Carbapenem-resistant hypervirulent ST23 Klebsiella pneumoniae with a highly transmissible dual-carbapenemase plasmid in Chile.

BACKGROUND: The convergence of hypervirulence and carbapenem resistance in the bacterial pathogen Klebsiella pneumoniae represents a critical global health concern. Hypervirulent K. pneumoniae (hvKp) strains, frequently from sequence type 23 (ST23) and having a K1 capsule, have been associated with severe community-acquired invasive infections. Although hvKp were initially restricted to Southeast Asia and primarily antibiotic-sensitive, carbapenem-resistant hvKp infections are reported worldwide. Here, within the carbapenemase production Enterobacterales surveillance system headed by the Chilean Public Health Institute, we describe the isolation in Chile of a high-risk ST23 dual-carbapenemase-producing hvKp strain, which carbapenemase genes are encoded in a single conjugative plasmid. RESULTS: Phenotypic and molecular tests of this strain revealed an extensive resistance to at least 15 antibiotic classes and the production of KPC-2 and VIM-1 carbapenemases. Unexpectedly, this isolate lacked hypermucoviscosity, challenging this commonly used hvKp identification criteria. Complete genome sequencing and analysis confirmed the K1 capsular type, the KpVP-1 virulence plasmid, and the GIE492 and ICEKp10 genomic islands carrying virulence factors strongly associated with hvKp. Although this isolate belonged to the globally disseminated hvKp clonal group CG23-I, it is unique, as it formed a clade apart from a previously reported Chilean ST23 hvKp isolate and acquired an IncN KPC-2 plasmid highly disseminated in South America (absent in other hvKp genomes), but now including a class-I integron carrying blaVIM-1 and other resistance genes. Notably, this isolate was able to conjugate the double carbapenemase plasmid to an E. coli recipient, conferring resistance to 1st -5th generation cephalosporins (including combinations with beta-lactamase inhibitors), penicillins, monobactams, and carbapenems. CONCLUSIONS: We reported the isolation in Chile of high-risk carbapenem-resistant hvKp carrying a highly transmissible conjugative plasmid encoding KPC-2 and VIM-1 carbapenemases, conferring resistance to most beta-lactams. Furthermore, the lack of hypermucoviscosity argues against this trait as a reliable hvKp marker. These findings highlight the rapid evolution towards multi-drug resistance of hvKp in Chile and globally, as well as the importance of conjugative plasmids and other mobile genetic elements in this convergence. In this regard, genomic approaches provide valuable support to monitor and obtain essential information on these priority pathogens and mobile elements.

Glutathione catabolism by Enterobacteriaceae species to hydrogen sulphide adversely affects the viability of host systems in the presence of 5'fluorodeoxyuridine.

Reduced glutathione (GSH) plays an essential role in relieving oxidative insult from the generation of free radicals via normal physiological processes. However, GSH can be exploited by bacteria as a signalling molecule for the regulation of virulence. We describe findings arising from a serendipitous observation that when GSH and Escherichia coli were incubated with 5'fluorodeoxyuridine (FUdR)-synchronised populations of Caenorhabditis elegans, the nematodes underwent rapid death. Death was mediated by the production of hydrogen sulphide mainly through the action of tnaA, a tryptophanase-encoding gene in E. coli. Other Enterobacteriaceae species possess similar cysteine desulfhydrases that can catabolise l-cysteine-containing compounds to hydrogen sulphide and mediate nematode killing when worms had been pre-treated with FUdR. When colonic epithelial cell lines were infected, hydrogen sulphide produced by these bacteria in the presence of GSH was also able to inhibit ATP synthesis in these cells particularly when cells had been treated with FUdR. Therefore, bacterial production of hydrogen sulphide could act in concert with a commonly used genotoxic cancer drug to exert host cell impairment. Hydrogen sulphide also increases bacterial adhesion to the intestinal cells. These findings could have implications for patients undergoing chemotherapy using FUdR analogues that could result in intestinal damage.

A hydrophilic polyimidazolium antibiotic targeting the membranes of Gram-negative bacteria.

