Two novel species of rapidly growing mycobacteria: Mycobacterium lehmannii sp. nov. and Mycobacterium neumannii sp. nov.

Two rapidly growing mycobacteria with identical 16S rRNA gene sequences were the subject of a polyphasic taxonomic study. The strains formed a well-supported subclade in the mycobacterial 16S rRNA gene tree and were most closely associated with the type strain of Mycobacterium novocastrense . Single and multilocus sequence analyses based on hsp65, rpoB and 16S rRNA gene sequences showed that strains SN 1900T and SN 1904T are phylogenetically distinct but share several chemotaxonomic and phenotypic features that are are consistent with their classification in the genus Mycobacterium . The two strains were distinguished by their different fatty acid and mycolic acid profiles, and by a combination of phenotypic features. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values for strains SN 1900T and SN 1904T were 61.0 % and 94.7 %, respectively; in turn, the corresponding dDDH and ANI values with M. novocastrense DSM 44203T were 41.4 % and 42.8 % and 89.3 % and 89.5 %, respectively. These results show that strains SN1900T and SN 1904T form new centres of taxonomic variation within the genus Mycobacterium . Consequently, strains SN 1900T (40T=CECT 8763T=DSM 43219T) and SN 1904T (2409T=CECT 8766T=DSM 43532T) are considered to represent novel species, for which the names Mycobacterium lehmannii sp. nov. and Mycobacterium neumannii sp. nov. are proposed. A strain designated as ‘ Mycobacterium acapulsensis’ was shown to be a bona fide member of the putative novel species, M. lehmannii

Mycobacterium eburneum sp. nov., a non-chromogenic, fast-growing strain isolated from sputum

A polyphasic study was undertaken to establish the taxonomic position of a non-chromogenic, rapidly growing Mycobacterium strain that had been isolated from sputum. The strain, CECT 8775T, has chemotaxonomic and cultural properties consistent with its classification in the genus Mycobacterium and was distinguished from the type strains of closely related mycobacterial species, notably from Mycobacterium paraense DSM 46749T, its nearest phylogenetic neighbour, based on 16S rRNA, hsp65 and rpoB gene sequence data. These organisms were also distinguished by a broad range of chemotaxonomic and phenotypic features and by a digital DNA–DNA relatedness value of 22.8 %. Consequently, the strain is considered to represent a novel species of Mycobacterium for which the name Mycobacterium eburneum sp. nov is proposed; the type strain is X82T (CECT 8775T=DSM 44358T)

Conservation of cell shape and mycolic acids by formaldehyde-fixation.

<p>(A) Incubation of formaldehyde-fixed MACB on TSB medium. Formaldehyde-fixed MACB from 2 ml culture were inoculated on TSB agar medium and incubated at 30°C for 5 days. (B) SEM images of formaldehyde-fixed MACB. (C) TLC analysis of total fatty acid methylesters extracted from live or formaldehyde-fixed MACB. (D) MALDI mass spectra of mycolic acid methylesters from formaldehyde-fixed cells and non-fixed cells. Numbers on arrows represent (0) A-1 MA, (1) A-2 MA, (2) A-3 MA, (3) A-4 MA, (4) A-5 MA, and (5) A-6 MA (MA: mycolic acid). Definition of subtype (A-1 to A-6) are described in supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142372#pone.0142372.s002" target="_blank">S1 Table</a>. Abbreviations; Tp: <i>Tsukamurella pulmonis</i> TP-B0596, Ro: <i>Rhodococcus opacus</i> B4, Re: <i>Rhodococcus erythropolis</i> PR4, Cg: <i>Corynebacterium glutamicum</i> ATCC13869.</p

Co-aggregation of <i>S</i>. <i>lividans</i> and MACB observed using SEM.

<p>(A) Co-aggregation of <i>S</i>. <i>lividans</i> and live <i>R</i>. <i>erythropolis</i>. Adhesion of rod-shaped cells of <i>R</i>. <i>erythropolis</i> was observed entirely on pellets of <i>S</i>. <i>lividans</i> formed in liquid culture. (B) Co-aggregaton of <i>S</i>. <i>lividans</i> and live <i>R</i>. <i>opacus</i>. Adhesion of rod-shaped cells of <i>R</i>. <i>opacus</i> were observed entirely on pellets of <i>S</i>. <i>lividans</i> formed in liquid culture. Bacteria were cultured in YGGS medium for 5 days at 180 rpm, 30°C.</p

Conservation of cell shape and mycolic acids by γ-irradiation.

