Genomic insight into transmission mechanisms of carbapenem-producing Citrobacter spp. isolates between the WWTP and connecting rivers.

Carbapenem-resistant Enterobacteriaceae (CRE) poses major health risks worldwide. Most studies have focused on carbapenem resistance in Klebsiella pneumoniae and Escherichia coli; however, the occurrence and transmission of carbapenem-resistant Citrobacter spp. (CRCS) are poorly understood. In this study, we investigated the occurrence and potential transmission patterns of CRCS in different functional areas of an urban wastewater treatment plant (WWTP) and connecting rivers during one-year monitoring in Shandong Province, China. In total, 14 CRCS were detected in 376 environmental samples, including those from the WWTP inlet (n = 7), WWTP anaerobic tank (n = 2), and rivers (n = 5). Citrobacter braakii (n = 6) was the dominant subtype among 14 CRCS isolates, followed by Citrobacter freundii (n = 5), Citrobacter sedlakii (n = 2), and Citrobacter werkmanii (n = 1). All CRCS were resistant to imipenem, meropenem, ampicillin, amoxicillin/clavulanic acid, cefotaxime, ceftazidime, trimethoprim/sulfamethoxazole, and ciprofloxacin. Plasmid analysis showed that the blaKPC-2 gene was located on IncN and IncFII (Yp) plasmids, whereas the blaNDM gene was located on IncX3 and IncN2 plasmids. Clonal transmission of CRCS harboring carbapenem genes occurred between the WWTP and connecting rivers on a temporal or spatial scale. High genomic relatedness of NDM-5-producing C. sedlakii was identified between river water and WWTP aerosol, suggesting a potential exposure risk of CRCS for workers and surrounding residents near the WWTP. Furthermore, NDM-5-producing C. sedlakii isolated from rivers was related to C. sedlakii isolated from soil and well water in different regions of China, indicating that NDM-5-producing C. sedlakii may be widespread in China. These findings indicate that rare healthcare-associated pathogens such as CRCS can contribute to widespread carbapenem production in the environment; thus, CRCS should be continuously monitored.

Functional metabolomics revealed functional metabolic-characteristics of chronic hepatitis that is significantly differentiated from acute hepatitis in mice.

Diagnosis and therapeutics of acute- and chronic- hepatitis (A-H and C-H) cannot be distinguished during clinical practice because functional molecular-characteristics of two conditions remains elusive. Here, we employed a functional metabolomics strategy to discover functional metabolites that can readily distinguish C-H from A-H in CCl4 treated mice. Metabolic-differentiation between A-H and C-H was identified as A-H was largely characterized by the dysregulated purine cycle and amino acid metabolism, while the disorders of hepatic taurine-conjugated bile acids and glycerolipid biosynthesis were observed with C-H. Excitingly, we found that the enhanced conversation of C18-22 PUFA-containing TAGs to MUFA-containing TAGs promoted the development of C-H, which was also closely associated with the changes of TCA intermediates regulated by gut microbiota (Muribaculaceae and Prevotellaceae). Such metabolic discovery on hepatitis was validated by the functional annotation of metabolic genes, as the decreased expressions of Slc27a2, Acaa1a and Acaa1b mostly account for the dysregulation of purine degradation with AH, then the lowered expressions of Cyp2e1, Cat, Slc27a5 and Klb are significantly related to the dysregulated bile acids with C-H. Collecting clinical samples from the patients with hepatitis to compare serum metabolomes with A-H and C-H mice, the determinant functional metabolites were identified to significantly distinguish C-H from A-H in both experimental and clinical settings, suggesting metabolic discovery with CCl4 treated mice could be further efficiently explored to guide clinical research of A-H and C-H. Collectively, our study is providing novel insight into distinctive metabolic-characteristics of A-H and C-H underlying the innovative diagnosis and therapeutics of hepatitis.

Emergence of IncX3 Plasmid-Harboring blaNDM-5 in a Citrobacter sedlakii Isolated from Outdoor Aerosol in Wastewater Treatment Plant.

A carbapenem-resistant Citrobacter sedlakii strain AA2CS carrying blaNDM-5 was detected in outdoor aerosols of a wastewater treatment plant (WWTP) in China and the whole genome was sequenced subsequently. AA2CS was captured in an aerobic tank with aerosol particles of sizes ranging from 4.7 to 7.0 µm. Besides blaNDM-5, AA2CS also harbored 21 other antibiotic resistance genes and displayed a high level of resistance to ampicillin, cefotaxime, ceftazidime, tetracycline, and meropenem. BlaNDM-5 was located on the IncX3 plasmid (pCSNDM-5) with an IS3000-IS5-blaNDM-5-bleMBL-trpF-dsbD-IS26 structure. pCSNDM-5 was highly homologous to other blaNDM-5-carrying IncX3 plasmids in China and can be transferred to the Escherichia coli recipient J53. To our knowledge, this is the first report of carbapenem-resistant Enterobacteriaceae in outdoor aerosols in WWTPs.

