Colistin-degrading proteases confer collective resistance to microbial communities during polymicrobial infections.

BACKGROUND: The increasing prevalence of resistance against the last-resort antibiotic colistin is a significant threat to global public health. Here, we discovered a novel colistin resistance mechanism via enzymatic inactivation of the drug and proposed its clinical importance in microbial communities during polymicrobial infections. RESULTS: A bacterial strain of the Gram-negative opportunistic pathogen Stenotrophomonas maltophilia capable of degrading colistin and exhibiting a high-level colistin resistance was isolated from the soil environment. A colistin-degrading protease (Cdp) was identified in this strain, and its contribution to colistin resistance was demonstrated by growth inhibition experiments using knock-out (Δcdp) and complemented (Δcdp::cdp) mutants. Coculture and coinfection experiments revealed that S. maltophilia carrying the cdp gene could inactivate colistin and protect otherwise susceptible Pseudomonas aeruginosa, which may seriously affect the clinical efficacy of the drug for the treatment of cystic fibrosis patients with polymicrobial infection. CONCLUSIONS: Our results suggest that Cdp should be recognized as a colistin resistance determinant that confers collective resistance at the microbial community level. Our study will provide vital information for successful clinical outcomes during the treatment of complex polymicrobial infections, particularly including S. maltophilia and other colistin-susceptible Gram-negative pathogens such as P. aeruginosa. Video abstract.

Fungal ß-Glycosidase Belonging to Subfamily 4 of Glycoside Hydrolase Family 30 with Transglycosylation Activity.

Fomitopsis palustris, a prominent wood decayer, is known to produce a variety of glycoside hydrolases (GHs). In this study, we characterized a fungal ß-glycosidase belonging to subfamily 4 of GH family 30 (GH30). The recombinant protein (FpGH30) showed the highest hydrolytic activity toward p-nitrophenyl-ß-d-fucopyranoside (pNPßFuc), followed by p-nitrophenyl-α-l-arabinopyranoside (pNPαAra) and p-nitrophenyl-ß-d-galactopyranoside (pNPßGal). FpGH30 also exhibited transglycosylation activities, which catalyzed the transfer of glycosyl moieties to different glycosides and alkyl alcohols. When pNPßFuc, pNPßGal, and pNPαAra were used as substrates, self-condensation reactions occurred, leading to the production of the corresponding transglycosylated products with yields of 21, 26, and 25%, respectively. The enzyme was also able to catalyze the transfucosylation of pNP derivatives of ß-d-glucose, ß-d-mannose, and ß-d-xylose and alkyl alcohols (C1-C6), producing the corresponding transfucosylated products and alkyl fucosides. Our study indicates that FpGH30 is the first characterized fungal ß-glycosidase belonging to subfamily 4 of GH30 with transglycosylation activities.

Mobile resistome of human gut and pathogen drives anthropogenic bloom of antibiotic resistance.

BACKGROUND: The impact of human activities on the environmental resistome has been documented in many studies, but there remains the controversial question of whether the increased antibiotic resistance observed in anthropogenically impacted environments is just a result of contamination by resistant fecal microbes or is mediated by indigenous environmental organisms. Here, to determine exactly how anthropogenic influences shape the environmental resistome, we resolved the microbiome, resistome, and mobilome of the planktonic microbial communities along a single river, the Han, which spans a gradient of human activities. RESULTS: The bloom of antibiotic resistance genes (ARGs) was evident in the downstream regions and distinct successional dynamics of the river resistome occurred across the spatial continuum. We identified a number of widespread ARG sequences shared between the river, human gut, and pathogenic bacteria. These human-related ARGs were largely associated with mobile genetic elements rather than particular gut taxa and mainly responsible for anthropogenically driven bloom of the downstream river resistome. Furthermore, both sequence- and phenotype-based analyses revealed environmental relatives of clinically important proteobacteria as major carriers of these ARGs. CONCLUSIONS: Our results demonstrate a more nuanced view of the impact of anthropogenic activities on the river resistome: fecal contamination is present and allows the transmission of ARGs to the environmental resistome, but these mobile genes rather than resistant fecal bacteria proliferate in environmental relatives of their original hosts. Video abstract.

Functional characterization of a thermostable endoglucanase belonging to glycoside hydrolase family 45 from Fomitopsis palustris.

A gene encoding an endoglucanase belonging to subfamily C of glycoside hydrolase family 45 (GH45) was identified in the brown rot fungus Fomitopsis palustris and functionally expressed in Pichia pastoris. The recombinant protein displayed hydrolytic activities toward various substrates such as carboxymethyl cellulose, phosphoric acid swollen cellulose, glucomannan, lichenan, and ß-glucan. In particular, the enzyme had a unique catalytic efficiency on ß-1,4-glucans rather than mixed ß-1,3/1,4-glucans as compared to other GH45 endoglucanases. The fungal enzyme was relatively thermostable, retaining more than 91.4% activity at 80 °C for 1 h. Site-directed mutagenesis studies revealed that the mutants N95D and D117N had significantly reduced enzymatic activities, indicating that both residues are essential for the catalytic reaction. Our study expands knowledge and understanding of the catalytic mechanism of GH45 subfamily C enzymes and also suggests that this thermostable endoglucanase from F. palustris has great potential in industrial applications.

Cohnella saccharovorans sp. nov., isolated from ginseng soil.

