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Background Citrobacter freundii is the most common class of pathogens in the genus Citrobacter and is an important pathogen associated with certain underlying diseases or immune dysfunction. The aim of this study was to elucidate the resistance mechanism of clinically derived carbapenem-resistant C. freundii isolate and to characterize the genetic environment and delivery pattern of the IncN1 plasmid carrying the blaIMP-4 gene from C. freundii isolate. Materials and Methods We identified a clinical isolate of C. freundii L91 carrying blaIMP-4 and performed phylogenetic analysis by whole-genome sequencing. The complete genomic sequence of L91 was obtained using the Illumina HiSeq 4000-PE150 and PacBio RS II platforms. Antimicrobial susceptibility testing was determined by the VITEK 2 system. Plasmid characteristics were presented by S1-pulsed-field gel electrophoresis (PFGE), Southern blotting and conjugation experiments. Results S1-PFGE, Southern blot and conjugation assay confirmed the presence of blaIMP-4 genes on a conjugative plasmid in this isolate. C. freundii L91 and transconjugant L91-E. coli 600 strains both showed resistance to carbapenems. In silico analysis further showed that pIMP-4-L91 is an IncN1 plasmid with a length of 51,042 bp. Furthermore, blaIMP-4 gene was found encoded in the blaIMP-4-qacG2-aacA4-catB3 cassette array within a class 1 integron. A conserved structure sequence (ΔISKpn27-blaIMP-4-ΔISSen2-hp-hp-IS6100) was found in the upstream and downstream of the blaIMP-4. Conclusion We performed a comprehensive phylogenetic analysis of carbapenemase-resistant C. freundii and elucidated the resistance mechanism of clinically derived C. freundii L91. Not only that, we also found that the blaIMP-4 gene is located on the IncN1 plasmid and has a horizontal transfer function and a certain ability to spread. To lower the risk of the dissemination of such C. freundii isolates in clinical settings, more surveillance is needed in the future.

To investigate the prevalence, probable mechanisms and serotype correlation of colistin resistance in clinical isolates of Salmonella from patients in China, Salmonella isolates were collected from fecal and blood samples of patients. In this study, 42.8% (136/318) clinical isolated Salmonella were resistant to colistin. MIC distribution for colistin at serotype level among the two most prevalent serotypes originating from humans in China indicated that Salmonella Enteritidis (83.9% resistance, 125/149) were significantly less susceptible than Salmonella Typhimurium (15.3% resistance, 9/59, P < 0.01). mcr genes and mutations in PmrAB confer little for rate of colistin resistant Salmonella isolated from human patients. Phylogenetic tree based on core-genome single nucleotide polymorphisms (SNPs) was separately by the serotypes and implied a diffused distribution of MICs in the same serotype isolates. Relatvie expression levels of colistin resistant related pmr genes were significantly higher in non-mcr colistin resistant S. Typhimurium than in colistin sensitive S. Typhimurium, but no discernable differences between colistin resistant and sensitive S. Enteritidis, indicating a different mechanism between colistin resistant S. Typhimurium and S. Enteritidis. In conclusion, colistin susceptibility and colistin resistant mechanism of clinical isolated Salmonella were closely associated with specific serotypes, at least in the two most prevalent serotype Enteritidis and Typhimurium. We suggest clinical microbiology laboratory interpreting Salmonella colistin MIC results in the serotype level. Funding Agencies|National Key R&D Program of China [2017YFC1600100]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81702040]; National Science Foundation of Zhejiang province, China [LY20H190002]; Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas)Swedish Research Council Formas [2016-00640]; Swedish Foundation for International Cooperate in Research and Higher Education (STINT) [CH2016-6707]

Background Carbapenem-resistant Acinetobacter baumannii (CRAB) is a rapidly emerging, life-threatening nosocomial infection. This study aimed to explore the risk factors, clinical features, antimicrobial therapy, and outcomes of CRAB bloodstream infections (BSIs). Methods This is a retrospective, comparative analysis of data from patients with A. baumannii BSI, treated from 2012 to 2015 at a tertiary teaching hospital. Risk factors associated with CRAB BSI and factors associated with the 28-day mortality were evaluated using logistic analyses. Results Data from 293 patients with confirmed A. baumannii BSI were included; 242 (82.6%) patients had CRAB BSI and 51 (17.4%) patients had non-CRAB BSI. Risk factors significantly associated with CRAB BSI were a previous intensive care unit (ICU) stay (P=0.029), cefoperazone-sulbactam use (P=0.030), and carbapenem use (P=0.004). Among 236 patients with A. baumannii BSI who were evaluable at 28 days after receiving antibacterial therapy, there were 86 deaths. Factors associated with the 28-day mortality were ICU stay after BSI (P=0.040), readmission within 90 days (P=0.029), Acute Physiology and Chronic Health Evaluation II (APACHE II) score at diagnosis >19 (P=0.012), tigecycline therapy (P=0.021), presence of septic shock (P=0.029), and multiple organ failure (P=0.016). Death rates in patients treated with tigecycline were 53.5% vs 24.1% in patients treated with other agents. Among 186 patients with CRAB BSI evaluable at 28 days, 84 patients died. The associated risk factors were an ICU stay after BSI (P=0.036), APACHE II score >19 at diagnosis (P = 0.002), presence of septic shock (P=0.030), and multiple organ failure (P=0.007). Conclusion This study demonstrated that an ICU stay and cefoperazone-sulbactam or carbapenem use were seen to be the risk factors associated with the development of CRAB BSI. Critical illness and tigecycline therapy were significantly associated with higher mortality of patients with A. baumannii BSI.

