Recovery and genetic characterization of clinically-relevant ST2 carbapenem-resistant Acinetobacter baumannii isolates from untreated hospital sewage in Zhejiang Province, China.

The global transmission of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant and grave threat to human health. To investigate the potential relationship between hospital sewage and the transmission of CRAB within healthcare facilities, isolates of Acinetobacter spp. obtained from untreated hospital sewage samples were subjected to antimicrobial susceptibility tests, genome sequencing, and bioinformatic and phylogenetic tree analysis, and that data were matched with those of the clinical isolates. Among the 70 Acinetobacter spp. sewage isolates tested, A. baumannii was the most prevalent and detectable in 5 hospitals, followed by A. nosocomialis and A. gerneri. Worryingly, 57.14 % (40/70) of the isolates were MDR, with 25.71 % (18/70) being resistant to carbapenem. When utilizing the Pasteur scheme, ST2 was the predominant type among these CRAB isolates, with Tn2006 (ΔISAba1-blaOXA-23-ATPase-yeeB-yeeA-ΔISAba1) and Tn2009 (ΔISAba1-blaOXA-23-ATPase-hp-parA-yeeC-hp-yeeB-ΔISAba1) being the key mobile genetic elements that encode carbapenem resistance. Seven A. gerneri isolates which harbored Tn2008 (ISAba1-blaOXA-23 -ATPase) and the blaPER-1 gene were also identified. Besides, an A. soil isolate was found to exhibit high-level of meropenem resistance (MIC ≥128 mg/L) and harbor a blaNDM-1 gene located in a core genetic structure of ISAba125-blaNDM-1-ble-trpF-dsbC-cutA. To investigate the genetic relatedness between isolates recovered from hospital sewage and those collected from ICUs, a phylogenetic tree was constructed for 242 clinical isolates and 9 sewage isolates. The results revealed the presence of two evolutionary clades, each containing isolates from both ICU and sewage water, suggesting that CRAB isolates in untreated sewage water were also the transmission clones or closely related evolutionary isolates recoverable in hospital settings. Findings in this work confirm that hospital sewage is a potential reservoir of CRAB.

Comparison of the NG-Test Carba 5, Colloidal Gold Immunoassay (CGI) Test, and Xpert Carba-R for the Rapid Detection of Carbapenemases in Carbapenemase-Producing Organisms.

Carbapenem-resistant Enterobacterales (CRE) are increasingly recognized as an urgent public health concern. The rapid and accurate identification of carbapenemases could provide insights into antimicrobial therapy and infection control. In this study, we evaluated the efficacy of three different methods, including the NG-test Carba 5, colloidal gold immunoassay (CGI) test, and Xpert Carba-R assay, for the rapid detection of five carbapenemases (KPC, NDM, IMP, OXA-48, and VIM). A total of 207 Gram-negative strains collected from patients and hospital sewages were tested. The presence or absence of carbapenemase genes in the whole-genome sequences was used as the gold standard for evaluating the accuracy of the above-mentioned three methods. Among the 192 strains carrying only one carbapenemase gene, the accuracies of the NG-Test Carba 5, CGI test, and Xpert Carba-R were 96.88% (95% CI, 93.01-98.72%), 96.88% (95% CI, 93.01-98.72%), and 97.92% (95% CI, 94.41-99.33%), respectively. Xpert Carba-R was able to detect all 13 types of KPC variants, including KPC-2, KPC-3, KPC-25, KPC-33, KPC-35, KPC-51, KPC-52, KPC-71, KPC-76, KPC-77, KPC-78, KPC-93, and KPC-123, with a detection sensitivity of 100.00% (95% CI, 96.50-100.00%), a specificity of 100.00% (95% CI, 92.38-100.00%), and a κ index of 1.00. For IMP, Carba 5 was superior to the other two methods, with a sensitivity of 100% (95% CI, 71.66-100.00%), a specificity of 100% (95% CI, 97.38-100.00%), and a κ index of 1.00. For the remaining 15 strains carrying two or three kinds of carbapenemase genes, Carba 5 performed the best, which accurately identified all the target genes, followed by Xpert Carba-R (12/15, 80.00%) and the CGI test (10/15, 66.67%). Therefore, all three assays demonstrated reliable performances in carbapenemase detection, and Xpert Carba-R should be recommended for the detection of KPC variants, especially for patients at a high risk of infections caused by ceftazidime/avibactam-resistant strains. IMPORTANCE: CRE was listed as one of the top three pathogens that are in critical need of new antibiotics by the WHO. The rapid and accurate identification of carbapenemases is important for antimicrobial therapy and infection control. In recent years, new beta-lactam/beta-lactamase inhibitor combinations such as ceftazidime/avibactam (CZA) have been approved by the Food and Drug Administration (FDA) to cope with CRE challenges. CZA was effective against class A, class C, and some class D enzymes such as OXA-48-like. However, CZA-resistant KPC variants emerged at an alarming speed, which posed a new challenge for the accurate identification of KPC variants. In this study, we evaluated the performance of two lateral flow immunochromatographic assays, namely, NG-test Carba 5 and the CGI test, and the automated real-time quantitative PCR Xpert Carba-R in the rapid detection of carbapenemases. Notably, 13 types of KPC variants were enrolled in this study, which covered most KPC variants discovered in China. Carba-R was superior to NG-teat Carba 5 and the CGI test; it was able to detect all of the included KPC variants, including KPC-2, KPC-3, KPC-25, KPC-33, KPC-35, KPC-51, KPC-52, KPC-71, KPC-76, KPC-77, KPC-78, KPC-93, and KPC-123.

