Emergence of an Extensive Drug Resistant Citrobacter portucalensis Clinical Strain Harboring bla SFO-1, bla KPC-2, and bla NDM-1.

Background: To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing bla SFO-1, bla NDM-1, and bla KPC-2. Methods: Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids. Results: The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying bla SFO-1, bla NDM-1, and bla KPC-2 on plasmids of various sizes. The uncommon ESBL gene bla SFO-1 coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS26-fosA3-IS26-ampR-bla SFO-1-IS26. The bla NDM-1 was found on an IncX3 plasmid, coexisting with bla SHV-12, displaying the sequence IS5-IS3000-IS3000-Tn2-bla NDM-1-ble-trpF-dsbD-cutA-gros-groL, lacking ISAa125. The bla KPC-2 is located on an unclassified plasmid, exhibiting the sequence Tn2-tnpR-ISKpn27-bla KPC-2-ISKpn6-korC. Conjugation assays confirmed the transferability of both bla NDM-1 and bla KPC-2. Conclusion: We discovered the coexistence of bla SFO-1, bla NDM-1, and bla KPC-2 in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains.

Paeoniflorigenone inhibits ovarian cancer metastasis through targeting the MUC1/Wnt/ß­catenin pathway.

Ovarian cancer (OC) is one of the most common gynecological malignancies. Currently, chemoradiotherapy is the primary clinical treatment approach for OC; however, it has severe side effects and a high rate of recurrence. Thus, there is an urgent need to develop innovative therapeutic options. Paeoniflorigenone (PFG) is a monoterpene compound isolated from the traditional Chinese medicine Paeoniae Radix Rubra. PFG can inhibit the proliferation of tumor cells; however, its anticancer activity against OC has yet to be elucidated. Mucin 1 (MUC1) is highly expressed in various malignant tumors, and is associated with tumor proliferation, metastasis and epithelial­mesenchymal transition (EMT). In addition, MUC1 affects numerous signaling pathways in tumor cells. In order to develop a possible treatment approach for metastatic OC, the antitumor activity of PFG in OC cells was investigated using Cell Counting Kit­8 assay, Edu assay, flow cytometry, Transwell assay and western blot analysis. In addition, it was assessed how PFG affects MUC1 expression and function. The experiments revealed that PFG significantly inhibited OC cell proliferation, migration, invasion and EMT. PFG also induced S­phase cell cycle arrest in OC cells. Furthermore, PFG inhibited MUC1 promoter activity, which led to a decrease in MUC1 protein expression. By contrast, MUC1 promoted OC progression, including cell proliferation, cell cycle progression and cell migration. Stable knockdown of MUC1 in OC cells improved the ability of PFG to block the Wnt/ß­catenin pathway, and to limit tumor cell invasion and migration, whereas MUC1 overexpression partially counteracted the antitumor effects of PFG. In conclusion, the present study demonstrated that PFG may inhibit the MUC1/Wnt/ß­catenin pathway to induce anti­metastatic, anti­invasive and anti­EMT effects on OC. Notably, MUC1 may be a direct target of PFG. Thus, PFG holds promise as a specific antitumor agent for the treatment of OC.

Regulation of hPCL3 isoforms' ubiquitination by TRIM21 in non-small cell lung cancer progression.

Non-small cell lung cancer (NSCLC) is the main subtype of lung cancer. The role of hPCL3 isoforms, hPCL3S and hPCL3L, remains ambiguous. This study examines the functional implications of these isoforms in NSCLC, using lung cancer cell lines A549 and NCI-H226c for in vivo and in vitro analyses. The results indicate that elevated expression of both hPCL3S and hPCL3L correlates with diminished overall survival, although only hPCL3S levels are augmented in clinical NSCLC specimens. Inhibition of either isoform leads to reduced cell proliferation, invasion, and migration, with hPCL3S knockdown displaying superior effectiveness. Moreover, the findings reveal that TRIM21 interacts with both isoforms and mediates hPCL3S degradation through K48-linked ubiquitination in NSCLC cells. Conversely, TRIM21 does not facilitate hPCL3L degradation, despite forming K63-linked polyubiquitin chains. These observations highlight the divergent roles of hPCL3 isoforms in NSCLC and underscore the potential therapeutic value of targeting hPCL3S.

