Whole-Genome Sequencing of Alcaligenes faecalis HZ01, with Potential to Inhibit Nontuberculous Mycobacterial Growth

ABSTRACT Alcaligenes faecalis is a Gram-negative rod that is ubiquitous in the environment and is an opportunistic human pathogen. Here, we report the whole-genome sequencing analysis of A. faecalis HZ01, which presents mycobacterial growth inhibitory activity and was isolated from a contaminated culture of Mycobacterium chubuense ATCC 27278.

chous bacterium (1). This opportunistic pathogen is widely distributed in the environment and is related to nosocomial diseases (2,3), with biotechnological potential in the pharmaceutical industry and in bioremediation of contaminated environments (4), such as the production of antibacterial substances (5)(6)(7). Although A. faecalis represents a promising source for new bioactive substances, there is limited literature on genomic approaches (8).
During the development of previous studies, we observed a contaminant microorganism that had grown on a Mycobacterium chubuense ATCC 27278 culture at 37°C on Middlebrook 7H10 medium and exhibited mycobacterial growth inhibitory activity ( Fig. 1A to C). In a similar study, it was verified that the antibacterial activity of A. faecalis is via a live-cell and contact-dependent mechanism (9). The ATCC strain was obtained from our mycobacterial collection. To isolate the contaminant microorganism, we selected three colonies showing a halo of mycobacterial growth inhibition, and then they were individually streaked on another Middlebrook 7H10 medium plate and incubated at 37°C for 48 h. The contaminant microorganism was identified as A. faecalis by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of pure cultures of the three isolates obtained originally and was stored at 280°C in nutrient broth supplemented with glycerol (final concentration of 15% [vol/vol]) (10).
Following the bacterial culture in MacConkey agar in a 37°C incubator for 48 h, we performed genomic DNA extraction using the QIAamp DNA minikit (Qiagen, Hilden, Germany) and library preparation using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). Whole-genome sequencing (WGS) was conducted on the Illumina NextSeq 500 platform with 2 Â 150-bp paired-end reads.
A total of 8,369,218 reads were obtained, and the genome coverage was 606Â. We obtained a total of 7,854,398 reads after quality trimming. By mapping the reads obtained against A. faecalis subsp. faecalis (ATCC 8750) (https://genomes.atcc.org/genomes/ a6829cff570e4f50) using the Burrows-Wheeler aligner (20), we observed that 86.68% of the reads were properly paired against the reference genome. After de novo assembly, we obtained 57 contigs; the largest contig had 848,880 bp. The draft genome obtained had a total length of 4,141,412 bp, with a GC content of 56.79% (Fig. 1D). The N 50 and N 75 values were 669,949 bp and 410,060 bp, respectively. There was no presence of plasmids. We found 7,873 complex variants, 191 deletions, 188 insertions, 993 multiple-nucleotide polymorphisms (MNPs), 4,7401 single-nucleotide polymorphisms (SNPs), and a total of 5,6647 variants.
Due to increasing challenges in treating multidrug-resistant infections, such as mycobacterial diseases, and the global shortage of successful drug therapy options, the discovery of new antimicrobial agents is necessary to improve patient outcomes.
Data availability. The A. faecalis HZ01 WGS data were deposited in DDBJ/ENA/ GenBank under accession number JAFMOE000000000 (the version described in this paper is JAFMOE010000000), BioSample accession number SAMN17762316, BioProject accession number PRJNA698913, and SRA accession number SRR13612681.

ACKNOWLEDGMENTS
We are grateful to Marlei Gomes da Silva for technical assistance during the bacterial isolation.