Collateral sensitivity to pleuromutilins in vancomycin-resistant Enterococcus faecium.

The acquisition of resistance to one antibiotic sometimes leads to collateral sensitivity to a second antibiotic. Here, we show that vancomycin resistance in Enterococcus faecium is associated with a remarkable increase in susceptibility to pleuromutilin antibiotics (such as lefamulin), which target the bacterial ribosome. The trade-off between vancomycin and pleuromutilins is mediated by epistasis between the van gene cluster and msrC, encoding an ABC-F protein that protects bacterial ribosomes from antibiotic targeting. In mouse models of vancomycin-resistant E. faecium colonization and septicemia, pleuromutilin treatment reduces colonization and improves survival more effectively than standard therapy (linezolid). Our findings suggest that pleuromutilins may be useful for the treatment of vancomycin-resistant E. faecium infections.

A rapid MALDI-TOF mass spectrometry-based method for colistin susceptibility testing in Escherichia coli.

Colistin is recognized as a last-resort treatment option against multi-drug resistant bacteria including carbapenem-resistant Enterobacteriaceae (CRE). However, the plasmid-mediated colistin-resistance gene mcr-1 has been reported globally resulting in an increase of colistin-resistant bacteria. A quick and accurate method for determining the pathogen resistance of colistin is therefore crucial in the clinic. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a potential tool forto be applied for antimicrobial susceptibility testing. We compared the growth of Escherichia coli strains in the presence or absence of colistin. Automated analyses of the spectra were performed with a prototype software tool written with package R. Three mcr-1-positive and six mcr-1-negative E. coli were used for establishing the model to obtain the optimal incubation time, the breakpoint concentration of colistin and cut-off of the relative growth (RG) value. The distinction between susceptible and resistant strains was already noticeable after 2 h of incubation. The best separation between the susceptible and resistant strains was achieved at a concentration of 4 µg ml-1 and a relative growth cut-off value of 0.6. Application of the model for the analysis of 128 E. coli isolates, a sensitivity of 97.4% and a specificity of 88.2% were achieved compared with colistin MIC results. The rapid MALDI-TOF MS-based method approach is simple to set-up, uses a short incubation time, and had excellent outcomes with respect to sensitivity and specificity for colistin sensitivity testing in Escherichia coli.

Clinical evolution of ST11 carbapenem resistant and hypervirulent Klebsiella pneumoniae.