OBJECTIVES: The rise of MDR Gram-negative bacteria (GNB), especially those resistant to last-resort drugs such as carbapenems and colistin, is a global health risk and calls for increased efforts to discover new antimicrobial compounds. We previously reported that polyimidazolium (PIM) compounds exhibited significant antimicrobial activity and minimal mammalian cytotoxicity. However, their mechanism of action is relatively unknown. We examined the efficacy and mechanism of action of a hydrophilic PIM (PIM5) against colistin- and meropenem-resistant clinical isolates. METHODS: MIC and time-kill testing was performed for drug-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. N-phenyl-1-naphthylamine and propidium iodide dyes were employed to determine membrane permeabilization. Spontaneous resistant mutants and single deletion mutants were generated to understand potential resistance mechanisms to the drug. RESULTS: PIM5 had the same effectiveness against colistin- and meropenem-resistant strains as susceptible strains of GNB. PIM5 exhibited a rapid bactericidal effect independent of bacterial growth phase and was especially effective in water. The polymer disrupts both the outer and cytoplasmic membranes. PIM5 binds and intercalates into bacterial genomic DNA upon entry of cells. GNB do not develop high resistance to PIM5. However, the susceptibility and uptake of the polymer is moderately affected by mutations in the two-component histidine kinase sensor BaeS. PIM5 has negligible cytotoxicity on human cells at bacterial-killing concentrations, comparable to the commercial antibiotics polymyxin B and colistin. CONCLUSIONS: PIM5 is a potent broad-spectrum antibiotic targeting GNB resistant to last-resort antibiotics.

Bacterial-induced cell fusion is a danger signal triggering cGAS-STING pathway via micronuclei formation.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease in the tropics and subtropics with high morbidity and mortality. The facultative intracellular bacterium induces host cell fusion through its type VI secretion system 5 (T6SS5) as an important part of its pathogenesis in mammalian hosts. This allows it to spread intercellularly without encountering extracellular host defenses. We report that bacterial T6SS5-dependent cell fusion triggers type I IFN gene expression in the host and leads to activation of the cGAMP synthase-stimulator of IFN genes (cGAS-STING) pathway, independent of bacterial ligands. Aberrant and abortive mitotic events result in the formation of micronuclei colocalizing with cGAS, which is activated by double-stranded DNA. Surprisingly, cGAS-STING activation leads to type I IFN transcription but not its production. Instead, the activation of cGAS and STING results in autophagic cell death. We also observed type I IFN gene expression, micronuclei formation, and death of chemically induced cell fusions. Therefore, we propose that the cGAS-STING pathway senses unnatural cell fusion through micronuclei formation as a danger signal, and consequently limits aberrant cell division and potential cellular transformation through autophagic death induction.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Dominant Carbapenemase-Encoding Plasmids in Clinical Enterobacterales Isolates and Hypervirulent Klebsiella pneumoniae, Singapore.

Dissemination of carbapenemase-encoding plasmids by horizontal gene transfer in multidrug-resistant bacteria is the major driver of rising carbapenem-resistance, but the conjugative mechanics and evolution of clinically relevant plasmids are not yet clear. We performed whole-genome sequencing on 1,215 clinical Enterobacterales isolates collected in Singapore during 2010-2015. We identified 1,126 carbapenemase-encoding plasmids and discovered pKPC2 is becoming the dominant plasmid in Singapore, overtaking an earlier dominant plasmid, pNDM1. pKPC2 frequently conjugates with many Enterobacterales species, including hypervirulent Klebsiella pneumoniae, and maintains stability in vitro without selection pressure and minimal adaptive sequence changes. Furthermore, capsule and decreasing taxonomic relatedness between donor and recipient pairs are greater conjugation barriers for pNDM1 than pKPC2. The low fitness costs pKPC2 exerts in Enterobacterales species indicate previously undetected carriage selection in other ecological settings. The ease of conjugation and stability of pKPC2 in hypervirulent K. pneumoniae could fuel spread into the community.

Genetic and epigenetic variation separately contribute to range expansion and local metalliferous habitat adaptation during invasions of Chenopodium ambrosioides into China.

