Gut microbiota is an assortment of microorganisms inhabiting the length and width of the mammalian gastrointestinal tract. The composition of this microbial community is host specific, evolving throughout an individual's lifetime and susceptible to both exogenous and endogenous modifications. Recent renewed interest in the structure and function of this "organ" has illuminated its central position in health and disease. The microbiota is intimately involved in numerous aspects of normal host physiology, from nutritional status to behavior and stress response. Additionally, they can be a central or a contributing cause of many diseases, affecting both near and far organ systems. The overall balance in the composition of the gut microbial community, as well as the presence or absence of key species capable of effecting specific responses, is important in ensuring homeostasis or lack thereof at the intestinal mucosa and beyond. The mechanisms through which microbiota exerts its beneficial or detrimental influences remain largely undefined, but include elaboration of signaling molecules and recognition of bacterial epitopes by both intestinal epithelial and mucosal immune cells. The advances in modeling and analysis of gut microbiota will further our knowledge of their role in health and disease, allowing customization of existing and future therapeutic and prophylactic modalities.

Gut Microbiota in Health and Disease

The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey’s Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.

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Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.

SUMMARY Strains of bacteria resistant to antibiotics, particularly those that are multiresistant, are an increasing major health care problem around the world. It is now abundantly clear that both Gram-negative and Gram-positive bacteria are able to meet the evolutionary challenge of combating antimicrobial chemotherapy, often by acquiring preexisting resistance determinants from the bacterial gene pool. This is achieved through the concerted activities of mobile genetic elements able to move within or between DNA molecules, which include insertion sequences, transposons, and gene cassettes/integrons, and those that are able to transfer between bacterial cells, such as plasmids and integrative conjugative elements. Together these elements play a central role in facilitating horizontal genetic exchange and therefore promote the acquisition and spread of resistance genes. This review aims to outline the characteristics of the major types of mobile genetic elements involved in acquisition and spread of antibiotic resistance in both Gram-negative and Gram-positive bacteria, focusing on the so-called ESKAPEE group of organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), which have become the most problematic hospital pathogens.

https://cmr.asm.org/content/cmr/31/4/e00088-17.full-text.pdf

Mobile Genetic Elements Associated with Antimicrobial Resistance

Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2001.02337.x

The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix.

An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

Medical microbiology is extremely reliant on the culture of bacteria from clinical specimens and their subsequent identification by biochemical and phenotypic characteristics for the diagnosis of disease. Following determination of the structure of DNA by Watson & Crick (1953), studies in bacteriology have seen a major shift from functional to molecular techniques for identifying bacteria (Towner & Cockayne, 1993). This review will deal with the 16s-23s spacer region of the rRNA operon (Fig. 1) and its use in the identification of micro-organisms at the species and strain (typing) levels.

New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region

Three structural classes of (1-->3)-beta-D-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (1-->3)-beta-glucans and side-chain-branched (1-->3,1-->2)-beta-glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (1-->3,1-->6)-beta-glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (1-->3)-beta-glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (1-->3)-beta-glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.

Curdlan and other bacterial (1→3)-β-d-glucans

Summary: Three Pseudomonas aeruginosa phages specific for bacteria harbouring the P-group plasmid RP1 have been isolated, and their properties compared with those of a previously described sex-specific phage, PRR1 (Olsen & Shipley, 1973). These phages are distinguishable from each other by various criteria, although in terms of host range to FP+ and RP+ lines of P. aeruginosa
pao they comprise broadly two groups. Thus all the phages infect bacteria harbouring any of a group of plasmids with similar properties to those of RP1, but whereas the filamentous phage Pf3 is specific for this group, the host ranges of PRR1, PR3 and PR4 are considerably wider. Nevertheless, with one exception, this does not extend to plasmids isolated outside the United Kingdom, which suggests that all these plasmids share a common ancestry even though by other criteria they constitute three fairly discrete subgroups. Of the plasmids that fail to allow phage propagation, three, when present in the same cell as RP1, reduce its susceptibility to phage infection. This inhibition may reflect a relationship between these elements, similar to that found among plasmids of Enterobacteria. A correlation is observed between the susceptibility of bacteria to phage infection and their ability to mediate plasmid transfer, such that these phages can conveniently be used to isolate both derepressed or transfer-defective mutants of various R factors.

The properties and host range of male-specific bacteriophages of Pseudomonas aeruginosa.

Genes essential for the production of a linear, bacterial (1-->3)-beta-glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)-beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.

/pdf/detection-of-two-loci-involved-in-1-3-b-glucan-curdlan-289potx81w.pdf

Detection of two loci involved in (1→3)-β-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

Publisher Summary Many bacteria of diverse type and habitat are now known to harbour plasmid DNA. This observation lends credence to the view that these elements are ubiquitous among procaryotes and are likely to be detected in any species in which a thorough search for them is made. In recent years, the demonstration of plasmids in an increasing variety of bacteria has been striking and directly the result of the development of techniques that allow the physical demonstration, isolation and molecular characterization of plasmid DNA. Thus, plasmids in bacteria in which genetic analysis is presently limited or impossible, are as amenable to detailed molecular study as those from genetically well-characterized bacteria. Such investigations are satisfactory where the primary concern is the molecular characterization, comparison and in vitro manipulation of plasmids for specific purposes such as the construction of cloning vectors. This chapter describes the analyses of the latter type and deals with the primarily genetic procedures that can be used, first to identify a plasmid-encoded phenotype, and then to characterize further the element involved.

Vilma A. Stanisich

Papers

New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region

Curdlan and other bacterial (1→3)-β-d-glucans

The properties and host range of male-specific bacteriophages of Pseudomonas aeruginosa.

Detection of two loci involved in (1→3)-β-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

2 Identification and Analysis of Plasmids at the Genetic Level