Carbapenem-resistant and hypervirulent K. pneumoniae (CR-HvKP) strains that have emerged recently have caused infections of extremely high mortality in various countries. In this study, we discovered a conjugative plasmid that encodes carbapenem resistance and hypervirulence in a clinical ST86 K2 CR-HvKP, namely 17ZR-91. The conjugative plasmid (p17ZR-91-Vir-KPC) was formed by fusion of a non-conjugative pLVPK-like plasmid and a conjugative blaKPC-2-bearing plasmid and is present dynamically with two other non-fusion plasmids. Conjugation of p17ZR-91-Vir-KPC to other K. pneumoniae enabled them to rapidly express the carbapenem resistance and hypervirulence phenotypes. More importantly, genome analysis provided direct evidence that p17ZR-91-Vir-KPC could be directly transmitted from K2 CR-HvKP strain, 17ZR-91, to ST11 clinical K. pneumoniae strains to convert them into ST11 CR-HvKP strains, which explains the evolutionary mechanisms of recently emerged ST11 CR-HvKP strains.

Emergence and Genomic Characterization of a KPC-2-, NDM-1-, and IMP-4-Producing Klebsiella michiganensis Isolate.

A rectal swab sample was collected from a patient with Guillain-Barré syndrome and enriched in lysogeny broth. Carbapenem-resistant bacteria were selected by China Blue agar plates containing 0.3 µg/ml meropenem. Carbapenemase-producing Klebsiella michiganensis was identified and characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), immune colloidal gold technique, a conjugation experiment, PCR analysis, and antimicrobial susceptibility testing. The genome of K. michiganensis was determined by whole genome sequencing. Antimicrobial susceptibility testing showed that the K. michiganensis was resistant to imipenem, meropenem, ertapenem, cefmetazole, ceftazidime, cefotaxime, piperacillin/tazobactam, sulbactam/cefoperazone, ceftazidime/avibactam, cefepime, and aztreonam while susceptible to polymyxin B, ciprofloxacin, tigecycline, and amikacin. Immune colloidal gold technique suggested that this strain co-produced three different carbapenemases [Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), and Imipenem (IMP)]. Whole genome sequencing analysis indicated that this strain belonged to ST91, and bla KPC-2, bla NDM-1, and bla IMP-4 were carried on different conjugative plasmids. Besides, the co-existence and transferability of bla KPC-2, bla NDM-1, and bla IMP-4 in K. michiganensis facilitates the potential horizontal dissemination and nosocomial spread of resistance genes among multidrug-resistant organisms.

Conjugation of Virulence Plasmid in Clinical Klebsiella pneumoniae Strains through Formation of a Fusion Plasmid.

The rapid dissemination of non-conjugative virulence plasmids among non-K1/K2 types of Klebsiella pneumoniae poses an unprecedented threat to human health, yet the underlying mechanisms governing dissemination of such plasmids is unclear. In this study, a novel 68 581 bp IncFIA plasmid is discovered that can be fused to a hypervirulence-encoding plasmid to form a hybrid conjugative virulence plasmid in conjugation experiments; such fusion events involve homologous recombination between a 241 bp homologous region located in each of the two plasmids. The fusion hypervirulence-encoding plasmid can be conjugated to both classic and blaKPC-2 -bearing carbapenem-resistant K. pneumoniae strains through conjugation, enabling such strains to acquire the ability to express the hypervirulence phenotype. Dissemination of this fusion virulence plasmid will impose an enormous burden on current efforts to control and treat infections caused by multidrug resistant and hypervirulent K. pneumoniae.

Application of CRISPR/Cas9-Based Genome Editing in Studying the Mechanism of Pandrug Resistance in Klebsiella pneumoniae.

In this study, a CRISPR/Cas9-mediated genome editing method was used to study the functions of the mgrB, tetA, and ramR genes in mediating colistin and tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). Inactivation of the tetA or ramR gene or the mgrB gene by CRISPR/Cas9 affected bacterial susceptibility to tigecycline or colistin, respectively. This study proved that the CRISPR/Cas9-based genome editing method could be effectively applied to K. pneumoniae and should be further utilized for genetic characterization.

Dynamic Colonization of Klebsiella pneumoniae Isolates in Gastrointestinal Tract of Intensive Care Patients.