BACKGROUND AND AIMS: Invasive plants often colonize wide-ranging geographical areas with various local microenvironments. The specific roles of epigenetic and genetic variation during such expansion are still unclear. Chenopodium ambrosioides is a well-known invasive alien species in China that can thrive in metalliferous habitats. This study aims to comprehensively understand the effects of genetic and epigenetic variation on the successful invasion of C. ambrosioides. METHODS: We sampled 367 individuals from 21 heavy metal-contaminated and uncontaminated sites with a wide geographical distribution in regions of China. We obtained environmental factors of these sampling sites, including 13 meteorological factors and the contents of four heavy metals in soils. Microsatellite markers were used to investigate the demographic history of C. ambrosioides populations in China. We also analysed the effect of epigenetic variation on metalliferous microhabitat adaptation using methylation-sensitive amplified polymorphism (MSAP) markers. A common garden experiment was conducted to compare heritable phenotypic variations among populations. KEY RESULTS: Two distinct genetic clusters that diverged thousands of years ago were identified, suggesting that the eastern and south-western C. ambrosioides populations in China may have originated from independent introduction events without recombination. Genetic variation was shown to be a dominant determinant of phenotypic differentiation relative to epigenetic variation, and further affected the geographical distribution pattern of invasive C. ambrosioides. The global DNA unmethylation level was reduced in metalliferous habitats. Dozens of methylated loci were significantly associated with the heavy metal accumulation trait of C. ambrosioides and may contribute to coping with metalliferous microenvironments. CONCLUSIONS: Our study of C. ambrosioides highlighted the dominant roles of genetic variation in large geographical range expansion and epigenetic variation in local metalliferous habitat adaptation.

Remediation of copper-contaminated soils using Tagetes patula L., earthworms and arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) and earthworms have potential uses in the bioremediation of contaminated soils. In recent years, heavy metal-contaminated sites have been remediated by adding plants and AMF or earthworms to the soil. However, there are few studies on remediation using combinations of plants, animals, and microbes, especially for the remediation of Cu-contaminated soil. The present study investigated the separate and combined effects of AMF and earthworms on Cu-contaminated soil in which Tagetes patula L. was grown. The results show that the combined application of AMF and earthworms markedly increased the biomass of plant shoots and roots by more than 100%. It also increased Cu extraction by T. patula by 270%. The combined treatment was effective in increasing the CEC, contents of OM, and available Cu, P and K, but reduced the soil pH. Furthermore, the combined treatment significantly increased the abundance and diversity of the soil microbial community. In particular, the abundances of the bacteria Bacteroides, Proteobacteria, and Actinobacteria were increased, with the genera Flavobacterium, Pedobacter, Algoriphagus, Gaetbulibacter, Pseudomonas, Luteimonas, and Arthrobacter dominating. Meanwhile, the abundance of the fungus Zygomycota was increased, with Mortierella dominating. Moreover, inoculation with earthworms greatly improved the structure of the soil microbial community.

Compared with phytoremediation or microbial remediation alone, combined remediation was considered to be a more efficient remediation strategy. This study describes the effects of the combined remediation of T. patula, AMF, and earthworms on the physiochemical properties and microbial community of Cu-contaminated soil. This provides a comprehensive strategy to improve the phytoremediation efficiency in Cu-contaminated soil.

A meta-analysis about the accumulation of heavy metals uptake by Sedum alfredii and Sedum plumbizincicola in contaminated soil.

Sedum alfredii and Sedum plumbizincicola typically have high heavy metal (such as Zn and Cd) accumulation capacities with fast growth rates and relatively high Pb tolerance in contaminated soils. We compared the accumulation characteristics of heavy metals in Sedum species through meta-analysis. Furthermore, we analyzed the effects of soil organic matter (SOM) and soil pH on Cd, Pb and Zn accumulation by S. alfredii and S. plumbizincicola and the correlation between various metals. Results showed that the accumulations of Cd and Zn in shoots were higher than that of roots, but Pb accumulated in roots more than shoots. Moreover, there is a significant positive correlation between the accumulation of Zn and Cd in shoots. We found that the heavy metal accumulation rate in shoots was higher with lower soil pH. Sedum species had the highest Cd adsorption capacity in 20-30 g/kg SOM and the highest Zn adsorption capacity in SOM less than 20 g/kg. The accumulation rate of Cd in shoots of S. plumbizincicola was increased with exposure time, while the accumulation rate of Zn was slightly decreased.

