Description of Chloramphenicol Resistant Kineococcus rubinsiae sp. nov. Isolated From a Spacecraft Assembly Facility

Abstract

A Gram-positive, coccoid, motile, aerobic bacterium, designated strain B12^T was isolated from a Jet Propulsion Laboratory spacecraft assembly cleanroom, Pasadena, CA, United States. Strain B12^T was resistant to chloramphenicol (100 μg/mL), and is a relatively slow grower (3-5 days optimal). Strain B12^T was found to grow optimally at 28 to 32°C, pH 7 to 8, and 0.5% NaCl. Fatty acid methyl ester analysis showed that the major fatty acid of the strain B12^T was anteiso C_{15 : 0} (66.3%), which is also produced by other Kineococcus species. However, arachidonic acid (C_{20 : 4} ω6,9,12,16c) was present in strain B12^T and Kineococcus glutinatus YIM 75677^T but absent in all other Kineococcus species. 16S rRNA analysis revealed that strain B12^T was 97.9% similar to Kineococcus radiotolerans and falls within the Kineococcus clade. Low 16S rRNA gene sequence similarities (<94%) with other genera in the family Kineosporiaceae, including Angustibacter (93%), Kineosporia (94% to 95%), Pseudokineococcus (93%), Quadrisphaera (93%), and Thalassiella (94%) demonstrated that the strain B12^T does not belong to these genera. Phylogenetic analysis of the gyrB gene show that all known Kineococcus species exhibited <86% sequence similarity with B12^T. Multi-locus sequence and whole genome sequence analyses confirmed that B12^T clades with other Kineococcus species. Average nucleotide identity of strain B12^T were 75-78% with other Kineococcus species, while values ranged from 72-75% with species from other genera within family Kineosporiaceae. Average amino-acid identities were 66-72% with other Kineococcus species, while they ranged from 50-58% with species from other genera. The dDDH comparison of strain B12^T genome with members of genera Kineococcus showed 20-22% similarity, again demonstrating that B12^T is distantly related to other members of the genus. Furthermore, analysis of whole proteome deduced from WGS places strain B12^T in order Kineosporiales, confirming that strain B12^T is a novel member of family Kineosporiaceae. Based on these analyses and other genome characteristics, strain B12^T is assigned to a novel species within the genus Kineococcus, and the name Kineococcus rubinsiae sp. nov., is proposed. The type strain is B12^T (=FJII-L1-CM-PAB2^T; NRRL B-65556^T, DSM 110506^T).

Keywords: Kineococcus rubinsiae; antibiotic resistant bacteria; cleanroom; genome; spacecraft assembly facility.

FIGURE 1

SEM micrographs of Kineococcus rubinsiae B12^T. The compact clustering might be due to the production of extracellular polysaccharide (EPS) seen in abundance (thread-like structures). Upon vigorous agitation the EPS are removed and individual coccoid cells are visualized (inset).

FIGURE 2

Maximum-likelihood 16S rRNA phylogeny for Kineococcus rubinsiae B12^T and other members of class Actinobacteria reconstructed from 16S rRNA sequences found in public databases. The 16S rRNA sequence from Piscicoccus intestinalis NBRC 104926^T was used for the out-group. Bootstrapping values are included on internal nodes in the phylogeny, and represent the number of trials (out of 100) that included that particular branching pattern. Strain numbers followed by GenBank accession numbers are included for each sequence in the phylogeny. Black dots on internal nodes represent branch agreement between this maximum-likelihood phylogeny, a neighbor-joining phylogeny, and a maximum-parsimony phylogeny generated from the same sequence alignment.

FIGURE 3

Maximum-likelihood gyrB phylogeny for Kineococcus rubinsiae B12^T and other members of family Kineosporiaceae reconstructed from full-length gyrB sequences found in public databases. The gyrB sequence from Nakamurella multipartita DSM 4423^T was used for the out-group. Bootstrapping values are included on internal nodes in the phylogeny, and represent the number of trials (out of 100) that included that branching pattern. Strain numbers followed by GenBank accession numbers are included for each sequence in the phylogeny.

FIGURE 4

Maximum-likelihood phylogeny for Kineococcus rubinsiae B12^T and other members of family Kineosporiaceae reconstructed from concatenated, full-length nucleotides sequences of genes frr, gyrB, nusA, rplS, rpsB, and tsf. The concatenated sequence from Nakamurella multipartita DSM 4423^T was used for the out-group. Bootstrapping values are included on internal nodes in the phylogeny, and represent the number of trials (out of 100) that included that branching pattern. Strain numbers followed by GenBank accession numbers are included for each sequence in the phylogeny.

FIGURE 5

Maximum-likelihood phylogeny for Kineococcus rubinsiae B12^T and other members of family Kineosporiaceae reconstructed from full-length genome sequences. The genome of Nakamurella multipartita DSM 4423^T was used for the out-group. Strain numbers followed by GenBank accession numbers are included for each sequence in the phylogeny.