<p>(A) Incubation of γ-irradiated MACB on TSB medium. γ-irradiated MACB from 2 ml culture were inoculated on TSB agar medium, and incubated at 30°C for 5 days. (B) SEM images of γ-irradiated MACB. (i) non-irradiated cells of <i>R</i>. <i>opacus</i>. (ii) γ-irradiated (5 kGy) cells of <i>R</i>. <i>opacus</i>. (iii) non-irradiated cells of <i>R</i>. <i>erythropolis</i>. (iv) γ-irradiated (5 kGy) cells of <i>R</i>. <i>erythropolis</i>. (v) non-irradiated cells of <i>T</i>. <i>pulmonis</i>. (vi) γ-irradiated (5 kGy) cells of <i>T</i>. <i>pulmonis</i>. (C) TLC analysis of total fatty acid methylesters extracted from live or γ-irradiated MACB. (D) MALDI mass spectra of mycolic acid methylesters from γ-irradiated MACB and live cells. Numbers on arrows represent (0) A-1 MA, (1) A-2 MA, (2) A-3 MA, (3) A-4 MA, (4) A-5 MA, and (5) A-6 MA (MA: mycolic acid). Definition of subtype (A-1 to A-6) are described in supporting information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142372#pone.0142372.s002" target="_blank">S1 Table</a>.</p

Pigments inducing effect by γ-irradiated MACB.

<p>(A) Induction activity of γ-irradiated MACB was tested on solid medium culture. Both bacteria were inoculated on YGGS agar and incubated for 5 days at 30°C. (B-D) Induction activity of γ-irradiated MACB were tested in liquid medium. SEM images were obtained from mixed-cultures in YGGS medium (5 days at 180 rpm, 30°C): (B) <i>S</i>. <i>lividans</i> with live <i>R</i>. <i>erythropolis</i> (left) or γ-irradiated <i>R</i>. <i>erythropolis</i> (right). (C) <i>S</i>. <i>lividans</i> with live <i>R</i>. <i>opacus</i> (left) or γ-irradiated <i>R</i>. <i>opacus</i> (right). (D) <i>S</i>. <i>lividans</i> with live <i>T</i>. <i>pulmonis</i> (left) or γ-irradiated <i>T</i>. <i>pulmonis</i> (right).</p

Killing of Mycolic Acid-Containing Bacteria Aborted Induction of Antibiotic Production by <i>Streptomyces</i> in Combined-Culture

<div><p>Co-culture of <i>Streptomyces</i> with mycolic acid-containing bacteria (MACB), which we termed “combined-culture,” alters the secondary metabolism pattern in <i>Streptomyces</i> and has been a useful method for the discovery of bioactive natural products. In the course of our investigation to identify the inducing factor(s) of MACB, we previously observed that production of pigments in <i>Streptomyces lividans</i> was not induced by factors such as culture extracts or mycolic acids. Although dynamic changes occurred in culture conditions because of MACB, the activation of pigment production by <i>S</i>. <i>lividans</i> was observed in a limited area where both colonies were in direct contact. This suggested that direct attachment of cells is a requirement and that components on the MACB cell membrane may play an important role in the response by <i>S</i>. <i>lividans</i>. Here we examined whether this response was influenced by dead MACB that possess intact mycolic acids assembled on the outer cell membrane. Formaldehyde fixation and γ-irradiation were used to prepare dead cells that retain their shape and mycolic acids of three MACB species: <i>Tsukamurella pulmonis</i>, <i>Rhodococcus erythropolis</i>, and <i>Rhodococcus opacus</i>. Culturing tests verified that <i>S</i>. <i>lividans</i> does not respond to the intact dead cells of three MACB. Observation of combined-culture by scanning electron microscopy (SEM) indicated that adhesion of live MACB to <i>S</i>. <i>lividans</i> mycelia were a significant interaction that resulted in formation of co-aggregation. In contrast, in the SEM analysis, dead cells were not observed to adhere. Therefore, direct attachment by live MACB cells is proposed as one of the possible factors that causes <i>Streptomyces</i> to alter its specialized metabolism in combined-culture.</p></div