To assess the genomic profiles of tigecycline (Tgc)-resistant Acinetobacter baumannii, including antibiotic resistance (AR) genes and virulence factors (VF), whole-genome shotgun sequencing was performed on 19 Tgc-resistant (TgcR) A. baumannii strains collected in a tertiary hospital during the early phase of the clinical introduction of Tgc in China from late 2012 to mid-2014. The major sample types containing TgcR strains were sputum and drain fluid. Data from an average of 624 Mbp of sequence was generated on each bacterial genome, with Q30 quality of 90%, and an average coverage of 96.6%. TCDC-AB0715 was used as a reference genome. The genome sequences were annotated for functional elements including AR genes, VFs, genome islands, and inserted sequences before they were comparatively analyzed. The antibiotic susceptibility phenotypes of the strains were examined by a broth microdilution method to determine the minimal inhibitory concentration (MIC) of strains against major clinical antibiotics. The AR genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database (CARD). Thirty-three ARGs were shared by all 19 TgcR strains, and 24 ARGs were distributed differently among strains. A total of 391 VFs were found to be diversely distributed in all TgcR strains. Based on ARG number distribution, the 19 TgcR strains were divided into several groups. Highly differentiated genes included gpi, mphG, armA, msrE, adec, catB8, aadA, sul1, blaOXA–435, aph3i, and blaTEM–1, which may represent gene markers for TgcR A. baumannii sub-types. In addition, when compared with Tgc-sensitive (TgcS) strains collected during the same period, TgcR strains featured enrichment of ARGs including aph6id, aph3ib, and teta. Compared with 26 other whole-genome sequences of A. baumannii deposited in GeneBank, TgcR strains in this study commonly lacked the EF-Tu mutation for elfamycin resistance. Previous investigation of three A. baumannii strains isolated from one patient indicated genomic exchange and a homologous recombination event associated with generation of tigecycline resistance. This study further analyzed additional TgcR strains. Phylogenetic analysis revealed a close evolutionary relationship between 19 TgcR strains and to isolates in East and Northeast China. In short, the comprehensive functional and comparative genomic analysis of 19 clinical TgcR A. baumannii strains isolated in the early stage of Tgc usage in China revealed their close phylogenetic relationship yet variable genetic background involving multiple resistance mechanisms. Using a simple ARG or VF gene number diversity method and marker genes, TgcR strain sub-types can be identified. The distinct characteristics of TgcR A. baumannii strains with versatile genomic resistance and regulation patterns raise concern regarding prediction and control of Tgc resistance in the clinic.

We report the characterization of six carbapenem-resistant Raoultella spp. (CRRS) in our hospital and a genomic analysis of 58 publicly available isolates. CRRS isolates are sporadically identified around the world, and different transposons carrying carbapenemases were the resistant mechanisms. Mobile genetic elements play an important role in acquiring antibiotic resistance genes from the hospital., We report the characterization of six carbapenem-resistant Raoultella spp. (CRRS) in our hospital and a genomic analysis of 58 publicly available isolates. CRRS isolates are sporadically identified around the world, and different transposons carrying carbapenemases were the resistant mechanisms. Mobile genetic elements play an important role in acquiring antibiotic resistance genes from the hospital. An improved understanding of these transposon and targeted control measures will be very valuable to prevent CRRS dissemination.

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.

Metallo-β-lactamases (MBLs) are a group of enzymes that can inactivate most commonly used β-lactam-based antibiotics. Among MBLs, New Delhi metallo-β-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.

Objectives The blaKPC gene is rarely reported in Citrobacter koseri. Here we report the first draft genome sequence of a blaKPC-2-carrying C. koseri isolate from a patient with diarrhoea. Methods Transferability of the blaKPC-2-bearing plasmid was determined by the filter mating method. The whole genome sequence of C. koseri L168 was determined using an Illumina HiSeq platform. The genome was de novo assembled using Velvet 1.2.10. Acquired antimicrobial resistance genes and plasmid replicons were identified using ResFinder 2.1 and PlasmidFinder 1.3, respectively. Results Antimicrobial susceptibility testing (AST) showed that C. koseri L168 was resistant to multiple antibiotics but was susceptible to ciprofloxacin, gentamicin, tobramycin, amikacin, tigecycline and colistin. A KPC-2-harbouring plasmid was conjugative and the transconjugants conferred increased resistance to carbapenems confirmed by conjugation experiments and AST. In silico analysis revealed the presence of the β-lactam resistance genes blaKPC-2 and blaMAL-1. Additionally, plasmids of incompatibility groups IncFII and IncX4 were identified in the genome by PlasmidFinder. BLAST analysis revealed that blaKPC-2 was located on a Tn3 transposon element in C. koseri L168 with the conserved linear structure ISKpn27–blaKPC-2–ΔISKpn6–korC–klcA. Conclusions To our knowledge, this is only the second report of C. koseri producing KPC-2, and we report the first draft genome sequence of a blaKPC-2-carrying C. koseri isolate from a patient with diarrhoea in China. This work may facilitate our understanding of the pathogenesis, multidrug resistance mechanisms and genomic features of this species. Further monitoring of bacteria carrying carbapenemase genes in patients’ gut microbiota is warranted.

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.

The increasing prevalence of KPC-producing Klebsiella pneumoniae (KPC-Kp) strains poses a serious threat to patients. Therapeutic options are limited to colistin, fosfomycin, tigecycline and selected aminoglycosides. Although the combination of fosfomycin with other antimicrobials is recommended, data regarding possible synergistic activity in vitro and in vivo appear inconsistent. Here we report that five drug combinations (fosfomycin combined with imipenem, ertapenem, tigecycline, colistin or amikacin) had a significant additive effect against 136 KPC-Kp strains in an in vitro chequerboard assay. In addition, time-kill assays revealed that fosfomycin enhanced the bactericidal activity of the five other antimicrobial agents. Moreover, owing to its persistent bactericidal effect, the combination of fosfomycin plus amikacin is an effective therapeutic candidate for infections by KPC-producing organisms.