S. alfredii and S. plumbizincicola are two common Cd and Zn hyperaccumulators. We systematically compared the accumulation characteristics of heavy metals in Sedum plants and the effects of soil organic matter and pH on the accumulation of Cd, Pb and Zn in S. alfredii and S. plumbizincicola through meta-analysis. This provides certain theoretical knowledge for the application of sedum plants to the phytoremediation of heavy metal contaminated soils.

Screening of Foliar Barrier Agents and Reduces the Absorption and Transport of Cd in Wheat.

Different foliar barrier agents (FBA) were used by foliar spraying in first season field and pot experiments to compare their effects on Cadmium (Cd) reduction in wheat grains. The best two FBA (50 µM SNP and 2 mM Na2EDTA) can significantly reduce Cd concentration in wheat grains, and the filling period was the most effective period for FBA application. Compared with the control (H2O), foliar spraying 50 µM SNP or 2 mM Na2EDTA inhibited the moving of Cd from the lower tissue to upper tissue in stem and also significantly reduced the Cd accumulation in grains. Furthermore, compared with normal wheat variety (AK58), foliar spraying 50 µmol SNP or 2 mM Na2EDTA as the best two FBA significantly reduced Cd concentration in shoots of Cd low accumulation varieties (HZB and HJBY), which can be used for the safe production of wheat in Cd-contaminated farmlands.

Cell envelope defects of different capsule-null mutants in K1 hypervirulent Klebsiella pneumoniae can affect bacterial pathogenesis.

Hypervirulent Klebsiella pneumoniae (hvKP) causes Klebsiella-induced liver abscess. Capsule is important for the pathogenesis of Klebsiella in systemic infection, but its role in gut colonisation is not well understood. By generating ΔwcaJ, Δwza and Δwzy capsule-null mutants in a prototypical K1 hypervirulent isolate, we show that inactivation of wza (capsule exportase) and wzy (capsule polymerase) confer cell envelope defects in addition to capsule loss, making them susceptible to bile salts and detergent stress. Bile salt resistance is restored when the initial glycosyltransferase wcaJ was inactivated together with wzy, indicating that build-up of capsule intermediates contribute to cell envelope defects. Mouse gut colonisation competition assays show that the capsule and its regulator RmpA were not required for hvKP to persist in the gut, although initial colonisation was decreased in the mutants. Both ΔrmpA and ΔwcaJ mutants gradually outcompeted the wild type in the gut, whereas Δwza and Δwzy mutants were less fit than wild type. Together, our results advise caution in using the right capsule-null mutant for determination of capsule's role in bacterial pathogenesis. With the use of ΔwcaJ mutant, we found that although the capsule is important for bacterial survival outside the gut environment, it imposes a fitness cost in the gut.

Phytotoxicity and accumulation of Cu in mature and young leaves of submerged macrophyte Hydrilla verticillata (L.f.) Royle.

Phytotoxicity and accumulation of Cu in mature and young leaves of submerged macrophyte Hydrilla verticillata (L.f.) Royle were investigated by analyzing the chlorophyll contents, chloroplast ultrastructure and leaf surface structure under different Cu treatments (0, 0.01, 0.05 and 0.1 f mg L-1). The results showed that 0.05 and 0.1 mg L-1 Cu treatment decreased the contents of Chl a and Chl b, and caused damage on leaf surface structure and chloroplast ultrastructure compared with control (0 mg L-1 Cu treatment). Higher concentration of Cu induced Chlorophyll decreases and the damages on the leaf surface structure and chloroplast ultrastructure were more pronounced in mature than in young leaves. It was observed that leaf Cu concentration increased almost linearly with exposure time and majority of the Cu accumulated in the cell walls. Among different cell wall fractions, the majority of Cu accumulated in cell walls was bound to the hemicellulose 1 and cellulose, followed by the pectin, hemicellulose 2. Mature leaves had significantly higher the concentrations of total Cu and bound-Cu in cell walls due to higher uronic acid content in their cell wall fractions (pectin, hemicellulose 1 and cellulose) than young leaves. Distinct cell wall composition might partially contribute to the different Cu toxicity and accumulation between mature and young leaves of submerged macrophyte H. verticillata. Our results show that mature leaves are more efficient in the uptake and accumulation of Cu than young leaves, which might explain why mature leaves sustain more severe damage.

