The aggregation of mycobacterial cells in a definite order, forming microscopic structures that resemble cords, is known as cord formation, or cording, and is considered a virulence factor in the Mycobacterium tuberculosis complex and the species Mycobacterium marinum. In the 1950s, cording was related to a trehalose dimycolate lipid that, consequently, was named the cord factor. However, modern techniques of microbial genetics have revealed that cording can be affected by mutations in genes not directly involved in trehalose dimycolate biosynthesis. Therefore, questions such as "How does mycobacterial cord formation occur?" and "Which molecular factors play a role in cord formation?" remain unanswered. At present, one of the problems in cording studies is the correct interpretation of cording morphology. Using optical microscopy, it is sometimes difficult to distinguish between cording and clumping, which is a general property of mycobacteria due to their hydrophobic surfaces. In this work, we provide a new way to visualize cords in great detail using scanning electron microscopy, and we show the first scanning electron microscopy images of the ultrastructure of mycobacterial cords, making this technique the ideal tool for cording studies. This technique has enabled us to affirm that nonpathogenic mycobacteria also form microscopic cords. Finally, we demonstrate that a strong correlation exists between microscopic cords, rough colonial morphology, and increased persistence of mycobacteria inside macrophages.Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), killed 1.5 million people in 2006. A further 200,000 HIV-positive people died from HIV-associated TB (http://www.who.int/tb/en/index.html). Intensive research into the virulence factors that determine the pathogenicity of M. tuberculosis have been carried out since the tubercle bacillus was discovered. Unfortunately, despite the knowledge obtained, the factors that make M. tuberculosis virulent have not yet been identified (23,29). One of the first phenotypic characteristics linked to virulence was the microscopic formation of cords. When M. tuberculosis cells grow in a liquid medium without detergent, they form tight bundles, or cords, consisting of bacilli in which the orientation of the long axis of each cell is parallel to the long axis of the cord. M. tuberculosis microscopic cords were first observed by Robert Koch in 1882, but knowledge of their significance increased in 1947 with studies by Middlebrook et al. (16). These authors compared the virulent H37Rv and avirulent H37Ra M. tuberculosis strains and found that the formation of cords took place only in the virulent strain, whereas cells from the avirulent H37Ra strain were not oriented and merely formed irregular clumps. In 1953, Bloch isolated a toxic glycolipid from M. tuberculosis and related it to the virulence of the tubercle bacillus and to cording. Bloch named the glycolipid cord factor, and later, it was identified as trehalose dimycolate (TDM) (2, 17). However, 56...

Motility in mycobacteria was described for the first time in 1999. It was reported that Mycobacterium smegmatis and Mycobacterium avium could spread on the surface of solid growth medium by a sliding mechanism and that the presence of cell wall glycopeptidolipids was essential for motility. We recently reported that Mycobacterium vaccae can also spread on growth medium surfaces; however, only smooth colonies presented this property. Smooth colonies of M. vaccae do not produce glycopeptidolipids but contain a saturated polyester that is absent in rough colonies. Here, we demonstrate that Mycobacterium chubuense, Mycobacterium gilvum, Mycobacterium obuense, and Mycobacterium parafortuitum, which are phylogenetically related to M. vaccae, are also motile. Such motility is restricted to smooth colonies, since natural rough mutants are nonmotile. Thin-layer chromatography analysis of the content of cell wall lipids confirmed the absence of glycopeptidolipids. However, compounds like the above-mentioned M. vaccae polyester were detected in all the strains but only in smooth colonies. Scanning electron microscopy showed great differences in the arrangement of the cells between smooth and rough colonies. The data obtained suggest that motility is a common property of environmental mycobacteria, and this capacity correlates with the smooth colonial morphotype. The species studied in this work do not contain glycopeptidolipids, so cell wall compounds or extracellular materials other than glycopeptidolipids are implicated in mycobacterial motility. Furthermore, both smooth motile and rough nonmotile variants formed biofilms on glass and polystyrene surfaces.The genus Mycobacterium contains more than 100 species of nontuberculous mycobacteria (NTM) (28). Unlike the members of Mycobacterium tuberculosis complex and Mycobacterium leprae, NTM species are not obligated pathogens and are inhabitants of the environment. They can be found in natural water, water distribution systems, soil, protozoans, plants, and animals (24, 27). The emergence, in the last decade, of opportunistic NTM infections in humans has prompted the study of characteristics that allow mycobacteria to persist in their natural reservoirs. Motility has been related to the capacity of bacteria to colonize and persist in the environment. Mycobacteria were considered nonmotile microorganisms until 1999, when Martínez et al. (17) reported that Mycobacterium smegmatis and Mycobacterium avium spread on the surface of a growth medium by sliding motility. In these species, motility was related to colonial morphology and the presence of glycopeptidolipids (GPLs), a class of glycosylated peptidolipids located in the cell wall of some mycobacteria (for a recent review on GPLs, see the report of Chatterjee and Khoo [5]). Smooth colonies were motile and contained GPLs, but spontaneous rough colonies lacked GPLs and were nonmotile (17). Genetic evidence for the requirement of GPLs for sliding motility was later provided for M. smegmatis but not for M. avium (25). The latter...

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Incorporation of propidium monoazide indicates that there is an increase in H. pylori cells with the damaged membrane over the study, leading to the manifestation of cellular degeneration and death. Consequently, quantitative PCR combined with propidium monoazide contributes to our understanding of the role of H. pylori cells, under adverse environmental conditions.

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Mycobacterium vaccae is of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae 15483 ATCC(T) strain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccae switches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 degrees C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-gamma and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccae may affect the immunomodulatory effects induced from M. vaccae preparations.

Currently, one of the most challenged points to expand the use of viability PCR technique is achieving the complete exclusion of dead cells amplification signals, thus avoiding the overestimation of live cells population. Considering that, and based on the hypothesis that DNA may be retained by microtube walls, the impact of the microtube was addressed on signals from live and heat-killed cells. A double-dye reagent, PEMAX™, which comprises a mix of photo-reactive azide forms of phenanthridium, was used in this work. We found that if both the incubation and the photoactivation steps are carried out in different microtubes, the dead cell signal is greatly reduced than when those steps are done in the same tube. Therefore, the strategy depicted in this study presents a simple and efficient step in minimizing false-positive signal when employing viability PCR.