The safety of probiotics is tied to their intended use, which includes consideration of potential vulnerability of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. Unique to probiotics is that they are alive when administered, and unlike other food or drug ingredients, possess the potential for infectivity or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. The presence of transferable antibiotic resistance genes, which comprises a theoretical risk of transfer to a less innocuous member of the gut microbial community, must also be considered. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicogenicity must be assessed depending on the characteristics of the genus and species of the microbe being used. Immunological effects must be considered, especially in certain vulnerable populations, including infants with undeveloped immune function. A few reports about negative probiotic effects have surfaced, the significance of which would be better understood with more complete understanding of the mechanisms of probiotic interaction with the host and colonizing microbes. Use of readily available and low cost genomic sequencing technologies to assure the absence of genes of concern is advisable for candidate probiotic strains. The field of probiotic safety is characterized by the scarcity of studies specifically designed to assess safety contrasted with the long history of safe use of many of these microbes in foods.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T ⅐ A-to-A ⅐ T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.Oligonucleotides can bind to duplex DNA and form triple helices in a sequence-specific manner (2,3,5,12,25,39). Progress in elucidating the third-strand binding code has raised the possibility of developing nucleic acids as sequence-specific reagents for research and possibly clinical applications. Oligonucleotide-mediated triplex formation has been shown to prevent transcription factor binding to promoter sites and to block mRNA synthesis in vitro and in vivo (4,9,11,17,18,21,26,29,33,41). Such inhibition of expression, however, is transient, depending on the sustained presence of the oligonucleotides. It also depends on the stability of the triple helix, which can be disrupted by transcription initiated at nearby sites (37). To overcome these problems, methods to prolong oligonucleotide-duplex interactions using DNA intercalating or cross-linking agents have been explored in experiments to block transcription initiation or elongation (17,18,39,40).Instead of using triplex formation to transiently block gene expression, however, we reasoned that it would be advantageous to use triple helix formation to target m...

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.In recent years, DNA and oligonucleotide microchip (microarray) technology has played an increasingly important role in genomic studies, drug discovery, and toxicological research. Unlike other hybridization formats (hybridization with microplates or dot blot hybridization with membrane-bound probes), glass microchips allow significant miniaturization so that thousands of individual probes can be arranged on one glass slide. As a result, this technology is ideal for an extensive parallel identification of nucleic acids and analysis of gene expression. Simultaneous analysis for the presence of multiple markers makes it possible to determine a complete genetic profile of a single strain or distinguish one strain from a very large collection of possible alternatives in one experiment. Therefore, this approach is potentially useful for the screening of multiple microbial isolates in a diagnostic assay.Oligonucleotide microchips containing multiple oligonucleotides are spotted on the chip surface. DNA samples for analysis are labeled with fluorescent dyes and hybridized with the oligonucleotide spots on the chip. The fluorescence pattern is then recorded by a scanner, quantified, and analyzed. While DNA microchips have been used mostly for gene expression studies, the technique has great potential to be used for the discrimination of genotypes, point mutants, and other closely related sequences by employing oligonucleotides specific for each sequence variant.Microarray technology has great potential for use in diagnostic microbiology. Microbial pathogens are currently identified by using surrogate biochemical and immunological marker...