Dissemination of blaNDM-5 via IncX3 plasmids in carbapenem-resistant Enterobacteriaceae among humans and in the environment in an intensive vegetable cultivation area in eastern China.

The environment of a large-scale vegetable production area can be exposed to antibiotic residues and antibiotic-resistant bacteria (ARB) via animal manure and irrigation with contaminated water, which can facilitate the dissemination of ARB. However, the occurrence of ARB in plantation areas and their dissemination in this environment remain largely unexplored. In total, 382 samples including those from vegetable (n = 106), soil (n = 87), well water (n = 24), river water (n = 20), river sediments (n = 20), farmer feces (n = 58) and farmer hands (n = 67) were collected in 2019 from a large-scale cultivation area in Shandong, China. Selective agar plates were used to screen for carbapenem-resistant Enterobacteriaceae (CRE) and whole-genome sequencing and Southern blotting were used to characterise isolates and mobile genetic elements carrying carbapenem resistance determinants. A total of nine NDM-5-producing isolates of Escherichia coli, Klebsiella pneumoniae, and Citrobacter spp. were identified from environmental sources and human feces, all of which were multidrug-resistant. Single nucleotide polymorphism analysis suggested clonal transmission of carbapenem-resistant Citrobacter sedlakii within greenhouse soils in the area. Eight of the isolates carried closely related or identical IncX3 plasmids carrying blaNDM-5, which were shown to be conjugative via filter mating experiments, indicating the highly transmissible nature of this genetic element. Isolates of E. coli and Citrobacter freundii were detected in the feces of local farm workers and contained similar IncX3 plasmids with blaNDM-5 environmental isolates, suggesting a potential risk of CRE transfer from the work environment to the farm workers. Thus, further research is required to investigate the potential health risks associated with environmental exposure to CRE in vegetable cultivation areas.

Genetic characterization and virulence of a carbapenem-resistant Raoultella ornithinolytica isolated from well water carrying a novel megaplasmid containing blaNDM-1.

Infections caused by carbapenem-resistant Enterobacteriaceae are a growing concern worldwide. Raoultella ornithinolytica is a species in the Enterobacteriaceae family which can cause hospital-acquired infections and is sporadically reported as carbapenem-resistant from human and environmental sources. In this study, we firstly report on an NDM-1-producing R. ornithinolytica, Rao166, isolated from drinking water in an animal cultivation area in China. In addition to carbapenem-resistance, Rao166 was resistant to several other antibiotics including gentamicin, sulfamethoxazole-trimethoprim, tetracycline and fosfomycin. Rao166 carried a novel IncFIC-type megaplasmid, 382,325 bp in length (pRAO166a). A multidrug resistance region, 60,600 bp in length, was identified in the plasmid containing an aac(3)-IId-like gene, aac(6')-Ib-cr, blaDHA-1, blaTEM-1B, blaCTX-M-3, blaOXA-1, blaNDM-1, qnrB4, catB3, arr-3, sul1, and tet(D). Results from virulence assays implied that Rao166 has considerable pathogenic potential. Although pRAO166a was found to be non-transmissible, dissemination of the NDM-1 producing strain may occur from well water to humans or animals through cross-contamination during food preparation or directly via drinking water, and potentially lead to difficult-to-treat infections. Thus, contamination of well water by this carbapenem-resistant and presumptively virulent strain of R. ornithinolytica should be considered a potential public health risk.

Characterization of Chromosome-Mediated BlaOXA-894 in Shewanella xiamenensis Isolated from Pig Wastewater.

A new variant of the blaOXA-546 gene, namely blaOXA-894, was identified on the chromosome of Shewanella xiamenensis isolated from pig wastewater in rural China. OXA-894 differs from OXA-546 (A46V, I219del) and OXA-48 (T167I, I219del) with two amino acid substitutions, respectively. The isolate was resistant to ampicillin, aztreonam, imipenem, meropenem and fosfomycin. Carba NP test confirmed S. xiamenensis strain sx20 as a carbapenemase-producer. The blaOXA-894 gene was located between the gene encoding a LysR family transcriptional regulator and the C15 gene. Its gene environment was similar to other S. xiamenensis with chromosome-located blaOXA-48-like genes. The T24H and T94V amino acid substitutions of LuxS protein were predicted to be deleterious, which may affect the virulence phenotype. The occurrence and potential health risk of carbapenem-resistant S. xiamenensis in a water environment is of concern.

