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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 organism's 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 ⌬upp 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 ⌬slpX 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.Lactobacillus acidophilus NCFM is a commercially established probiotic bacterium that is widely used in dietary supplements and in milk and fermented dairy products (47). Originally a human intestinal isolate from the 1970s (8), this strain has since been examined extensively for various desirable traits. Due to the importance of understanding the molecular mechanisms involved in probiotic functions, the complete genome sequence of L. acidophilus NCFM was determined (2). The NCFM genome sequence serves as a blueprint for in silico identification of candidate gene loci and gene regulatory networks that may play essential roles in the survival and host interactions of this microorganism in the gastrointestinal (GI) tract, including genes involved in acid tolerance (5), bile tolerance (38, 42), adherence factors (20), environmental sensing and response (7), prebiotic sugar utilization (9), polysaccharide biosynthesis, oxalate degradation (6), and bacteriocin production (22). In addition, ongoing microarray gene expression studies have revealed specific gene sets of interest that are being investigated further. The fundamental approach for establishing functional roles for important gene features involves inactivation of the genes ...

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 LTAnegative 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 + CD45RB high T 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.antiinflammatory | lactobacilli | Toll-like receptor 2 | innate immunity

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 phageenriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-Staphylococcus activity from the cream on plates and in broth.

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

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 methicillinresistant Staphylococcus aureus. LysK thus has potential as an antimicrobial for applications in the prevention and/or treatment of infections caused by staphylococci.

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.

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