Sauton's Media Research Articles

Corrigendum: The TbD1 Locus Mediates a Hypoxia-Induced Copper Response in Mycobacterium bovis

[This corrects the article DOI: 10.3389/fmicb.2022.817952.].

First isolation of Mycobacterium setense from hospital water

Objective: To present the findings of a study on isolation of four unrelated environmental strains of Mycobacterium setense (M. setense) from hospital environment and help to assess the natural habitat and the mode of transmission in man. Methods: The water samples were collected from hospital departments and cultured on Lowenstein-Jensen and Sauton's media. The isolates, i.e., AW3-2, AW5, AW11 and AW18 were subjected to identification by conventional and molecular tests including sequencing analysis of 16S rRNA. Results: The water isolates revealed the phenotypic and molecular features which were consistent with M. setense including a genus specific amplicon of the hsp65 gene and 99.6% similarities with those of M. setense CIP: 109395T 16S rRNA gene sequences. Conclusions: The current report will contribute to a better understanding of the pathogenesis and path of transmission of this opportunistic pathogen to human.

First report of isolation of Mycobacterium canariasense from hospital water supplies.

Mycobacterium canariasense was first isolated as a novel species in 2004 from clinical specimens in Spain. Since then there have only been a few additional reports from Spain, the USA, and Lebanon on the isolation of this rare species from clinical specimens. We herein present the first report on isolation of this organism from hospital water, which provides evidence for determining the natural habitat of this rare species. The water samples were collected from hospital departments and cultured on Löwenstein-Jensen and Sauton's media. The isolates, i.e. WP5, WP20, and AW2-3, were subjected to identification by conventional and molecular tests including sequencing analysis of 16S rRNA. The water isolates revealed phenotypic and molecular features consistent with M. canariasense including a genus-specific amplicon of the hsp65 gene and 100% similarities with those of M. canariasense CIP: 107998(T) 16S rRNA gene sequences. The current report might be of value in tracing the probable source of infection in patients.

Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis

Organized bacterial communities, or biofilms, provide an important reservoir for persistent cells that are inaccessible or tolerant to antibiotics. Curli pili are cell-surface structures produced by certain bacteria and have been implicated in biofilm formation in these species. In order to determine whether these structures, which were suggested to be encoded by the Rv3312A (mtp) gene, have a similar role in Mycobacterium tuberculosis, we generated a Δmtp mutant and a mtp-complemented strain of a clinical isolate of M. tuberculosis and analyzed these strains for their ability to produce pili in comparison to the wild-type strain. Phenotypic analysis by transmission electron microscopy proved the essentiality of mtp for piliation in M. tuberculosis. We then compared biofilm formation of the derived strains in detergent-free Sauton's media. Biofilm mass was quantified spectrophotometrically using crystal violet. Furthermore, we examined mtp gene expression by quantitative real-time PCR in wild-type cells grown under biofilm versus planktonic growth conditions. We found a 68.4% reduction in biofilm mass in the mutant compared to the wild-type strain (P=0.002). Complementation of the mutant resulted in a restoration of the wild-type biofilm phenotype (P=0.022). We, however, found no significant difference between mtp expression in cells of the biofilm to those growing planktonically. Our findings highlight a crucial, but non-specific, role of pili in the biofilm lifestyle of M. tuberculosis and indicate that they may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing persistence.

In vitro culture medium influences the vaccine efficacy of Mycobacterium bovis BCG

The varied rates of protection induced by Mycobacterium bovis BCG vaccine against tuberculosis has been attributed to many factors such as genetic variability among BCG strains, rapid clearance of BCG in some populations, and different levels of previous exposure of vaccinated populations to environmental mycobacteria. However, the methods and conditions employed to prepare this vaccine for human usage by various manufacturers have not been investigated as potential factors contributing to the variation in vaccine efficacy. A review of the literature indicates discrepancies between the approach for growing BCG vaccine in the laboratory to assess immune responses and protective ability in animal models, and that employed for production of the vaccine for administration to humans. One of the major differences is in the growth medium used for routine propagation in the laboratory and the one used for bulk vaccine production by manufacturers. Here we compared the immunogenicity of the BCG vaccine grown in Middlebrook 7H9 medium, the most commonly used medium in laboratory studies, against that grown in Sauton medium, which is used for growing BCG by most manufacturers. Our results showed clear differences in the behavior of BCG grown in these different culture media. Compared to BCG grown in Middlebrook 7H9 medium, BCG grown in Sauton media was more persistent inside macrophages, more effective at inhibiting apoptosis of infected cells, induced stronger inflammatory responses and stimulated less effective immunity against aerosol challenge with a virulent Mtb strain. These findings suggested that the growth medium used for producing BCG vaccine is an important factor that deserves increased scrutiny in ongoing efforts to produce more consistently effective vaccines against Mtb.

A genomic study on the cultivable and nerve invading Mycobacterium HI-75 after the recovery 3 months of the inoculation to nude mice.

The sequence of the polymerase chain reaction (PCR) product of 16S ribosomal RNA (16S rRNA) of the leproma-derived and cultivable Mycobacterium HI-75 (M. HI-75) which was obtained from the infected regions of inoculated mice, was examined and compared with that of the cultured bacteria by the direct sequencing techniques. The sequence was completely consistent with the cultured bacilli in the comparable 837 bases of 16S rRNA. The mycobacterium examined in this study was originally isolated as M. leprae (ML) by Skinsnes, et al. in 1975 from leproma of a lepromatous type Hansen's disease patient and therefore named as Mycobacterium leprae HI-75 by them, and was maintained from 1984 using either Ogawa's or Sauton's media in the beginning and Ogawa's medium enriched with glucronic acid and N-acetyl-D-glucosamine recently. Sasaki and Hamit reported the nerve invasion and the growth of the inoculated bacilli either to the nude mice or the I131 treated immunocompromised Swiss mice. We previously reported that cultured HI-75 was most similar to M. scrofulaceum by the direct sequencing of the gene of 16S rRNA. The 16S rRNA obtained from the mouse tissue in the present study indicated that M. HI-75 would be a variant of M. scrofulaceum possessing an ability to invade into peripheral nerve. The results suggest that the HI-75 strain claims a nature as a pathogen to develop a leproma-like lesion.