Clostridium difficile infection (CDI) is associated with risk for severe disease and high mortality. Little is known about the extent of hospital-acquired CDI in Mainland China. In this study, we aimed to investigate the annual CDI incidence, bacterial genotypes, risk factors for severe CDI and survival over a 7-year period. A total of 307 hospital-acquired CDI patients were enrolled, and 70.7% of these cases were male. CDI incidence was 3.4 per 10,000 admissions. Thirty-three different sequence types (STs) were identified, among which ST-54 (18.2%), ST-35 (16.6%) and ST-37 (12.1%) were the most prevalent. During the follow-up period, 66 (21.5%) patients developed severe CDI and 32 (10.4%) patients died in 30 days. Multivariate analysis revealed that bloodstream infection, pulmonary infection and C-reactive protein were significantly associated with severe CDI. After adjustment for potential confounders, old age, bloodstream infection, fever, mechanical ventilation, connective tissue disease, macrolide use and hypoalbuminaemia were independently associated with 30-day mortality in patients with CDI. The CDI prevalence has been low and stable in our center, and STs of Clostridium difficile were different from dominant STs in Western countries. Our data emphasize the need of continued education and surveillance of CDI to reduce the CDI burden in China.

Background: The emergence and spread of ST11 carbapenem-resistant, hypervirulent K. pneumonia (ST11-CR-HvKP) in China generated great concern from the public health community. The identification of ST11-CR-HvKP strain is expected to become a serious public health issue in China, considering the carbapenem resistance and virulence had converged in an epidemic clone. However, the underlying mechanism that enables its wide dissemination in China remains unclear.Results: Here, we investigate the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) carriage by inpatients in a teaching hospital over a 1-year period, to identify ST11-CR-HvKP reservoirs, and to understand the transmission of these pathogens across healthcare networks. We identified a high colonization prevalence of CPE (12.4%) among inpatients with diarrhea. Correlations were detected between antibiotic exposure, surgical history, and being CPE positive. A genomic investigation of 65 CRKP isolates indicated a shared bacterial population among various wards. Maximum-likelihood phylogenetic tree demonstrated that these isolates were partitioned into three major clades. An analysis of the wzi locus revealed three different K types (KL105, KL47, and K64) among the ST11 isolates, indicating genetic diversity among these isolates. Our review of the cases showed that these patients had no contact with each other, indicating nosocomial transmission. Genetic and sequence mapping revealed complexity in the existence of virulence plasmids and resistance plasmids in the ST11-CRKP isolates. These data indicate that this process was more complicated than was earlier anticipated, as it may have involved multiple ST11 K. pneumoniae lineages and a variety of virulence plasmids. Conclusions: Collectively, this work represents the first evidence of gut microbiota may act as the source of ST11-CR-HvKP isolate. Active surveillance approaches, particularly in ICUs based on the results of this study, should be implemented to combat the spread of ST11-CR-HvKP and to improve patient outcomes. Key words: gut microbiota; hypervirulent; KPC-2; reservoir; genomic characterization

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., 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-β-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.

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, bla

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 blaCTX-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. Conclusions: 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.

It is becoming increasingly recognized that the environment plays an important role both in the emergence and in dissemination of antibiotic-resistant bacteria (ARB), Mechanisms and factors facilitating this development are, however, not yet well understood. The high detection rate of CTX-M genes in environmental sources provides an opportunity to explore this issue. In this study, 88 CTX-M-producing

Objectives The aim of this study was to investigate the characteristics and complete genome sequence of an IMP-8, CTX-M-14, CTX-M-3 and QnrS1 co-producing multidrug-resistant Enterobacter asburiae isolate (EN3600) from a patient with wound infection. Methods Species identification was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Carbapenemase genes were identified by PCR and Sanger sequencing. The complete genome sequence of E. asburiae EN3600 was obtained using a PacBio RS II platform. Genome annotation was done by Rapid Annotation using Subsystem Technology (RAST) server. Acquired antimicrobial resistance genes (ARGs) and plasmid replicons were detected using ResFinder 2.1 and PlasmidFinder 1.3, respectively. Results The genome of E. asburiae EN3600 consists of a 4.8-Mbp chromosome and five plasmids. The annotated genome contains various ARGs conferring resistance to aminoglycosides, β-lactams, fluoroquinolones, fosfomycin, macrolides, phenicols, rifampicin and sulfonamides. In addition, plasmids of incompatibility (Inc) groups IncHI2A, IncFIB(pECLA), IncFIB(pQil) and IncP1 were identified. The genes blaIMP-8, blaCTX-M-14 and blaCTX-M-3 were located on different plasmids. The blaIMP-8 gene was carried by an 86-kb IncFIB(pQil) plasmid. The blaCTX-M-3 and qnrS1 genes were co-harboured by an IncP1 plasmid. In addition, blaCTX-M-14 was associated with blaTEM-1B, blaOXA-1, catB3 and sul1 genes in a 116-kb non-typeable plasmid. Conclusion To our knowledge, this is the first complete genome sequence of an E. asburiae isolate co-producing IMP-8, CTX-M-14, CTX-M-3 and QnrS1. This genome may facilitate the understanding of the resistome, pathogenesis and genomic features of Enterobacter cloacae complex (ECC) and will provide valuable information for accurate identification of ECC.