Emergence of a NDM-1-producing ST25 Klebsiella pneumoniae strain causing neonatal sepsis in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) seriously threaten the efficacy of modern medicine with a high associated mortality rate and unprecedented transmission rate. In this study, we isolated a clinical K. pneumoniae strain DY1928 harboring bla NDM-1 from a neonate with blood infection. Antimicrobial susceptibility testing indicated that DY1928 was resistant to various antimicrobial agents, including meropenem, imipenem, ceftriaxone, cefotaxime, ceftazidime, cefepime, piperacillin-tazobactam, and amoxicillin-clavulanate. S1 nuclease-pulsed field gel electrophoresis (S1-PFGE), southern blot and conjugation experiment revealed that the bla NDM-1 gene was located on a conjugative plasmid of IncA/C2 type with a 147.9 kb length. Whole-genome sequencing showed that there was a conservative structure sequence (bla NDM-1-ble-trpF-dsbD) located downstream of the bla NDM-1 gene. Multilocus sequence typing (MLST) classified DY1928 as ST25, which was a hypervirulent K. pneumoniae type. Phylogenetic analysis of genomic data from all ST25 K. pneumoniae strains available in the NCBI database suggested that all bla NDM-1 positive strains were isolated in China and had clinical origins. A mouse bloodstream infection model was constructed to test the virulence of DY1928, and 11 K. pneumoniae strains homologous to DY1928 were isolated from the feces of infected mice. Moreover, we found that DY1928 had a tendency to flow from the blood into the intestine in mice and caused multiple organ damage. To our knowledge, this is the first study to report an infection caused by bla NDM-1-positive ST25 K. pneumoniae in the neonatal unit. Our findings indicated that stricter surveillance and more effective actions were needed to reduce the risk of disseminating such K. pneumoniae strains in clinical settings, especially in neonatal wards.

Characterizations of heavy metal contamination, microbial community, and resistance genes in a tailing of the largest copper mine in China.

Copper mine tailings are causing great environmental concern nowadays due to their high contents of heavy metals. These hazards may release to air, water, and soil, posing great threat to the living organisms in the surroundings. In the present work, we profiled the heavy metal contents, microbiome and resistome of a mine tailing in Dexing Copper Mine, which is the largest open-pit copper mine in China. A total of 39.75 Gb clean data was generated by metagenomics sequencing and taxonomy analysis revealed Actinobacteria, Proteobacteria, Acidobacteria, Euryarchaeota, and Nitrospirae as the most abundant phylum in this tailing. In general, 76 heavy metal resistance genes (HMRGs) and 194 antimicrobial resistance genes (ARGs) were identified with merA and rpoB2 as the most abundant HMRG and ARG, respectively. We also compared the differences of heavy metal concentrations among the six sampling sites in the same tailing and found that significant differences exited in copper and zinc. Hierarchical cluster analysis showed that the samples from the six sampling sites were clustering in two groups based on heavy metal concentrations. Accordingly, clustering based on microbial composition and relative abundances of resistance genes exhibited the same clustering pattern, indicating a possible shaping influence of heavy metals on the microbiome and resistome in this tailing. Our work presented heavy metal contents, microbial composition and resistance genes in a copper mine tailing of the largest copper mine in China, and these data will of great use in the surveillance, maintenance, and remediation of this tailing.

Occurrence and distribution of antimicrobial resistance genes in the soil of an industrial park in China: A metagenomics survey.

As zoned areas of industries, industrial parks have great impacts on the environment. Several studies have demonstrated that chemical compounds and heavy metals released from industrial parks can contaminate soil, water, and air. However, as an emerging pollutant, antimicrobial resistance genes (ARGs) in industrial parks have not yet been investigated. Here, we collected soil samples from 35 sites in an industrial park in China and applied a metagenomics strategy to profile the ARGs and virulence factors (VFs). We further compared the relative abundance of ARGs between the sites (TZ_31-35) located in a beta-lactam antimicrobial-producing factory and other sites (TZ_1-30) in this industrial park. Metagenomic sequencing and assembly generated 14, 383, 065 contigs and 17, 631, 051 open reading frames (ORFs). Taxonomy annotation revealed Proteobacteria and Actinobacteria as the most abundant phylum and class, respectively. The 32 pathogenic bacterial genera listed in the virulence factor database (VFDB) were all identified from the soil metagenomes in this industrial park. In total, 685,354 ARGs (3.89% of the ORFs) and 272,694 virulence factors (VFs) (1.55% of the ORFs) were annotated. These ARGs exhibited resistance to several critically important antimicrobials, such as rifampins, fluroquinolones, and beta-lactams. In addition, no significant difference in the relative abundance of ARGs was observed between sites TZ_31-35 and TZ_1-30, indicating that ARGs have already disseminated widely in this industrial park. The present study gave us a better understanding of the whole picture of the resistome and virulome in the soil of the industrial park and suggested that we should treat the industrial park as a whole in the surveillance and maintenance of ARGs.