Genomic Analysis Of A KPC-2-Producing Klebsiella Pneumoniae ST11 Outbreak From A Teaching Hospital In Shandong Province, China.

PURPOSE: Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bacteria causes nosocomial infections worldwide. However, KPC-producing K. pneumoniae outbreak has never been reported in Shandong Province, China. The purpose of our study was to elucidate the epidemiological and drug resistance mechanisms of KPC-producing K. pneumoniae strains collected from a large teaching hospital in Shandong during the outbreak. Moreover, we attempted to characterize the genetic environment and phylogenetic analysis of bla KPC-2 in outbreak isolates. METHODS: We monitored a 64-day outbreak of infection in a general hospital in Shandong Province, and the bacteria causing the infection were all ST11-type K. pneumoniae. The genotype correlation of KPC-producing K. pneumoniae isolates was assessed by whole-genome sequencing (WGS) phylogenetic analysis. Subsequent studies included antibiotic susceptibility testing, multilocus sequence typing (MLST) and S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blot hybridization. RESULTS: From February 1, 2018 to April 5, 2018, 14 KPC-producing K. pneumoniae isolates from different wards were collected. All 14 isolates were resistant to carbapenems and carried the extended-spectrum ß-lactamase (ESBL) gene as well as fosA, and sul genes. Whole-genome analysis showed that all 14 the outbreak isolates were all ST11 type. The bla KPC-2 carrying plasmids were all belong to IncFIIK2 type, and the size ranged from 94 kb to 368 kb. CONCLUSION: As far as we know, this report first describes the genomics characterization of KPC-2-producing K. pneumoniae outbreak isolates from Shandong Province, China. In our study, these isolates appeared to be cloned, and ST11 K. pneumoniae was the major clone caused the outbreak. Therefore, routine surveillance of such strains in this region is urgently warranted.

PARSUC: A Parallel Subsampling-Based Method for Clustering Remote Sensing Big Data.

Remote sensing big data (RSBD) is generally characterized by huge volumes, diversity, and high dimensionality. Mining hidden information from RSBD for different applications imposes significant computational challenges. Clustering is an important data mining technique widely used in processing and analyzing remote sensing imagery. However, conventional clustering algorithms are designed for relatively small datasets. When applied to problems with RSBD, they are, in general, too slow or inefficient for practical use. In this paper, we proposed a parallel subsampling-based clustering (PARSUC) method for improving the performance of RSBD clustering in terms of both efficiency and accuracy. PARSUC leverages a novel subsampling-based data partitioning (SubDP) method to realize three-step parallel clustering, effectively solving the notable performance bottleneck of the existing parallel clustering algorithms; that is, they must cope with numerous repeated calculations to get a reasonable result. Furthermore, we propose a centroid filtering algorithm (CFA) to eliminate subsampling errors and to guarantee the accuracy of the clustering results. PARSUC was implemented on a Hadoop platform by using the MapReduce parallel model. Experiments conducted on massive remote sensing imageries with different sizes showed that PARSUC (1) provided much better accuracy than conventional remote sensing clustering algorithms in handling larger image data; (2) achieved notable scalability with increased computing nodes added; and (3) spent much less time than the existing parallel clustering algorithm in handling RSBD.

Identification of alternative 5'/3' splice sites based on the mechanism of splice site competition.

Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5'/3' splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified approximately 70% of the splice sites into alternative and constitutive, as well as approximately 80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.

The effect of U1 snRNA binding free energy on the selection of 5' splice sites.

The importance of U1 snRNA binding free energy in the regulation of alternative splicing has been studied in some genes with site-directed mutagenesis. Here we report a large-scale analysis of its impact on 5' splice site (5'ss) selection in human genome. The results show that free energy exerts different effects on alternative 5'ss choice in different situations and -8.1 kcal/mol is a threshold. When both free energies of two competing 5'ss are larger than -8.1 kcal/mol, the 5'ss with lower free energy is more frequently used. However, in other pairs of 5'ss, lower-free-energy 5'ss does not seem to be favored and even the other 5'ss is used more frequently, which suggests that very low binding free energy would impair splicing. Some observations hold true only for those alternative 5' splicing with short alternative exons (<50nt), which implies a complex mechanism of 5'ss selection involving both U1 snRNA binding free energy and regulatory factors.