Background: China was one of the major antibiotics consumers globally and has significant bacterial resistance to antibiotics. To contain antimicrobial resistance, the Chinese government initiated a Special National antibiotics-stewardship Campaign (SNC) in hospitals in 2011. The impact of this campaign on antibiotics use in quantity and quality is reported. Methods: In 2011-2016, by the protocol of the SNC, all public hospitals should establish an antibiotics-stewardship task force. They each produced an antibiotics formulary and applied clinical antibiotics guidelines, hired clinical pharmacists to review antibiotics prescriptions, and established bacterial-resistance monitoring system. Data from national hospital surveillance networks from 2010 to 2016 served to determine the impact of the SNC on the antibiotics consumption of and bacterial resistance in Chinese hospitals. Findings: By 2016, all hospitals had established an antimicrobial-stewardship task force; 88.8%-97.5% hospitals implemented the strategies issued by the National Health and Family Planning Commission. The amount of money spent on antibiotics relative to total drug procurement costs dropped from 22.4% in 2010 to 12.0% in 2016. There were sharp drops in the median rates of antibiotics use in hospitalized patients (from 67.8% in 2010 to 40.8% in 2016) and outpatients (19.5% to 8.5%), the rate of prophylactic antibiotics use in clean surgeries (97.9% to 38.3%), and the antibiotics use intensity (85.3±29.8 to 48.5 ±8.0 defined daily dosages/100 patient days). But, guideline compliance by surgeons was still very limited in 2016 (12.6%). Clinicians continued to favor broad-spectrum antibiotics. Methicillin-resistant Staphylococcus aureus prevalence decreased from 52.6% to 34.4%. However, the carbapenem-resistant Klebsiella pneumoniae prevalence rose rapidly from 2.7% to 8.7%. Interpretation: The SNC associated with great reductions in the antibiotics consumption of Chinese hospitals in quantity, their antibiotics use without indications, and their prevalence of major resistant bacteria. However, factors that associate with irrational antibiotics use remained prevalent, including clinician preference for broad-spectrum antibiotics and low guideline compliance. To establish a sustainable antimicrobial-stewardship system in China to improve the rationality of antibiotics use, these factors must be tackled urgently. Funding Statement: The study was granted in part by the National Key Research and Development Program of China (2017YFC1200203), the National Natural Science Foundation of China from Ministry of Science and Technology of China (81361138021, 81711530049), Key Research and Development Program of Zhejiang Province (2015C03032). Declaration of Interests: The authors declare no competing interests.

The co-occurrence of hypervirulence and KPC-2 carbapenem resistant phenotypes in a highly-transmissible ST11 clone of Klebsiella pneumoniae has elicited a deep concern from public health and social communities. To address this puzzle, we conducted a large-scale epidemiological, clinical and genomic study of K. pneumonia ST11 clones with both hypervirulence and carbapenam resistance in two tertiary hospitals in Zhejiang Province. The majority of the patients (16/24) were diagnosed to be infected with exclusively carbapenem-resistant K. pneumoniae (CRKP) while eight cases had mixed infections. Consequently, ten death cases were recorded, some of which were due to a failure to respond to antibiotic therapies. We identified two new rare sequence types (ST437 & ST449) to KPC-2-producing CRKP, in addition to the dominant ST11. These clinical isolates of K. pneumoniae are multi-drug resistant, and heterogeneous in virulence factor profiles. Experimental infections of wax moth larvae suggested that they are in various level of hypervirulence. Moreover, we completed full genome sequencing of 23 plasmids comprising 13 rmpA2-positive virulence plasmids and 10 blaKPC-2-harboring resistance plasmids. In addition to the prevalent pLVKP-like virulence plasmid variants (~178kb), we found an unexpected diversity among KPC-2-producing plasmids whose dominant form is IncFII-IncR type (~120kb), rather than the previously-anticipated version of ~170kb. Additionally, we are first to report a novel blaKPC-2-carrying ~9.8kb mini-plasmid, designated pK186-KPC. These findings illustrate a more comprehensive picture of convergence of hypervirulence and carbapenem resistance in ST11 K. pneumoniae, providing extensive genomic insights into the diversity of dominant clones in Zhejiang Province. Funding Statement: This work was supported by National Natural Science Foundation of China (31830001, 31570027 & 81772142, YF and 81873946, MH), and National Key R&D Program of China (2017YFD0500202, YF). Declaration of Interests: The authors declare that no conflict of interest is present. Ethics Approval Statement: Clinical data were recorded and approved by the local ethics committee from both the Second Affiliated Hospital, Zhejiang University School of Medicine (2018-01-F) and the Lishui People’s Hospital (2017-005-01).

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.

The objective of this study was to understand the impact of implementation of the Essential Medicine Policy (EMP) on the rational use of antibiotics in primary medical institutions in China. A retrospective survey was conducted in 39 primary medical institutions to compare the efficacy of EMP in rational antibiotic use. All institutions completed the survey 1 year before and 1 year after implementation of the EMP. In particular, antibiotic use and its rationality were closely examined. The institutions mainly dealt with common diseases, especially non-infectious chronic diseases. Antibiotic usage was very inappropriate both before and after EMP implementation. Before and after EMP implementation, respectively, the median outpatient cost was US$6.34 and US$5.05, 52.50% (2005/3819) and 53.41% (1865/3492) of the outpatient prescriptions contained antibiotics, and 76.23% (1132/1485) and 78.83% (1106/1403) of inpatients were administered antibiotics. In addition, 98.38% (425/432) and 97.52% (512/525) of surgical inpatients were administered antibiotics, respectively, and 80.76% (638/790) and 75.19% (503/669) of patients with a cold were prescribed antibiotics, respectively. The most commonly used antibiotics were broad-spectrum and injectable agents, including cephalosporins, fluoroquinolones and penicillins. This profile showed little change following implementation of the EMP. In conclusion, inappropriate antibiotic use is a serious problem in primary medical institutions in China. Whilst enforcing the EMP reduced the cost of medical services, it had little effect on promoting the rational use of antibiotics.

