Genome statistics of UM_WWY and three closely related species.

<p>Genome statistics of UM_WWY and three closely related species.</p

Supermatrix phylogenetic tree by concatenating <i>rpoB</i>, <i>tuf</i>, <i>sodA</i>, <i>16S rRNA</i> and <i>hsp65</i> gene sequences.

<p>Our results indicated that UM_WWY is closely related to <i>M</i>. <i>brisbanense</i> and other members in the <i>M</i>. <i>fortuitum</i> complex.</p

Schematic circular diagram of the UM_WWY genome.

<p>The following color bars represent: Dark red and light red are contigs, pink and orange represent forward and reverse CDS respectively, purple is tRNA, dark blue is genomic island, green is prophage, grey is virulence gene and the last track is the percentage of GC content.</p

The pathway for urea cycle derived from KEGG pathway analysis.

<p>UM_WWY possess a gene encoding for a putative enzyme arginase (EC 3.5.3.1) which hydrolyzes arginine into ornithine and urea. EC 6.3.4.5 = Argininosuccinate synthetase; EC 4.3.2.1 = Argininosuccinate lyase; EC 2.1.3.3 = Ornithine transcarbamoylase.</p

Whole-Genome Sequencing and Comparative Analysis of <i>Mycobacterium brisbanense</i> Reveals a Possible Soil Origin and Capability in Fertiliser Synthesis

<div><p><i>Mycobacterium brisbanense</i> is a member of <i>Mycobacterium fortuitum</i> third biovariant complex, which includes rapidly growing <i>Mycobacterium</i> spp. that normally inhabit soil, dust and water, and can sometimes cause respiratory tract infections in humans. We present the first whole-genome analysis of <i>M</i>. <i>brisbanense</i> UM_WWY which was isolated from a 70-year-old Malaysian patient. Molecular phylogenetic analyses confirmed the identification of this strain as <i>M</i>. <i>brisbanense</i> and showed that it has an unusually large genome compared with related mycobacteria. The large genome size of <i>M</i>. <i>brisbanense</i> UM_WWY (~7.7Mbp) is consistent with further findings that this strain has a highly variable genome structure that contains many putative horizontally transferred genomic islands and prophage. Comparative analysis showed that <i>M</i>. <i>brisbanense</i> UM_WWY is the only <i>Mycobacterium</i> species that possesses a complete set of genes encoding enzymes involved in the urea cycle, suggesting that this soil bacterium is able to synthesize urea for use as plant fertilizers. It is likely that <i>M</i>. <i>brisbanense</i> UM_WWY is adapted to live in soil as its primary habitat since the genome contains many genes associated with nitrogen metabolism. Nevertheless, a large number of predicted virulence genes were identified in <i>M</i>. <i>brisbanense</i> UM_WWY that are mostly shared with well-studied mycobacterial pathogens such as <i>Mycobacterium tuberculosis</i> and <i>Mycobacterium abscessus</i>. These findings are consistent with the role of <i>M</i>. <i>brisbanense</i> as an opportunistic pathogen of humans. The whole-genome study of UM_WWY has provided the basis for future work of <i>M</i>. <i>brisbanense</i>.</p></div

Venn diagram showing the gene distribution of <i>M</i>. <i>fortuitum</i> complex members and UM_WWY.

<p>The four genomes shared 3,388 gene clusters and UM_WWY contains the highest number of strain-specific genes.</p

L-Arginine Destabilizes Oral Multi-Species Biofilm Communities Developed in Human Saliva