Complete Genome Sequences of Leclercia sp. W6 and W17 Isolated from a Gastric Cancer Patient.

Leclercia sp. W6 and W17, which belong to the Enterobacteriaceae, were isolated from a stomach sample from a 78-year-old female gastric cancer patient, and genomic sequencing and analysis were performed. The genome of Leclercia sp. W6 consists of one chromosome with a size of 4,945,486 bp, while that of Leclercia sp. W17 contains one chromosome and two plasmids with a total size of 5,125,645 bp. Average nucleotide identity (ANI) calculations indicated that strains W6 and W17 exhibited similarities < 91.0% to other strains within the Enterobacteriaceae, except for six Leclercia strains. Phylogenomic analysis based on core-genome showed that strains W6 and W17 belong to the genus Leclercia, and phylogenetic analysis based on ANI values revealed that strains W6 and W17 formed an independent clade from those six Leclercia strains. Furthermore, comparative genomic analysis revealed that strains W6 and W17 had 5086 orthologous clusters (OCs) in their pan-genomes, and 59 exclusive OCs which were absent in their closest relatives. Genomic annotations revealed that the genomes of strains W6 and W17 encoded genes related to multidrug resistance clusters, multiple antibiotic resistance loci, and multidrug efflux pumps and had an identical urease gene cluster and a dissimilatory nitrate reduction pathway. Bioinformatic analyses indicated that strains W6 and W17 represented a novel species within the genus Leclercia. Genomic annotations revealed that these strains encoded genes related to multidrug resistance, nitrate reduction, and urease activity, which contribute to gastric malignant transformation. This will broaden our knowledge of the genetic mechanisms of the Enterobacteriaceae and help improve the clinical conditions of gastric cancer patients.

Adipose-tissue derived porcine mesenchymal stem cells efficiently ameliorate CCl4-induced acute liver failure in mice.

Adipose tissue derived mesenchymal stem cells (ADMSCs) may be an attractive therapeutic source for acute liver injury because of their high accessibility and non-invasiveness. Here, we investigated the therapeutic potentials of porcine ADMSCs for acute liver failure (ALF). The morphology, differentiation potential, expression patterns of cell surface markers and liver-specific genes were compared between the ADMSCs derived from the pigs with or without ALF. For therapeutic studies, the expanded porcine ADMSCs from either ALF pig (ALF-ADMSCs) or healthy control pig (Nor-ADMSCs) of passage 3 were transplanted into CCl4-induced ALF mice, and the liver histology and functional tests were performed at days 1, 7, 14, and 21 after cell transplantation. ALF-ADMSCs expressed higher mRNA level of hepatic growth factor (HGF) than the Nor-ADMSCs. Both ALF-ADMSCs and Nor-ADMSCs improved liver histology, functions, and mouse survival rate. Higher level of porcine hepatocyte-specific genes was seen in the livers of ALF-ADMSCs transplanted mice as compared to the Nor-ADMSCs transplanted mice. In particular, ALF-ADMSCs transplanted mice expressed significantly higher level of albumin and cytokeratin 18 in the liver tissues as compared to the Nor-ADMSCs transplanted mice. ALF-ADMSCs might be superior to Nor-ADMSCs in the treatment of ALF as the former possesses stronger hepatic differentiation potential.

PHD Finger Protein 19 Enhances the Resistance of Ovarian Cancer Cells to Compound Fuling Granule by Protecting Cell Growth, Invasion, Migration, and Stemness.

Ovarian cancer is one of the most common gynecological malignancies in women worldwide with a poor survival rate. We have previously reported that compound fuling granule (CFG), a traditional Chinese medicinal preparation used to treat ovarian cancer in China for over 20 years, significantly promotes cell cycle arrest, apoptosis, senescence, TGFß-induced invasion and migration, tumor growth, and distant metastasis in ovarian cancer cells. However, the underlying mechanisms are not clear. In the present study, we found that PHF19 expression in ovarian cancer cells positively correlated with their resistance ability to CFG. In addition, PHF19 overexpression increased the resistance of HEY-T30 and SKOV3 cells to CFG, while knockdown of PHF19 enhanced their sensitivity to CFG. Moreover, CFG significantly inhibited the expression of PHF19 both in mRNA and protein levels in these cells. Gain of function and loss of function experiments further proved that PHF19 is a crucial mediator involved in the ovarian cancer progression, including cell proliferation, invasion, migration, and stemness. Importantly, rescue the expression of PHF19 reverted CFG-induced suppression in ovarian cancer cell growth, EMT and stemness, while PHF19 knockdown accelerated CFG's anti-tumor effect. Overall, our results provide a series of evidence to reveal that PHF19 is critical suppressor for CFG's anti-tumor effect in ovarian cancer.