Sortase mediated transpeptidation reactions play a significant role in covalent attachment of surface proteins to the cell wall of Gram-positive bacteria. Earlier studies have shown that sortase A (StrA) is required for the virulence of Staphylococci. The human pathogen Staphylococcus simulans CJ16 carries a putative sortase A (SsiStrA) encoding gene, but neither transpeptidation activity nor biochemical characteristics of SsiStrA have been investigated. Here, we identified and characterized StrA from coagulase-negative Staphylococci. SsiStrA was cloned and overexpressed in Escherichia coli BL21 in a soluble form. Size-exclusion chromatography, cross-linking and dynamic light scattering demonstrated that SsiStrA existed as monomer-dimer equilibrium in vitro. We further demonstrated that SsiStrA has sortase activity, and it recognized and cleaved the sorting motif LXPTG. H117, C180 and R193 residues were critical for enzyme activity, and calcium ions enhanced activity.

In the present study, we report the whole genome sequences of two species, Ornithinibacillus contaminans DSM22953(T) isolated from human blood and Ornithinibacillus californiensis DSM 16628(T) isolated from marine sediment, in genus Ornithinibacillus. Comparative genomic study of the two species was conducted together with their close relative Ornithinibacillus scapharcae TW25(T), a putative pathogenic bacteria isolated from dead ark clam. The comparisons showed O. contaminans DSM22953(T) had the smallest genome size of the three species indicating that it has a relatively more stable habitat. More stress response and heavy metal resistance genes were found in the genome of O. californiensis DSM 16628(T) reflecting its adaption to the complex marine environment. O. scapharcae TW25(T) contained more antibiotic resistance genes and virus factors in the genome than the other two species, which revealed its pathogen potential.

Fecal microbial transplantation provides a high curative rate for recurrent Clostridium difficile infection (CDI). However, limitations associated with FMT drive the need to identify key taxa for selective probiotic therapy for prevention, treatment and cure of human CDI. CDI-associated changes in gut microbiota were investigated in adult patients in the Western countries and among infant population in China. However, there has been no such study involving adult patients in China. Therefore, using high throughput sequencing of the 16S ribosomal RNA V3 region and real-time quantitative polymerase chain reaction, we identified CDI-associated key taxa by comparing the fecal microbiota composition of 15 adult patients with CDI with those of 18 individuals with C. difficile-negative nosocomial diarrhea (CDN) and 25 healthy control subjects. Reduced fecal bacterial diversity and dramatic shifts of intestinal microbial composition in CDI and CDN groups were observed compared with healthy controls. Putative butyrate-producing anaerobic bacteria were significantly depleted whereas endotoxin-producing opportunistic pathogens and lactate-producing phylotypes increased dramatically in patients with CDI compared with healthy controls. Further screening of specific microbes causing diarrheal diseases and resistance against CDI is necessary.

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.

Background Carbapenem-resistant Acinetobacter baumannii (CRAB) is a rapidly emerging, life-threatening nosocomial infection. This study aimed to explore the risk factors, clinical features, antimicrobial therapy, and outcomes of CRAB bloodstream infections (BSIs). Methods This is a retrospective, comparative analysis of data from patients with A. baumannii BSI, treated from 2012 to 2015 at a tertiary teaching hospital. Risk factors associated with CRAB BSI and factors associated with the 28-day mortality were evaluated using logistic analyses. Results Data from 293 patients with confirmed A. baumannii BSI were included; 242 (82.6%) patients had CRAB BSI and 51 (17.4%) patients had non-CRAB BSI. Risk factors significantly associated with CRAB BSI were a previous intensive care unit (ICU) stay (P=0.029), cefoperazone–sulbactam use (P=0.030), and carbapenem use (P=0.004). Among 236 patients with A. baumannii BSI who were evaluable at 28 days after receiving antibacterial therapy, there were 86 deaths. Factors associated with the 28-day mortality were ICU stay after BSI (P=0.040), readmission within 90 days (P=0.029), Acute Physiology and Chronic Health Evaluation II (APACHE II) score at diagnosis >19 (P=0.012), tigecycline therapy (P=0.021), presence of septic shock (P=0.029), and multiple organ failure (P=0.016). Death rates in patients treated with tigecycline were 53.5% vs 24.1% in patients treated with other agents. Among 186 patients with CRAB BSI evaluable at 28 days, 84 patients died. The associated risk factors were an ICU stay after BSI (P=0.036), APACHE II score >19 at diagnosis (P = 0.002), presence of septic shock (P=0.030), and multiple organ failure (P=0.007). Conclusion This study demonstrated that an ICU stay and cefoperazone–sulbactam or carbapenem use were seen to be the risk factors associated with the development of CRAB BSI. Critical illness and tigecycline therapy were significantly associated with higher mortality of patients with A. baumannii BSI.