A novel bacterial strain, CJ22T, was isolated from soil of a ginseng field located in Anseong, Korea. Cells of strain CJ22T were aerobic, Gram-stain-positive, endospore-forming, motile, oxidase- and catalase-positive and rod-shaped. The isolate grew optimally at pH 7 and 30 °C. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain CJ22T belonged to the genus Cohnella, displaying highest sequence similarity of 97.3% with Cohnella panacarvi Gsoil 349T. DNA-DNA relatedness between strain CJ22T and its closest relative was 35.5 % (reciprocal value, 23.8%). The phenotypic features of strain CJ22T also distinguished it from related species of the genus Cohnella. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major isoprenoid quinone was menaquinone MK-7 and the major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, lysyl-phosphatidylglycerol, two unidentified phospholipids and two unidentified aminophospholipids. The predominant cellular fatty acids of strain CJ22T were anteiso-C15 : 0, iso-C16:0 and C16:0. The DNA G+C content was 63.1 mol%. Based on data from this polyphasic taxonomic study, strain CJ22T is considered to represent a novel species of the genus Cohnella, for which the name Cohnella saccharovorans sp. nov. is proposed. The type strain is CJ22T (=KACC 17501T=JCM 19227T).

Lysobacter panacisoli sp. nov., isolated from ginseng soil.

A novel bacterial strain, designated CJ29(T), was isolated from ginseng soil of Anseong in South Korea. Cells of strain CJ29(T) were Gram-stain-negative, facultatively anaerobic, rod-shaped and non-motile. Strain CJ29(T) grew optimally at 28-30 °C and pH 7.0. Based on 16S rRNA gene sequence analysis, strain CJ29(T) was shown to belong to the genus Lysobacter within the class Gammaproteobacteria and was related most closely to Lysobacter soli DCY21(T) (98.5% similarity) and Lysobacter niastensis GH41-7(T) (98.2%). DNA-DNA relatedness between strain CJ29(T) and its closest relatives was below 55.6%. The predominant cellular fatty acids of strain CJ29(T) were iso-C15 : 0, iso-C16 : 0 and iso-C17 : 1ω9c. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major isoprenoid quinone was ubiquinone 8 (Q-8). The G+C content of the genomic DNA was 65.6 mol%. Phenotypic, genotypic and phylogenetic characteristics strongly supported the differentiation of strain CJ29(T) from related species of the genus Lysobacter. On the basis of data from this polyphasic taxonomic study, strain CJ29(T) is considered to represent a novel species of the genus Lysobacter, for which the name Lysobacter panacisoli sp. nov. is proposed. The type strain is CJ29(T) ( = KACC 17502(T) = JCM 19212(T)).

Stakelama sediminis sp. nov., isolated from tidal flat sediment.

A novel bacterial strain designated CJ70(T) was isolated from tidal flat sediment in Korea. A polyphasic approach was used to identify this strain taxonomically. The isolate was Gram-stain-negative, strictly aerobic, yellow-pigmented, rod-shaped and non-motile. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain CJ70(T) was related most closely to Stakelama pacifica JLT832(T) with 95.7 % similarity and formed an independent phyletic line from recognized species of the genus Sphingomonas, comprising a clade with Stakelama pacifica, which is the only recognized species of the genus Stakelama. The predominant cellular fatty acids of strain CJ70(T) were C(18 : 1)ω7c (60.0 %), C(16 : 0) (21.2 %) and C(14 : 0) 2-OH (5.8 %). The major isoprenoid quinone was ubiquinone-10. The G+C content of the genomic DNA was 61.4 mol%. The results obtained from this study suggested that strain CJ70(T) represents a novel species of the genus Stakelama, for which the name Stakelama sediminis sp. nov. is proposed. The type strain is CJ70(T) ( = KACC 16559(T) = JCM 18079(T)).

Gordonia alkaliphila sp. nov., an actinomycete isolated from tidal flat sediment.

A Gram-stain-positive, non-motile, aerobic actinobacterium, designated strain CJ10(T), was isolated from tidal flat sediment from the Yellow Sea in South Korea. Strain CJ10(T) grew on tryptic soy agar in the presence of 0-4 % (w/v) NaCl (optimum growth in the absence of NaCl) and at pH 6-11 (optimum pH 9). On the basis of 16S rRNA gene sequence analysis, strain CJ10(T) belonged to the genus Gordonia and showed the highest sequence similarity to Gordonia hirsuta DSM 44140(T) (97.9 %) and Gordonia hydrophobica DSM 44015(T) (97.6 %). DNA-DNA relatedness levels of strain CJ10(T) were 47.4 % (CJ10(T) as probe) and 42.2 % (G. hirsuta DSM 44140(T) as probe) to G. hirsuta DSM 44140(T) and 8.6 % (CJ10(T) as probe) and 9.3 % (G. hydrophobica DSM 44015(T) as probe) to G. hydrophobica DSM 44015(T). The major isoprenoid quinone was MK-9(H(2)). The polar lipid profile of strain CJ10(T) consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside. The predominant cellular fatty acids were C(18 : 1)ω9c (38.0 %), C(16 : 0) (30.1 %) and summed feature 3 (C(16 : 1)ω6c and/or C(16 : 1)ω7c; 17.4 %). The DNA G+C content was 67.7 mol%. Therefore, the results from our polyphasic taxonomic study suggest that strain CJ10(T) represents a novel species in the genus Gordonia, for which the name Gordonia alkaliphila sp. nov. is proposed; the type strain is CJ10(T) (= KACC 16561(T) = JCM 18077(T)).