Characterization of a carbapenem- and colistin-resistant Enterobacter cloacae carrying Tn6901 in blaNDM-1 genomic context

Tam-Duong Le-Ha,1 Lien Le,1 Hong-Ngoc Le-Vo,1 Mizue Anda,2 Daisuke Motooka,3 Shota Nakamura,3 Linh Khanh Tran,1 Phuong Thi-Bich Tran,1 Tetsuya Iida,2 Van Cao11Department of Immunology and Microbiology, Pasteur Institute in Ho Chi Minh City, Ho Chi Minh City, Vietnam; 2Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; 3Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, JapanAbstract: We report a clinical strain of Enterobacter cloacae, PIMB10EC27, isolated in Vietnam in 2010 that was resistant to 21 of 26 tested antibiotics, including carbapenems (MICs >64 μg/mL) and colistin (MIC >128 μg/mL). The complete genome of strain PIMB10EC27 was sequenced by PacBio RSII and the Illumina Miseq system. Whole-genome analysis revealed that PIMB10EC27 contains a chromosome of the ST513 group (PIMBEC27, length 5,272,177 bp) and two plasmids, pEC27-1 of the IncX3 group (length 62,470 bp) and pEC27-2 of the IncHI1 group (length 84,602 bp). It also revealed that strain PIMB10EC27 carries 15 genes that confer resistance to at least 10 antibiotic groups. Particularly, the insertion of ISKpn19 and Tn6901 into the genomic context of blaNDM-1 was first identified and described. In another context, amino acid mutations G273D in PmrB and F515S in PmrC were first identified on the chromosome of PIMB10EC27, which may confer resistance to colistin in this strain.Keywords: blaNDM-1, colistin, Enterobacter cloacae, multidrug-resistance, Tn690

Validity and Reliability of the Counseling Center Assessment of Psychological Symptoms‐Japanese

To identify students who are struggling with mental distress and provide them with early and appropriate support, a valid and reliable multidimensional measure is required. The aim of this study was to investigate the convergent validity and the test–retest reliability of the Counseling Center Assessment of Psychological Symptoms-Japanese (CCAPS-Japanese). For the validity examination, 1,627 undergraduate students were randomized into five groups. Each group completed one of five questionnaires, comprised of the CCAPS-Japanese along with one, two, or three validation scales depending on the group. For the reliability examination, a total of 184 and 106 students completed the CCAPS-Japanese at one-week and two-week intervals, respectively. In the validity study, the highest correlation for each CCAPS-Japanese subscale was found to exist with its referent measure except for the Generalized Anxiety subscale. In the reliability study, correlations for subscale scores at test and retest were significant, ranging from .66 to .88. These findings suggest that the 55-item CCAPS-Japanese is applicable for use with Japanese university students.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/175456/1/jpr12345_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/175456/2/jpr12345.pd

Tuberculostearic Acid Controls Mycobacterial Membrane Compartmentalization

ABSTRACT The intracellular membrane domain (IMD) is a laterally discrete region of the mycobacterial plasma membrane, enriched in the subpolar region of the rod-shaped cell. Here, we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. The putative gene cfa showed the most significant effect on recovery from membrane compartment disruption by dibucaine. Enzymatic analysis of Cfa and lipidomic analysis of a cfa deletion mutant (Δcfa) demonstrated that Cfa is an essential methyltransferase for the synthesis of major membrane phospholipids containing a C19:0 monomethyl-branched stearic acid, also known as tuberculostearic acid (TBSA). TBSA has been intensively studied due to its abundant and genus-specific production in mycobacteria, but its biosynthetic enzymes had remained elusive. Cfa catalyzed the S-adenosyl-l-methionine-dependent methyltransferase reaction using oleic acid-containing lipid as a substrate, and Δcfa accumulated C18:1 oleic acid, suggesting that Cfa commits oleic acid to TBSA biosynthesis, likely contributing directly to lateral membrane partitioning. Consistent with this model, Δcfa displayed delayed restoration of subpolar IMD and delayed outgrowth after bacteriostatic dibucaine treatment. These results reveal the physiological significance of TBSA in controlling lateral membrane partitioning in mycobacteria. IMPORTANCE As its common name implies, tuberculostearic acid is an abundant and genus-specific branched-chain fatty acid in mycobacterial membranes. This fatty acid, 10-methyl octadecanoic acid, has been an intense focus of research, particularly as a diagnostic marker for tuberculosis. It was discovered in 1934, and yet the enzymes that mediate the biosynthesis of this fatty acid and the functions of this unusual fatty acid in cells have remained elusive. Through a genome-wide transposon sequencing screen, enzyme assay, and global lipidomic analysis, we show that Cfa is the long-sought enzyme that is specifically involved in the first step of generating tuberculostearic acid. By characterizing a cfa deletion mutant, we further demonstrate that tuberculostearic acid actively regulates lateral membrane heterogeneity in mycobacteria. These findings indicate the role of branched fatty acids in controlling the functions of the plasma membrane, a critical barrier for the pathogen to survive in its human host