Co-Existence of mcr-1 and bla NDM-5 in an Escherichia coli Strain Isolated from the Pharmaceutical Industry, WWTP.

ABSTRACT: The emergence of the plasmid-borne colistin-resistant gene (mcr-1) poses a great threat to human health. What is worse, the recent observations of the co-existence of mcr-1 with other antimicrobial resistance genes in some bacteria cause further concern. Here, we present the first report of a wild Escherichia coli strain that co-carries an mcr-1 encoding phage-like IncY plasmid (pR15_MCR-1) and a bla NDM-5 encoding IncX3 plasmid (pR15_NDM-5) from a pharmaceutical industry, wastewater treatment plant, in China. This study highlights the spreading of E. coli carrying both mcr-1 and bla NDM-5 genes in the pharmaceutical industry. IMPORTANCE: Escherichia coli strains that carry both mcr-1 and bla NDM-5 genes are of great health concern and are already found in humans and animals worldwide, yet there is a paucity of observations of this resistant strain in the environment. Here we present the first isolation of an E. coli strain (R15) that co-carries mcr-1 and bla NDM-5 genes from a wastewater treatment plant in China. Whole-genome sequencing indicated that R15 harbored two plasmids, pR15_MCR-1 and pR15_NDM-5, that carry mcr-1 and bla NDM-5, respectively. The observation of this wild-derived E. coli strain that carries mcr-1 and bla NDM-5 genes simultaneously calls for the urgency to improve monitoring and reducing its further spreading.

Occurrence and Genomic Characterization of Two MCR-1-Producing Escherichia coli Isolates from the Same Mink Farmer.

The spread of colistin resistance gene mcr-1 at the animal-human interface remains largely unknown. This work aimed to investigate the molecular characteristics of two extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli strains with mcr-1, i.e., strains H8 and H9, isolated from the same mink farmer. In this study, five mcr-positive E. coli strains were isolated from the mink farm. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) identified two genetically unrelated MCR-1 producers (H8 and H9) from the same farmer and two clonally related MCR-1-positive isolates (M5 and M6) from two different mink samples. Additionally, a mcr-1 variant, designated mcr-1.12, was identified in isolate M4. MIC determination revealed that all of the MCR-producing strains exhibited multiresistant phenotypes but showed susceptibility to imipenem, meropenem, amikacin, and tigecycline. Replicon typing showed that mcr-1 was associated with IncHI2 plasmids in 4 cases, while the gene was located on an IncI2 plasmid in 1 case. PacBio sequencing and plasmid analysis confirmed that the mcr-1 gene was located on an ∼204-kb IncHI2 plasmid in H8 and was carried by an ∼61-kb IncI2 plasmid in H9. To our knowledge, this work represents the first report of the occurrence of MCR-producing isolates from mink. Moreover, our report also describes the coexistence of two different MCR-1 producers in the same farmer. It highlights that fur farms can be reservoirs of mcr-1 genes. The identification of mcr-carrying plasmids on a fur farm is of potential public health importance, as it suggests that mcr is widespread in the animal husbandry industry.IMPORTANCE Colistin resistance is a real threat for both human and animal health. The mobile colistin resistance gene mcr has contributed to the persistence and transmission of colistin resistance at the interfaces of animals, humans, and ecosystems. Although mcr genes have usually been recovered from food animals, patients, and healthy humans, transmission of mcr genes at the animal-human interface remains largely unknown. This was the first study to isolate and characterize MCR-producing isolates from mink, as well as to report the coexistence of two different MCR-1 producers in the same farmer. The characterization and analysis of two MCR-1-producing E. coli isolates may have important implications for comprehension of the transmission dynamics of these bacteria. We emphasize the importance of improved multisectorial surveillance of colistin-resistant E. coli in this region.

Genetic characterization of a novel sequence type of multidrug-resistant Citrobacter freundii strain recovered from wastewater treatment plant.