Evaluation of biogas production potential of trace element-contaminated plants via anaerobic digestion.

Within the domain of phytoremediation research, the proper disposal of harvestable plant parts, that remove pollutants from contaminated soil, has been attracted extensive attention. Here, the bioenergy generation capability of trace metals (Cu, Pb, Zn, Cd, Mn, and As) polluted plants was assessed. The biogas production potential of accumulators or hyperaccumulator plants, Elsholtzia haichowensis, Sedum alfredii, Solanum nigrum, Phytolacca americana and Pteris vittata were 259.2 ± 1.9, 238.7 ± 4.2, 135.9 ± 0.9, 129.5 ± 2.9 and 106.8 ± 2.1 mL/g, respectively. The presence of Cu (at approximately 1000 mg/kg) increased the cumulative biogas production, the daily methane production and the methane yield of E. haichowensis. For S. alfredii, the presence of Zn (≥500 mg/kg) showed a significant negative impact on the methane content in biogas, and the daily methane production, which decreased the biogas and methane yield. The biogas production potential increased when the content of Mn was at 5 000-10,000 mg/kg, subsequently, decreased when the value of Mn at 20,000 mg/kg. However, Cd (1-200 mg/kg), Pb (125-2000 mg/kg) and As (1250-10,000 mg/kg) showed no distinctive change in the cumulative biogas production of S. nigrum, S. alfredii and P. vittata, respectively. The methane yield showed a strong positive correlation (R2 =0.9704) with cumulative biogas production, and the energy potential of the plant residues were at 415-985 kWh/ton. Thus, the anaerobic digestion has bright potential for the disposal of trace metal contaminated plants, and has promising prospects for the use in energy production.

Enhanced vacuole compartmentalization of cadmium in root cells contributes to glutathione-induced reduction of cadmium translocation from roots to shoots in pakchoi (Brassica chinensis L.).

Our previous studies showed that exogenous glutathione (GSH) decreased cadmium (Cd) concentration in shoots and alleviated the growth inhibition in pakchoi (Brassica chinensis L.) under Cd stress. Nevertheless, it is largely unknown how GSH decreases Cd accumulation in edible parts of pakchoi. This experiment mainly explored the mechanisms of GSH-induced reduction of Cd accumulation in shoot of pakchoi. The results showed that compared with sole Cd treatment, Cd + GSH treatment remarkably increased the expression of BcIRT1 and BcIRT2, and further enhanced the concentrations of Cd and Fe in root. By contrast, GSH application declined the concentration of Cd in the xylem sap. However, these results were not caused by xylem loading process because the expression of BcHMA2 and BcHMA4 had not significant difference between sole Cd treatment and Cd + GSH treatment. In addition, exogenous GSH significantly enhanced the expression of BcPCS1 and promoted the synthesis of PC2, PC3 and PC4 under Cd stress. At the same time, exogenous GSH also significantly improved the expression of BcABCC1 and BcABCC2 in the roots of seedling under Cd stress, suggesting that more PCs-Cd complexes may be sequestrated into vacuoles by ABCC1 and ABCC2 transporters. The results showed that exogenous GSH could up-regulate the expression of BcIRT1/2 to increase the Cd accumulation in root, and the improvement of PCs contents and the expression of BcABCC1/2 enhanced the compartmentalization of Cd in root vacuole of pakchoi under Cd stress. To sum up, exogenous GSH reduce the concentration of free Cd2+ in the cytoplast of root cells and then dropped the loading of Cd into the xylem, which eventually given rise to the reduction of Cd accumulation in edible portion of pakchoi.

Analysis of the long-term effectiveness of biochar immobilization remediation on heavy metal contaminated soil and the potential environmental factors weakening the remediation effect: A review.

