polyvalent Anti-Staphylococcus Bacteriophage K for Control of Antibiotic-Resistant Staphylococci from Hospitals

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

Genome of Staphylococcal Phage K: a New Lineage of Myoviridae Infecting Gram-Positive Bacteria with a Low G+C Content

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

The Recombinant Phage Lysin LysK Has a Broad Spectrum of Lytic Activity against Clinically Relevant Staphylococci, Including Methicillin-Resistant Staphylococcus aureus

This study concerns the cloning, characterization, and expression of the lysin (LysK) from staphylococcal phage K in Lactococcus lactis. Lactococcal lysates containing recombinant LysK were found to inhibit a range of different species of staphylococci isolated from bovine and human infection sources, including methicillin-resistant Staphylococcus aureus. LysK thus has potential as an antimicrobial for applications in the prevention and/or treatment of infections caused by staphylococci.

Analysis of the Genome Sequence of Lactobacillus gasseri ATCC 33323 Reveals the Molecular Basis of an Autochthonous Intestinal Organism

ABSTRACT This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.

Development and Application of a upp-Based Counterselective Gene Replacement System for the Study of the S-Layer Protein SlpX of Lactobacillus acidophilus NCFM

ABSTRACT In silico genome analysis of Lactobacillus acidophilus NCFM coupled with gene expression studies have identified putative genes and regulatory networks that are potentially important to this organisms survival, persistence, and activities in the gastrointestinal tract. Correlation of key genotypes to phenotypes requires an efficient gene replacement system. In this study, use of the upp-encoded uracil phosphoribosyltransferase (UPRTase) of L. acidophilus NCFM was explored as a counterselection marker to positively select for recombinants that have resolved from chromosomal integration of pORI-based plasmids. An isogenic mutant carrying a upp gene deletion was constructed and was resistant to 5-fluorouracil (5-FU), a toxic uracil analog that is also a substrate for UPRTase. A 3.0-kb pORI-based counterselectable integration vector bearing a upp expression cassette, pTRK935, was constructed and introduced into the Δupp host harboring the pTRK669 helper plasmid. Extrachromosomal replication of pTRK935 complemented the mutated chromosomal upp allele and restored sensitivity to 5-FU. This host background provides a platform for a two-step plasmid integration and excision strategy that can select for plasmid-free recombinants with either the wild-type or mutated allele of the targeted gene in the presence of 5-FU. The efficacy of the system was demonstrated by in-frame deletion of the slpX gene (LBA0512) encoding a novel 51-kDa secreted protein associated with the S-layer complex of L. acidophilus. The resulting ΔslpX mutant exhibited lower growth rates, increased sensitivity to sodium dodecyl sulfate, and greater resistance to bile. Overall, this improved gene replacement system represents a valuable tool for investigating the mechanisms underlying the probiotic functionality of L. acidophilus.

Inhibition of bacteriophage K proliferation on Staphylococcus aureus in raw bovine milk

Aims:  To assess the ability of staphylococcal bacteriophage K to inhibit Staphylococcus aureus in raw milk.

Functional Genomics of Probiotic Lactobacilli

Lactic acid bacteria (LAB) have been used in fermentation processes for millennia. Recent applications such as the use of living cultures as probiotics have significantly increased industrial interest. Related bacterial strains can differ significantly in their genotype and phenotype, and features from one bacterial strain or species cannot necessarily be applied to a related one. These strain or family-specific differences often represent unique and applicable traits. Since 2002, the complete genomes of 13 probiotic LABs have been published. The presentation will discuss these genomes and highlight probiotic traits that are predicted, or functionally linked to genetic content. We have conducted a comparative genomic analysis of 4 completely sequenced Lactobacillus strains versus 25 lactic acid bacterial genomes present in the public database at thetime of analysis. Using Differential Blast Analysis, each genome is compared with 3 other Lactobacillus and 25 other LAB genomes. Differential Blast Analysis highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Lactobacillus-specific genes include mucus-binding proteins involved in cell-adhesion and several transport systems for carbohydrates and amino acids. Comparative genomic analysis has identified gene targets in Lactobacillus acidophilus for functional analysis, including adhesion to mucin and intestinal epithelial cells, acid tolerance, bile tolerance, and quorum sensing. Whole genome transcriptional profiling of L. acidophilus, and isogenic mutants thereof, has revealed the impact of varying conditions (pH, bile, carbohydrates) and food matrices on the expression of genes important to probiotic-linked mechanisms.