Objectives: Monte Carlo simulation (MCS) was used to evaluate optimal dosage for cefepime (FEP), moxalactam (MOX), and cefperazone/sulbactam (CFZ/SBT) against extended-spectrum β-lactamase (ESBL) producers isolated from the Blood Bacterial Resistant Investigation Collaborative System. Methods: Minimum inhibitory concentration (MIC) was tested by agar dilution, and ESBL producers were identified by modified Clinical and Laboratory Standards Institute tests. Pharmacokinetic parameters were derived from data on healthy individuals, and probability of target attainment (PTA) and cumulative fraction of response (CFR) %fT >MIC values were estimated by MCS. Results: A total of 2032 Escherichia coli (875 ESBL-producing) and Klebsiella pneumoniae (157 ESBL-producing) strains, and 371 other Enterobacteriaceae strains, were isolated from patients with bloodstream infections (BSIs). MIC90 values for FEP, MOX, and CFZ/SBT against ESBL-producing E. coli and K. pneumoniae were 64/64 mg/L, 2/32 mg/L, and 64/128 mg/L, respectively. Conventional MOX and CFZ/SBT doses failed to reach 90% PTA against isolates with MICs ≥8 mg/L and ≥4 mg/L, respectively. Against ESBL producers, neither FEP nor CFZ/SBT achieved ≥90% CFR, while CFRs for MOX (1 g iv q6h, 2 g iv q12h, and 2 g iv q8h) exceeded 90% against ESBL-producing E. coli. Simulated CFRs for FEP and MOX were similar (>90%) against non-ESBL-producing Enterobacteriaceae, and higher than CFRs for CFZ/SBT. Conclusion: ESBL producers from BSIs were highly susceptible to MOX, and PTA values were generally higher for MOX than FEP or CFZ/SBT for conventional dosing regimens. This large MCS analysis shows that MOX but not FEP or CFZ/SBT can be used empirically to treat BSIs caused by ESBL-producing E. coli strains.

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.

Clostridium difficile infection (CDI) is a major cause of infectious diarrhea among hospitalized patients. Probiotics could be instrumental in restoring the intestinal dysbiosis caused by CDI. Here, we examined the protective effect of Pediococcus pentosaceus LI05 in a mouse CDI model. C57BL/6 mice were administrated P. pentosaceus LI05 (LI05 group) or sterile anaerobic PBS (CDI group) everyday for 14 days. Mice were exposed to antibiotics cocktail for 5 days; then challenged with C. difficile strain VPI10463. Mice were monitored daily for survival and weight loss. Colonic tissue and serum samples were assessed for intestinal histopathology, intestinal barrier function and systemic inflammation. The oral administration of P. pentosaceus LI05 improved the survival rate and alleviated the histopathological impact of C. difficile. Compared to the CDI group, the levels of inflammatory mediators in the colon as well as inflammatory cytokines and chemokines in serum were substantially attenuated in the LI05 group. P. pentosaceus LI05 alleviated the CDI-induced of disruption of ZO-1, occludin and claudin-1. Additionally, fecal microbiome analysis showed an enrichment in the abundance of the Porphyromonadaceae and Rikenellaceae, while, the relative abundance of Enterobacteriaceae were decreased. Our results demonstrated that the preventive effect of P. pentosaceus LI05 against CDI was mediated via improving tight junction proteins and down-regulating the inflammatory response. Therefore, P. pentosaceus LI05 could be a promising probiotic in CDI.

A woman infected by carbapenem-resistant Klebsiella pneumoniae is reported in this study. Tigecycline and meropenem combination was used, and indeed, in vitro checkerboard synergy test confirmed the antagonism between the two antibiotics. Thus, meropenem was ceased and single high-dose tigecycline was successful against the infection. Subsequent experiments showed that the isolates of the KPC-2-producing K. pneumoniae ST11 clone caused the infection. Therefore, tigecycline and meropenem combination should be used with caution.

Clostridium difficile infection (CDI) is a major cause of infectious diarrhea among hospitalized patients. Probiotics could be instrumental in restoring the intestinal dysbiosis caused by CDI. Here, we examined the protective effect of Pediococcus pentosaceus LI05 in a mouse CDI model. C57BL/6 mice were administrated P. pentosaceus LI05 (LI05 group) or sterile anaerobic PBS (CDI group) everyday for 14 days. Mice were exposed to antibiotics cocktail for 5 days; then challenged with C. difficile strain VPI10463. Mice were monitored daily for survival and weight loss. Colonic tissue and serum samples were assessed for intestinal histopathology, intestinal barrier function and systemic inflammation. The oral administration of P. pentosaceus LI05 improved the survival rate and alleviated the histopathological impact of C. difficile. Compared to the CDI group, the levels of inflammatory mediators in the colon as well as inflammatory cytokines and chemokines in serum were substantially attenuated in the LI05 group. P. pentosaceus LI05 alleviated the CDI-induced of disruption of ZO-1, occludin and claudin-1. Additionally, fecal microbiome analysis showed an enrichment in the abundance of the Porphyromonadaceae and Rikenellaceae, while, the relative abundance of Enterobacteriaceae were decreased. Our results demonstrated that the preventive effect of P. pentosaceus LI05 against CDI was mediated via improving tight junction proteins and down-regulating the inflammatory response. Therefore, P. pentosaceus LI05 could be a promising probiotic in CDI.

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.

Background: China is a major antibiotics consumer globally and has significant bacterial resistance to antibiotics. To contain antimicrobial resistance, the Chinese government initiated a special national antibiotics-stewardship campaign (SNC) in all hospitals in 2011. The impact of this campaign on irrational antibiotics use is reported. Methods: In 2011-2016, all hospitals established an antibiotics-stewardship task force under SNC management. They each produced an antibiotics formulary and clinical antibiotics guidelines, hired clinical pharmacists to review antibiotics prescriptions, and established bacterial-resistance monitoring system Data from national hospital surveillance networks in 2010 and 2016 served to determine the impact of the SNC on the antibiotics consumption of and bacterial resistance in Chinese hospitals. Findings: By 2016, all hospitals had established an antimicrobialstewardship task force; 88.8-97.5% completed the other tasks. The amount of money spent on antibiotics relative to total drug procurement costs dropped from 22.4% in 2010 to 12.0% in 2016. There were sharp drops in the median rates of antibiotics use in hospitalized patients (from 67.8% in 2010 to 40.8% in 2016) and outpatients (19.5% to 8.5%), the rate of prophylactic antibiotics use in clean surgeries (97.9% to 38.3%), and the antibiotics use intensity (85.3±29.8 to 48.5 ±8.0 defined daily dosages/100 patient days). Guideline compliance by surgeons was still very limited in 2016 (12.6%). Clinicians continued to favor broadspectrum antibiotics. Methicillin-resistant Staphylococcus aureus prevalence decreased from 52.6% to 34.4%. However, the carbapenem-resistant Klebsiella pneumoniae prevalence rose rapidly from 2.7% to 8.7%. Interpretation: The SNC associated with great reductions in the antibiotics consumption of Chinese hospitals, their irrational antibiotics use, and their prevalence of resistant bacteria. However, factors that associate with irrational antibiotics use remained prevalent, including clinician preference for broad-spectrum antibiotics and low guideline compliance. To establish a sustainable antimicrobial-stewardship system in China, these factors must be tackled urgently. Funding Statement: The study was granted in part by the National Key Research and Development Program of China（2017YFC1200203） the National Natural Science Foundation of China (81361138021, 81711530049), Key Research and Development Program of Zhejiang Province (2015C03032). Declaration of Interests: We declare no competing interests.