Currently, the research and application of biochar in the remediation of heavy metal contaminated soil has become a hotspot, especially regarding the remediation of agricultural land. Biochar has been proved to be effective in reducing the content of available heavy metals in the soil as well as the heavy metals in plants. However, the long-term effectiveness of biochar immobilization has not been widely studied. In this review, retrospective search was carried out on the published literature results concerning remediation effects of biochar on different areas of heavy metal contaminated soil in the recent years, its application in field remediation (several years), and some potential abiotic and biotic factors that may weaken the immobilization effects of biochar. This results indicate that: (1) biochar is widely used in the remediation of heavy metal contaminated soil in different areas and has excellent immobilization effect. (2) Most of the research demonstrate that the immobilization effect of biochar is effective for 2-3 years or according to few results even for 5 years. However, there have been various reports claiming that the immobilization effect of biochar decreases with time. (3) Abiotic factors such as acid rain, flooded environment, changes in soil condition (pH, redox and dissolved organic matter) and changes in biochar (Cl- and alkali leaching) can significantly weaken the immobilization effect of biochar. (4) Biotic factors such as plant roots, earthworms and soil microorganisms can also significantly reduce the immobilization effect of biochar. Therefore, field experiments having longer time span with biochar need to be further carried out, and the developmental research of modified biochar with a more stable immobilization effect also needs further attention.

Promotion of Zinc Tolerance, Acquisition and Translocation of Phosphorus in Mimosa pudica L. Mediated by Arbuscular Mycorrhizal Fungi.

Heavy metal contamination of soil is of increasing concern because of its potential risk to human health. In this study, two AMFs (Rhizophagus intraradices and Funneliformis mosseae) substantially increased the biomass of bashfulgrass in Zn-contaminated soil, even at Zn levels of up to 600 mg kg-1. Zn uptake in R. intraradices- and F. mosseae-mycorrhizal bashfulgrass was increased by 40-fold and 7-fold, respectively, when plants grown in Zn-contaminated (400 mg kg-1) soil. Elemental analysis showed that neither AMF had an effect on Zn concentration in plant tissues, including the roots and shoots. However, a significant increase of phosphorus (P) concentration was observed, suggesting the increased is from the improved use efficiency of soil nutrients by AMFs. Comparing the two AMFs, better growth performance with more biomass occurred with R. intraradices-inoculated bashfulgrass in Zn-contaminated soil. This is consistent with R. intraradices being more tolerant to Zn than F. mosseae, indicated by a higher colonization percentage in bashfulgrass roots. Taken together, our data indicate that AMFs possibly improve acquisition and translocation of P to promote increased biomass. Moreover, mycorrhiza did not enhance Zn accumulation in shoots and roots of bashfulgrass at the same Zn level. In the future, developing AMF (especially R. intraradices) inoculation of plants might be a desirable means of safe production of ornamental plants in metal-polluted soil.

Acquisition of Plasmid with Carbapenem-Resistance Gene blaKPC2 in Hypervirulent Klebsiella pneumoniae, Singapore.

The convergence of carbapenem-resistance and hypervirulence genes in Klebsiella pneumoniae has led to the emergence of highly drug-resistant superbugs capable of causing invasive disease. We analyzed 556 carbapenem-resistant K. pneumoniae isolates from patients in Singapore hospitals during 2010-2015 and discovered 18 isolates from 7 patients also harbored hypervirulence features. All isolates contained a closely related plasmid (pKPC2) harboring blaKPC-2, a K. pneumoniae carbapenemase gene, and had a hypervirulent background of capsular serotypes K1, K2, and K20. In total, 5 of 7 first patient isolates were hypermucoviscous, and 6 were virulent in mice. The pKPC2 was highly transmissible and remarkably stable, maintained in bacteria within a patient with few changes for months in the absence of antimicrobial drug selection pressure. Intrapatient isolates were also able to acquire additional antimicrobial drug resistance genes when inside human bodies. Our results highlight the potential spread of carbapenem-resistant hypervirulent K. pneumoniae in Singapore.

Comparative transcriptomic analysis reveals novel insights into the response to Cr(VI) exposure in Cr(VI) tolerant ectomycorrhizal fungi Pisolithus sp. 1 LS-2017.