Control of acyl-CoA carboxylase activity in mycobacteria

Acyl-CoA carboxylase activity in four pathogenic mycobacteria and Mycobacterium smegmatis was shown with both acetyl-CoA and propionyl-CoA substrates. Only very low activity was detected in mycobacteria grown in host tissues or on egg-based media rich in lecithin and avidin. This appeared to be a result of severe depression of activity, as strains which could be grown both in host tissue and egg-based media, and in the relatively simple Dubos or Sauton's media showed 8 to 120-fold higher activity in the simpler media.

Serial subculture of BCG on solid and liquid media

To follow an earlier study of the effects on BCG of deep subculture in liquid media, transfers on the surface of Lowenstein-Jensen and Sauton's media have been made. When a vaccine preparation that yields on culture 99+% of spreading-type colonies, with a small minority of non-spreading forms, is transferred on Löwenstein-Jensen medium, the minority population can be readily selected out. Growth of BCG on this medium is not homogeneous, and the selection of either the minority or majority populations appears fortuitous, whereas it was previously demonstrated that deep subculture in Dubos medium tends to favour selection of a minority yielding non-spreading colonies. When such a minority population has been favoured during transfer on Löwenstein-Jensen medium, subsequent subculture in Dubos medium usually accentuates the effect. Subculture as a surface pellicle on Sauton's medium has a powerful effect in reducing the relative size of a minority population yielding non-spreading colonies, andthereafter maintaining 99+% of spreading forms. The relationship between transfers on Löwenstein-Jensen and Sauton's media and those on potato-Sauton medium is briefly discussed.

投酸性菌の物質代謝に関する研究

In order to study the enzymatic properties of human type tubercle bacilli, H37 Rv, H37 Ra and BCG, and avian type bacilli, Takeo's strain, Myc. ATCC 607 and Myc. phlei, the auther prepared the soluble fraction and particulate fraction of these cells by means of ultracentrifuge frnm the ground cells that were cultured in Sauton's media. Using the fractions thus obtained, the author observede nzyme activities of these fractions. And then, the author picked out the soluble fraction of H37 Ra; dialyzed it and studied the dehydrogenase activity of the dialyzed soluble fraction for lactate and malate with the addition of Cofactor by means of Thunberg's methylen blue reduction method. The following results were obtained.1) It was observed that the enzyme activities of two fractions, soluble and particulate, were distinctly different in any organisms.2) It could be comfirmed that the specilic enzyme activity of each fraction for ehe substrates and the intensities of that activities were varied as the strains.3) The dehydrogenase activity of the dialyzed soluble fraction of H37 Ra for lactate was raised by the addition of CoI. And riboflavin could substitute for CoI.4) The more increased dehydrogenase activity for malate was observed by the simultaneous addition of GoI and riboflavin to the dialyzed soluble fraction of H37 Ra compaired with the single addition of CoI to that fraction. However riboflavin failed to replace the role of CoI.

結核菌物質代謝に関する研究

With the use of intact cell suspensions of virulent and avirulent strains of 2 and 6 weeks old cultures in the Sauton media, comparative studies on the dehydrogenating reaction of each substrate, sugars, sugar alcoholes, organic acids, were carried out by Thunberg method. The results were as follows.1) In the case of the 2 weeks old cells of avirulent strain without addition of substrate, the rate of methylene blue reduction is faster than that in the case of the virulent strain; and the 6 weeks old cells of virulent strain without substrate do not cause decoloration of methylene blue.2) When sugar or sugar alcohole is used as a substrare, no significant difference in the reduction time can be seen between the virulent and avirulent strains, nor any marked difference among each of the substrates. The rate of reduction by the 2 weeks old cells of avirulent strain, however, is more accelerated than that by the virulent strain by the addition of such substrate as glucose or other hexose.3) Regardless of the virulence of bacteria, of all the substrates used, pyruvate and lactate accelerate the reduction most markedly.4) When pyruvate or lactate is used as a substrate, the rate of reduction by the 6 weeks old cells of virulent strain is markedly decreased compared with that by the 2 weeks old cells, but with avirulent strain the decrease in the rate of reduction is not or hardly noticeable.5) When succinate is used as a substrate, in the case of 2 weeks old cells, the avirulent strain seems to have somewhat stronger methylene blue reducing activity than the virulent strain.6) The rate of reduction by the 2 weeks old cells of virulent strain is accelerated when alanine is used as a substrate, but this does not occur in the avirulent strain.

Effect of Chlorophyll Derivatives and Related Compounds on the Growth of M. tuberculosis

ConclusionsChlorin-e and chlorophyll in in concentrations of .025 and .050% in glycerine broth and Sauton's media inhibited the growth of H-37 and avian tubercle bacilli. Copper chlorin-e, deuteroporphyrin, copper deuteroporphyrin, and pyrroporphyrin sulfonic acid-sodium salt showed no growth retarding effects in concentrations of .025 and .050%. Evidence is presented to suggest that the H-37 tuber-culo-protein of the living bacillus is capable of binding copper chlorin-e. The absorption spectrum of the copper chlorin-e-bacillary protein complex when compared with that of copper chlorin-e-sodium in aqueous solution lends support to the assumption that chemical union between protein and pigment exists.