<div><p>The amino acid L-arginine inhibits bacterial coaggregation, is involved in cell-cell signaling, and alters bacterial metabolism in a broad range of species present in the human oral cavity. Given the range of effects of L-arginine on bacteria, we hypothesized that L-arginine might alter multi-species oral biofilm development and cause developed multi-species biofilms to disassemble. Because of these potential biofilm-destabilizing effects, we also hypothesized that L-arginine might enhance the efficacy of antimicrobials that normally cannot rapidly penetrate biofilms. A static microplate biofilm system and a controlled-flow microfluidic system were used to develop multi-species oral biofilms derived from pooled unfiltered cell-containing saliva (CCS) in pooled filter-sterilized cell-free saliva (CFS) at 37^oC. The addition of pH neutral L-arginine monohydrochloride (LAHCl) to CFS was found to exert negligible antimicrobial effects but significantly altered biofilm architecture in a concentration-dependent manner. Under controlled flow, the biovolume of biofilms (μm³/μm²) developed in saliva containing 100-500 mM LAHCl were up to two orders of magnitude less than when developed without LAHCI. Culture-independent community analysis demonstrated that 500 mM LAHCl substantially altered biofilm species composition: the proportion of <i>Streptococcus</i> and <i>Veillonella</i> species increased and the proportion of Gram-negative bacteria such as <i>Neisseria</i> and <i>Aggregatibacter</i> species was reduced. Adding LAHCl to pre-formed biofilms also reduced biovolume, presumably by altering cell-cell interactions and causing cell detachment. Furthermore, supplementing 0.01% cetylpyridinium chloride (CPC), an antimicrobial commonly used for the treatment of dental plaque, with 500 mM LAHCl resulted in greater penetration of CPC into the biofilms and significantly greater killing compared to a non-supplemented 0.01% CPC solution. Collectively, this work demonstrates that LAHCl moderates multi-species oral biofilm development and community composition and enhances the activity of CPC. The incorporation of LAHCl into oral healthcare products may be useful for enhanced biofilm control.</p></div

A model showing the proposed biofilm destabilizing effects of short-term exposure (transient; minutes) and longer-term exposure (sustained; hours) to high millimolar (≥100mM) LAHCl concentrations in flowing saliva.

<p>The model shows the effect of LAHCl on a well-developed multi-species biofilm (with respect to architecture and species composition) although similar destabilizing effects would occur on multi-species biofilms of younger or older developmental age. Cell shapes and sizes are not to scale. Postulated effects are based on data presented in this manuscript and on previous observations of the effects of L-arginine on bacterial cells and cell-cell interactions, as discussed in the body of the text.</p

Effects of flowing saliva supplemented with different LAHCl concentrations on the development of oral biofilms for 20 h in the Bioflux system.

<p>The graph shows differences in biofilm biovolume and representative CLSM 3D renderings are included to highlight differences in biofilm architecture. Light blue colored star symbols embedded in the graph indicate a significant increase in average biofilm biovolume over CFS control and orange colored star symbols embedded in the graph indicate a significant decrease in average biofilm biovolume over CFS control. For the rendered biofilms within the embedded images, green colored cells indicate viable cells and red colored cells indicate damaged/dead cells. Bars represent 20 μm. The associated table shows, in addition to the biovolumes highlighted in the graph, changes in average biofilm thickness, average biofilm roughness, and cell viability. For data presented in the associated table, means are shown in bold and standard deviations are shown in parentheses (each derived from at least 18 images from six biological replicates). *P<0.05; **P<0.01; ***P<0.001: significant differences from the CFS control.</p

Differences in architecture of oral biofilms grown a static biofilm system containing different concentrations of L-arginine monohydrochloride (LAHCl).

<p>Images show representative 3D renderings of 22 h-old oral biofilms grown from a cell-containing saliva (CCS) inoculum in the static biofilm system containing cell free saliva (CFS) supplemented with different concentrations of LAHCl. Green signal (Syto 9) indicates viable cells and red signal (propidium iodide) indicates damaged/dead cells. Upper renderings (A₁–F₁) are of the x–y plane. Middle renderings (A₂–F₂) are of the x–z plane. Lower renderings (A₃–F₃) represent an angled view (x–y–z). Bars represent 50 μm. The associated table shows changes in percentage of cell viability with means presented in bold and standard deviations shown in parentheses (each derived from at least 27 images from nine biological replicates). *P<0.05; **P<0.01: significant differences from the CFS control.</p