BACKGROUND: Colistin resistance mediated by mcr-1-harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella. Based on its major contribution to the diarrhoea burden, the epidemic state and threat of mcr-1-harbouring Salmonella in community-acquired infections should be estimated. METHODS: This retrospective study analysed the mcr-1 gene incidence in Salmonella strains collected from a surveillance on diarrhoeal outpatients in Shanghai Municipality, China, 2006–2016. Molecular characteristics of the mcr-1-positive strains and their plasmids were determined by genome sequencing. The transfer abilities of these plasmids were measured with various conjugation strains, species, and serotypes. FINDINGS: Among the 12,053 Salmonella isolates, 37 mcr-1-harbouring strains, in which 35 were serovar Typhimurium, were detected first in 2012 and with increasing frequency after 2015. Most patients infected with mcr-1-harbouring strains were aged

In this study, a novel metallo-β-lactamases fold hydrolase PH-1 was identified from Pelagibacterium halotolerans B2(T). This novel member of the family Hyphomicrobiaceae was isolated from the East China Sea. In silico analysis demonstrated that PH-1 and its relative homologues cluster in a unique branch and constitute a new subgroup among MBLs. PH-1 was cloned and overexpressed in Escherichia coli BL21 in a soluble form. SDS-PAGE, MALDI-TOF/TOF-MS, and size-exclusion chromatography analysis demonstrated that the PH-1 was a monomer with molecular weight of about 29 kDa. Substrate specificity study showed PH-1 preferred penicillin type β-lactams and exhibited maximum activity toward penicillin-G. Additionally, our experiments also revealed that PH-1 was a halotolerant enzyme since it is active under 4 M NaCl. The enzyme activity of PH-1 was negatively affected by 1 mM Mn(2+) and EDTA. These observations lay a foundation for further study of MBLs from marine bacterium.

We report here the emergence of seven IMP-4-producing Raoultella ornithinolytica isolates obtained from one patient. All isolates carried the bla IMP-4 carbapenemase gene, five isolates also carried bla SHV-12 , four contained bla TEM-1 , and one contained bla OXA-1 . Notably, the R. ornithinolytica isolate Ro25724 also expressed Klebsiella pneumoniae carbapenemase (KPC)-2. The bla KPC-2 gene was located on a Tn 3 -Tn 4401 integration structure on a plasmid of ∼450 kb. This is the first description of the coexistence of bla KPC-2 and bla IMP-4 from the genus Raoultella .

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301(T)) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.

The cfr gene was detected in 14 meticillin-susceptible Staphylococcus aureus isolates recovered from outpatients with community-onset infections in a county hospital in China. The MIC of linezolid was 4 μg ml- 1 in eight isolates and 2 μg ml- 1 in six isolates. All isolates were susceptible to vancomycin and teicoplanin, but had elevated MICs for penicillin (0.5-128 μg ml- 1), chloramphenicol (2-32 μg ml- 1), clindamycin (0.5-128 μg ml- 1) and erythromycin (4-128 μg ml- 1). Nine isolates had mutations on domain V of 23S rRNA and/or the ribosomal L proteins that were not located close to the linezolid-binding pocket. Southern blotting experiments demonstrated that the cfr genes in all 14 isolates resided on plasmids. Sequence analysis of the 5.6 kb cfr-carrying plasmid segment revealed 99 % identity to the corresponding sequences in plasmid pSS-01 from animal staphylococci and plasmid pRM-01 from human staphylococci. Five isolates belonged to sequence type (ST)188 and three to ST965; the two ST types were previously reported in isolates of animal origin in some areas of China. These results indicate that the cfr-carrying plasmids in this study are likely of animal origin. The present study shows that cfr-harbouring S. aureus isolates have emerged in some areas of China and that cfr-carrying isolates may be transmitted between animals and humans.

The high frequency of fluoroquinolone resistance in Escherichia coli is a feature of clinical bacteriology in China, where the molecular epidemiology and genetic characteristics of this resistance in county hospitals remain unclear. A total of 590 nonduplicate E. coli isolates from 30 county hospitals located across seven Chinese regions were examined for plasmid-mediated quinolone resistance (PMQR) genes and mutations in quinolone resistance-determining regions (QRDRs). Multilocus sequence typing (MLST) and phylogenetic analysis of fluoroquinolone-resistant isolates were used to determine their genetic relatedness. The ciprofloxacin resistance rate of community-onset E. coli was 51.2%, and at least one PMQR gene was carried by 220 (37.3%) isolates. These included qnr (3.7%), aac(6 ′ )-Ib-cr (19.7%), qepA (14.4%), and oqxAB (3.8%). Two novel oqxB mutants were identified and named oqxB20 and oqxB29 . From 60 sequence types (STs) isolated, 5 novel STs (ST4499 to ST4503) were identified. ST1193 (7.9%) was the second most abundant ST among fluoroquinolone-resistant isolates (ST131 was the most common, with 14.6%), and this is the first report of it in China. This is also the first report of ST2115 and ST3014 isolates from human samples. Ciprofloxacin-resistant E. coli isolates fell mainly into phylogroups B2 and D. The rates of fluoroquinolone resistance and the prevalence of PMQR genes in community-onset E. coli isolates from Chinese county hospitals were high. The wide-ranging molecular epidemiology of E. coli isolates from scattered locations across China indicates that fluoroquinolone resistance evolved from different sources.