A multidrug-resistant Citrobacter freundii strain R17 was isolated from a wastewater treatment plant in China. Whole-genome sequencing of strain R17 revealed a new sequence type (ST412) chromosome (length 5,124,258 bp) and an Inc FII (Yp) group plasmid pCFR17_1 (length 206,820 bp). A total of 13 antibiotic-resistance genes (ARGs) that confer resistance to eight different antibiotic groups were encoded by strain R17 and 12 of them were carried by plasmid pCFR17_1. These data and analysis suggest that the environment-derived C. freundii strains may serve as potential sources of ARGs and highlight the need of further surveillance of this bacteria in the future.

Whole Genome Sequencing of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli Isolated From a Wastewater Treatment Plant in China.

BACKGROUND AND OBJECTIVES: Wastewater treatment plants (WWTPs) are one of the major reservoirs for antimicrobial resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) in the environment. Thus, the investigation on ARB and ARGs from WWTPs has attracted increasing attention in recent years. In order to uncover the resistome in a WWTP treating effluents from a pharmaceutical industry in China, the extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains were isolated and their whole genome sequences were obtained and analyzed. Moreover, metagenomic sequencing was applied to give a comprehensive view of antibiotic resistance in this WWTP. METHODS: 18 ESBL-producing E. coli strains were isolated from a WWTP located in Taizhou, China on April, 2017. All strains were sequenced using Illumina HiSeq 2000 sequencer. The whole genome sequences were assembled using SPAdes software and annotated with RAST server. Sequence types (STs), plasmids, ARGs and virulence genes were predicted from the genomes using MLST, Plasmid Finder, ResFinder and Virulence Finder, respectively. Metagenomic DNA of the same sample was extracted and sequenced using Illumina Hiseq X Ten platform. Metagenomic sequences were assembled using SOAPdenovo software. RESULTS: All 18 ESBL-producing E. coli strains were resistant to ampicillin, cefazolin, and ceftriaxone. Analysis of their genomes revealed that all strains carried beta-lactamase encoding genes and the most prevalent type was bla CTX-M . Various virulence genes and ARGs confronting resistance to other types of antimicrobial agents were also predicted. Further investigation on the metagenomics data indicated 11 ARGs with high amino acid identities to the known ARGs. Five of these ARGs, aadA1, aac(6')-lb-cr, flo(R), sul2 and sul1, were also present in the genomes of the ESBL-producing E. coli isolated from the same sample. CONCLUSION: Our study revealed the resistome of a pharmaceutical WWTP by both culture-dependent and metegenomic methods. The existence of ESBL-producing E. coli strains, indicating that pharmaceutical WWTP can play a significant role in the emergence of ARB. The occurrence of ARGs annotated from the metagenomic data suggests that pharmaceutical WWTP can play a significant role in the emergence of ARGs. Our findings highlight the need for strengthening the active surveillance of ARB and ARGs from pharmaceutical industry.

Detection and characterization of ESBL-producing Escherichia coli expressing mcr-1 from dairy cows in China.

Objectives: To investigate the prevalence and molecular characteristics of ESBL-producing Escherichia coli (ESBL-EC) in faecal samples from dairy cows in China. Methods: In total, 651 faecal samples were collected from cows distributed among the 10 provinces of China. Potential ESBL-EC isolates were cultured on selective medium. The clonal relatedness of the ESBL-EC isolates was assessed using MLST. WGS was conducted on 3 mcr-positive isolates and 14 additional randomly selected ESBL-EC isolates. Southern blot, S1-PFGE and conjugation were performed for mcr-1-carrying isolates. The genetic environment of the pMCR-JLF4 plasmid was also analysed. Results: In total, 290 unique ESBL-EC isolates were detected from 284 cows (43.6%). Alleles of CTX-M were observed in 94.1% (273/290) of all isolates. The most prevalent genotypes observed in this study were blaCTX-M-14, blaCTX-M-15, blaCTX-M-17 and blaCTX-M-55. Differentiation of 79 STs with a polyclonal structure was accomplished using MLST. Clonal complex 10 was the most prevalent major complex detected here. Furthermore, the mcr-1 gene was detected in three isolates. The complete sequence of the mcr-1-containing pMCR-JLF4 was determined. The plasmid was 66.7 kb in length, with a genetic structure of nikA-nikB-mcr-1-pap2. Conjugation analysis confirmed that the mcr-1 gene in pMCR-JLF4 was transferable without the assistance of the ISApl1 gene. Conclusions: The data presented here suggest high prevalence of ESBL-EC in Chinese cow farms. Furthermore, it was clearly demonstrated that commensal E. coli strains can be reservoirs of blaCTX-M genes, potentially contributing to the dissemination and transfer of the mcr-1 gene to pathogenic bacteria among cows.