Gastrointestinal carriage is regarded as a major reservoir of K. pneumoniae infections, especially in intensive care patients. A total of 101 (95.3%) KPC-producing carbapenem-resistant K. pneumoniae (CRKP) isolates were identified among 106 CRKP isolates collected from stool samples of inpatients performing active rectal screening for carbapenem-resistant Enterobacteriaceae during hospitalization in the ICUs of a tertiary hospital between 2016 and 2017. Among them, six KPC-producing CRKP isolates from three patients (two isolates for each patient) were identified with distinct antibacterial susceptibility. Our findings showed that: (1) bla KPC-2 gene is predominant in CRKP strains isolated from the intensive care patients and can be incorporated into various plasmids that are transmissible among multiple bacterial hosts in the human gastrointestinal tract; (2) the human gastrointestinal tract has a capacity to dynamically colonize multiple clones of CRKP strains with varied plasmids, diverse antimicrobial resistance genes and virulence genes. K. pneumoniae colonization is an important step in progression to extraintestinal infection, which provides the rationale for establishing intervention measures to prevent subsequent infection. Thus, close surveillance on CRKP colonization, together with effective infection prevention and control measures, should be put into practice.

Functionalized Nanoparticles Efficiently Enhancing the Targeted Delivery, Tumor Penetration, and Anticancer Activity of 7-Ethyl-10-Hydroxycamptothecin.

The enhanced permeability and retention (EPR) effect of tumors is much more complex than initially defined, and it alone is not sufficient for targeted delivery of nanosized agents. Meanwhile, poor tumor penetration is another major challenge for the treatment of solid tumors using nanoparticles. Development of delivery systems for SN38, the active metabolite of CPT-11 in human and a very potent anticancer molecule, has become an attractive research area. PEGx -p(HEMASN38)y (x and y are viable), a prodrug synthesized by using polyethylene glycol (PEG) as initiator and SN38 as monomer through atom transfer radical polymeration (ATRP) method, is previously reported. Using PEG2.4K -p(HEMASN38)3K as a model prodrug, herein an active-targeted strategy decorated with cys-arg-gly-asp-lys (CRGDK), a peptide specifically binds to neuropilin-1 overexpressed by tumor vessels and tumor cells, is successfully established to further improve the delivery and efficacy of SN38. CRGDK-functionalized PEG2.4K -p(HEMASN38)3K (C-SN38) nanoparticles and nonfunctionalized control (B-SN38) are prepared with two distinct sizes, 30 and 100 nm. Their physiochemical and biological characteristics are investigated in vitro and in vivo with multiple tumor models. It is demonstrated for the first time that CRGDK functionalization can be a promising strategy for efficient delivery of SN38, and C-SN38 is a potent drug candidate for the treatment of neuropilin-1 overexpressing tumors.

A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study.

BACKGROUND: Hypervirulent Klebsiella pneumoniae strains often cause life-threatening community-acquired infections in young and healthy hosts, but are usually sensitive to antibiotics. In this study, we investigated a fatal outbreak of ventilator-associated pneumonia caused by a new emerging hypervirulent K pneumoniae strain. METHODS: The outbreak occurred in the integrated intensive care unit of a new branch of the Second Affiliated Hospital of Zhejiang University (Hangzhou, China). We collected 21 carbapenem-resistant K pneumoniae strains from five patients and characterised these strains for their antimicrobial susceptibility, multilocus sequence types, and genetic relatedness using VITEK-2 compact system, multilocus sequence typing, and whole genome sequencing. We selected one representative isolate from each patient to establish the virulence potential using a human neutrophil assay and Galleria mellonella model and to establish the genetic basis of their hypervirulence phenotype. FINDINGS: All five patients had undergone surgery for multiple trauma and subsequently received mechanical ventilation. The patients were aged 53-73 years and were admitted to the intensive care unit between late February and April, 2016. They all had severe pneumonia, carbapenem-resistant K pneumoniae infections, and poor responses to antibiotic treatment and died due to severe lung infection, multiorgan failure, or septic shock. All five representative carbapenem-resistant K pneumoniae strains belonged to the ST11 type, which is the most prevalent carbapenem-resistant K pneumoniae type in China, and originated from the same clone. The strains were positive on the string test, had survival of about 80% after 1 h incubation in human neutrophils, and killed 100% of wax moth larvae (G mellonella) inoculated with 1 × 106 colony-forming units of the specimens within 24 h, suggesting that they were hypervirulent K pneumoniae. Genomic analyses showed that the emergence of these ST11 carbapenem-resistant hypervirulent K pneumoniae strains was due to the acquisition of a roughly 170 kbp pLVPK-like virulence plasmid by classic ST11 carbapenem-resistant K pneumoniae strains. We also detected these strains in specimens collected in other regions of China. INTERPRETATION: The ST11 carbapenem-resistant hypervirulent K pneumoniae strains pose a substantial threat to human health because they are simultaneously hypervirulent, multidrug resistant, and highly transmissible. Control measures should be implemented to prevent further dissemination of such organisms in the hospital setting and the community. FUNDING: Chinese National Key Basic Research and Development Program and Collaborative Research Fund of Hong Kong Research Grant Council.