We previously described the discovery of two Escherichia coli isolates (EC1002 and EC2474) co-harbouring mcr-1 and blaNDM-1 genes, which were recovered from bloodstream infection in China. More importantly, these antibiotic resistance genes were located on different plasmids and signaling the potential spread of pandrug-resistant bacteria. Here, the complete genome sequences of both isolates were determined using Pacbio RS II and Illumina HiSeq2000 systems. The genome of EC1002 consists of a 5,177,501 base pair chromosome and four circular plasmids, while the genome of EC2474 consists of a 5,013,813 base pair chromosome and three plasmids. The plasmid replicon type of pEC1002_NDM and pEC2474_NDM were identified as IncA/C2 and IncF, respectively. The genetic environment of blaNDM-1 in this study was similar to blaNDM-carrying plasmids detected in China, although the overall nucleotide identity and query coverage were variable. The plasmid replicon type of pEC1002_MCR and pEC2474_MCR were identified as IncI2 and IncHI2, respectively. Two different genetic strategies for mcr-1 gene spread were observed in this study and blaNDM-1 genes were also found transferred by two different mobile genetic elements in two plasmids. The findings of this study further support that the diversified transfer mechanisms of blaNDM-1 and mcr-1 present in Enterobacteriaceae.

Vibrio fluvialis is recognized as an emerging pathogen. However, not much is known about the mechanism of its pathogenesis, and its adaptation to a special niche such as the gall bladder. Here we describe two V. fluvialis strains that cause acute cholecystitis. It is noteworthy that both strains were susceptible to all antibiotics tested, which is in contrast to previous studies, suggesting substantial genetic diversity among V. fluvialis isolates. In agreement with their survival and growth in the gall bladder, the genomes of strains 12605 and 3663 contain a considerable number of genes that confer resistance to bile, including toxR, ompU, tolC, cmeABC, rlpB, yrbK, rpoS, damX and gltK. Furthermore, integrative and conjugative elements (ICEs), virulence factors and prophage regions were also detected in strains 12605 and 3663, reflecting their flexibility in recombination during the evolution of pathogenicity. Comparative analysis of nine available genomes of V. fluvialis revealed a core genome consisting of 3,147 genes. Our results highlight the association of V. fluvialis with a rare disease profile and shed light on the evolution of pathogenesis and niche adaptation of V. fluvialis.

Chiral metamirrors can reflect one circularly polarized light without changing its handedness, while almost completely absorb the other, which would have many potential applications in engineering. In this paper, recent progresses of our group on the chiral metamirrors are reviewed. Firstly, we discuss the general method to design the chiral metamirrors under the framework of Jones calculus. The structural requirement of such chiral metamirrors is the breaking in both the n-fold rotational (n ≥ 2) symmetry and mirror symmetry. According to the general design methodology, we propose and design some chiral metamirrors that absorb only one circularly polarized light. Our design includes a bi-layer chiral metamirror in the mid-infrared region, a single-layer chiral metamirror in microwave and a broadband chiral metamirror. Our findings may provide an efficient approach to boost the working bandwidth of the chiral metamirror and a variety of applications, including molecular spectroscopy and polarization-sensitive detector.

Background Shigellosis is the most common cause of gastrointestinal infections in developing countries. In China, the species most frequently responsible for shigellosis is Shigella flexneri. S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on biochemical and serological properties. Moreover, increasing numbers of ESBL-producing Shigella strains have been isolated from clinical samples. Despite this, only a few cases of ESBL-producing Shigella have been described in China. Therefore, a better understanding of ESBL-producing Shigella from a genomic standpoint is required. In this study, a S. flexneri type 1a isolate SP1 harboring blaCTX-M-14, which was recovered from the patient with diarrhea, was subjected to whole genome sequencing. Results The draft genome assembly of S. flexneri strain SP1 consisted of 4,592,345 bp with a G+C content of 50.46%. RAST analysis revealed the genome contained 4798 coding sequences (CDSs) and 100 RNA-encoding genes. We detected one incomplete prophage and six candidate CRISPR loci in the genome. In vitro antimicrobial susceptibility testing demonstrated that strain SP1 is resistant to ampicillin, amoxicillin/clavulanic acid, cefazolin, ceftriaxone and trimethoprim. In silico analysis detected genes mediating resistance to aminoglycosides, β-lactams, phenicol, tetracycline, sulphonamides, and trimethoprim. The bla CTX-M-14 gene was located on an IncFII2 plasmid. A series of virulence factors were identified in the genome. Conclusions In this study, we report the whole genome sequence of a blaCTX-M-14-encoding S. flexneri strain SP1. Dozens of resistance determinants were detected in the genome and may be responsible for the multidrug-resistance of this strain, although further confirmation studies are warranted. Numerous virulence factors identified in the strain suggest that isolate SP1 is potential pathogenic. The availability of the genome sequence and comparative analysis with other S. flexneri strains provides the basis to further address the evolution of drug resistance mechanisms and pathogenicity in S. flexneri. Electronic supplementary material The online version of this article (doi:10.1186/s12941-017-0212-2) contains supplementary material, which is available to authorized users.