Complete genome sequencing of Comamonas kerstersii 8943, a causative agent for peritonitis.

Because of poor differentiation among the members of genus Comamonas using phenotypic methods, human infections caused by C. kerstersii are sporadically reported in the literature. Here, we represent the first complete genome sequence of C. kerstersii 8943, which caused peritonitis in a patient with continuous ambulatory peritoneal dialysis (CAPD). The complete genome with no gaps was obtained using third-generation Pacific Biosciences (PacBio) RSII sequencing system with single-molecule real-time (SMRT) analysis. Protein-coding genes, rRNAs and tRNAs were predicted. Functional annotations of the genome using different databases revealed several genes related to pathogenicity including antibiotic resistance genes and prophages. Our work demonstrates that whole genome sequencing can enhance the resolution of clinical investigations and our data can be used as a reference genome during the rapid diagnosis of C. kerstersii infections in the future.

Biochemical and genetic characterization of a novel metallo-ß-lactamase from marine bacterium Erythrobacter litoralis HTCC 2594.

Metallo-ß-lactamases (MBLs) are a group of enzymes that can inactivate most commonly used ß-lactam-based antibiotics. Among MBLs, New Delhi metallo-ß-lactamase-1 (NDM-1) constitutes an urgent threat to public health as evidenced by its success in rapidly disseminating worldwide since its first discovery. Here we report the biochemical and genetic characteristics of a novel MBL, ElBla2, from the marine bacterium Erythrobacter litoralis HTCC 2594. This enzyme has a higher amino acid sequence similarity to NDM-1 (56%) than any previously reported MBL. Enzymatic assays and secondary structure alignment also confirmed the high similarity between these two enzymes. Whole genome comparison of four Erythrobacter species showed that genes located upstream and downstream of elbla2 were highly conserved, which may indicate that elbla2 was lost during evolution. Furthermore, we predicted two prophages, 13 genomic islands and 25 open reading frames related to insertion sequences in the genome of E. litoralis HTCC 2594. However, unlike NDM-1, the chromosome encoded ElBla2 did not locate in or near these mobile genetic elements, indicating that it cannot transfer between strains. Finally, following our phylogenetic analysis, we suggest a reclassification of E. litoralis HTCC 2594 as a novel species: Erythrobacter sp. HTCC 2594.

First detection and genomics analysis of KPC-2-producing Citrobacter isolates from river sediments.

The wide spread of carbapenemase-producing Enterobacteriaceae (CPE) in the environment is an emerging environmental issue with potentially-serious public health implications. However, carbapenemase-producing Citrobacter from environment has rarely been investigated. Here we report the isolation and comparative genomics of carbapenemase-producing Citrobacter isolates from river sediment in China. Potential CPE was isolated by selective MacConkey agar plates containing 2 mg/L meropenem. The presence of carbapenemase genes was detected by PCR and sequencing. The clonal relatedness of Klebsiella pneumoniae carbapenemase (KPC-2)-producing Citrobacter isolates was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Plasmid analysis of KPC-2-producing Citrobacter isolates was performed by S1-PFGE, Southern blotting, and whole genome sequencing. A total of four KPC-2-producing Citrobacter and three Aeromonas isolates were recovered from 54 sediment cultures of Shifeng River. Notably, all KPC-producing isolates were isolated from sampling sites near a waste water treatment plant. Antimicrobial susceptibility testing showed that three of the four sequenced isolates (C1710, C191, and C196) resistant to multiple antibiotics. Genotyping and pan-genome analyses revealed that the C191 and C196 C. freundii isolates exhibited a high level of genetic similarity. Plasmid analysis confirmed that the blaKPC-2 gene is located on either IncF or IncN3 plasmids in all isolates. The blaKPC-2 gene of C1710, C181 and C191 was successfully transferred with E. coli EC600 as the recipient strain. In silico analysis further suggested that pKPC-191 is a novel IncF plasmid, with 99% identity to two previously described IncFII plasmids at 71% coverage. We report here the presence of diverse conjugative blaKPC-2 plasmids from environmental Citrobacter isolates, which poses the possible dissemination of antimicrobial resistance into clinical isolates. To our knowledge, this is the first study to culture and characterize KPC-2-producing Citrobacter isolates from river sediments in China.