Genetic and Functional Characterization of blaCTX-M-199, a Novel Tazobactam and Sulbactam Resistance-Encoding Gene Located in a Conjugative mcr-1-Bearing IncI2 Plasmid.

We report the genetic and functional characterization of a novel CTX-M-199 ß-lactamase, which was encoded by a blaCTX-M-64 variant gene found in a conjugative mcr-1-bearing IncI2 plasmid and exhibited resistance to ß-lactamase inhibitors, tazobactam, and sulbactam.

MnO2/g-C3N4 nanocomposite with highly enhanced supercapacitor performance.

A novel sandwich-like MnO2/g-C3N4 nanocomposite (NC) based on the integration of high-density MnO2 nanorods (NRs) onto the surfaces of two-dimensional (2D) g-C3N4 sheets has been successfully fabricated through a facile soft chemical route at low temperature. The MnO2/g-C3N4 NC electrode enhanced the supercapacitor (SC) performance, benchmarked against both the bare MnO2 NRs electrode and the MnO2/graphene oxide (GO) NC electrode, exhibiting high specific capacitance of 211 F/g at a current density of 1 A/g, with good rate capacity and cycling stability. The sandwich-like hybrid structure, the unique 2D structure of the g-C3N4 sheets and the presence of nitrogen in the g-C3N4 all contributed to the promising SC performance of the MnO2/g-C3N4 NC. This work demonstrated the advantages of the g-C3N4 sheets over the commonly-used GO sheets in the design of novel hybrid composite for enhanced capacitance performance of MnO2-based electrochemical SCs, and the results could be extended to other electrode materials for SCs.

Comparative genetic characterization of Enteroaggregative Escherichia coli strains recovered from clinical and non-clinical settings.

The origin of pathogenic Enteroaggregative Escherichia coli (EAEC), a major causative agent of childhood diarrhea worldwide, remains ill-defined. The objective of this study was to determine the relative prevalence of EAEC in clinical and non-clinical sources and compare their genetic characteristics in order to identify strains that rarely and commonly cause human diarrhea. The virulence gene astA was commonly detectable in both clinical and non-clinical EAEC, while clinical isolates, but not the non-clinical strains, were consistently found to harbor other virulence factors such as aap (32%), aatA (18%) and aggR (11%). MLST analysis revealed the extremely high diversity of EAEC ST types, which can be grouped into three categories including: (i) non-clinical EAEC that rarely cause human infections; (ii) virulent strains recoverable in diarrhea patients that are also commonly found in the non-clinical sources; (iii) organisms causing human infections but rarely recoverable in the non-clinical setting. In addition, the high resistance in these EAEC isolates in particular resistance to fluoroquinolones and cephalosporins raised a huge concern for clinical EAEC infection control. The data from this study suggests that EAEC strains were diversely distributed in non-clinical and clinical setting and some of the clinical isolates may originate from the non-clinical setting.

Emergence of Carbapenem-Resistant Serotype K1 Hypervirulent Klebsiella pneumoniae Strains in China.

We report the emergence of five carbapenem-resistant K1 hypervirulent Klebsiella pneumoniae (hvKP) strains which caused fatal infections in hospital patients in Zhejiang Province, China, upon entry through surgical wounds. Genotyping results revealed the existence of three genetically related strains which exhibited a new sequence type, ST1797, and revealed that all strains harbored the magA and wcaG virulence genes and a plasmid-borne bla(KPC-2) gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible.

Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.

BACKGROUND: Until now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae. METHODS: The mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model. FINDINGS: Polymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10(-1) to 10(-3) cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011-14, and 16 (1%) of 1322 samples from inpatients with infection. INTERPRETATION: The emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria. FUNDING: Ministry of Science and Technology of China, National Natural Science Foundation of China.