Chromium (Cr) is one of the most toxic heavy metals and a health hazard to millions of people worldwide. Ectomycorrhizal (ECM) fungi can assist plants in phytoremediation of heavy metal contaminated soil. Cr tolerance differs among ECM fungal varieties, but the underlying molecular mechanisms of Cr tolerance in ECM fungi are not clear. This study identified, analysed and compared the Cr(VI)-induced transcriptional changes between Cr(VI)-tolerant strain (Pisolithus sp. 1 LS-2017) and Cr(VI)-sensitive strain (Pisolithus sp. 2 LS-2017) by de novo transcriptomic analysis. The results showed that 93,642 assembled unique transcripts representing the 22,353 (46.76%) unigenes matched the proteins we have known in the Nr database and 47,801 unigenes were got from the Pisolithus spp. For DEGs between the control and 10 mg/L Cr(VI) treatment, cyanoamino acid metabolic, type I diabetes mellitus metabolism, nitrogen metabolism and beta-Alanine metabolism pathways were significantly enriched (p < 0.05) in Pisolithus sp. 1 LS-2017. Two nitrate reductase family genes (nidD, niiA) provide Cr(VI) tolerance for Pisolithus sp. 1 LS-2017 by regulating Cr(VI) reduction. In addition, NO produced by nidD, niiA regulated denitrification can alleviate Cr(VI) induced oxidative stress. In Pisolithus sp. 2 LS-2017, the alcC, aldA and lcf2 gene may alleviate Cr(VI) induced oxidative stress by protecting SH groups and increasing secondary metabolism, reducing detoxify aldehydes to carboxylic acids and producing LCPUFAs respectively; .T gene regulate Cr(VI) induced wound healing by pigmentation and stability of melanin in spore; MKP2 gene accelerate Cr(VI) induced cell death and gpmA gene regulated Cr(VI) induced energy emergency.

Promotion of growth and phytoextraction of cadmium and lead in Solanum nigrum L. mediated by plant-growth-promoting rhizobacteria.

Plant growth-promoting rhizobacteria (PGPR) are a specific category of microbes that improve plant growth and promote greater tolerance to metal stress through their interactions with plant roots. We evaluated the effects of phytoremediation combining the cadmium accumulator Solanum nigrum L. and two Cd- and Pb-resistant bacteria isolates. To understand the interaction between PGPR and their host plant, we conducted greenhouse experiments with inoculation treatments at Nanjing Agricultural University (Jiangsu Province, China), in June 2018. Two Cd- and Pb-resistant PGPR with various growth-promoting properties were isolated from heavy metal-contaminated soil. 16S rRNA analyses indicated that the two isolates were Bacillus genus, and they were named QX8 and QX13. Pot experiments demonstrated that inoculation may improve the rhizosphere soil environment and promote absorption of Fe and P by plants. Inoculation with QX8 and QX13 also enhanced the dry weight of shoots (1.36- and 1.7-fold, respectively) and roots (1.42- and 1.96-fold) of plants growing in Cd- and Pb-contaminated soil, and significantly increased total Cd (1.28-1.81 fold) and Pb (1.08-1.55 fold) content in aerial organs, compared to non-inoculated controls. We also detected increases of 23% and 22% in the acid phosphatase activity of rhizosphere soils inoculated with QX8 and QX13, respectively. However, we did not detect significant differences between inoculated and non-inoculated treatments in Cd and Pb concentrations in plants and available Cd and Pb content in rhizosphere soils. We demonstrated that PGPR-assisted phytoremediation is a promising technique for remediating heavy metal-contaminated soils, with the potential to enhance phytoremediation efficiency and improve soil quality.

Recycling of Chemical Eluent and Soil Improvement After Leaching.

Ethylenediaminetetraacetic acid disodium salt (EDTA) was selected among various eluents due to its highest removal efficiency for lead (Pb) (43.7%) and zinc (Zn) (57.1%) leaching from Pb-Zn contaminated soil by soil column experiment. Compared with newly prepared EDTA eluent, using recycled EDTA eluent can still leaching down 71.1% of Pb and 63.2% of Zn respectively, which showed the reusable benefits of recycled EDTA eluent. After soils were leached by EDTA, soil quality decline, such as reducing of urease, catalase, invertase activities and microorganism numbers. However, adding 5% nutrition soil or earthworm fertilizer can significantly improve the quality of EDTA leached soil, and promote growth of peas and ryegrass compared with EDTA treatments. Overall, the improvement of EDTA leached soil by adding nutrition soil or earthworm fertilizer is important, and recycled EDTA eluent can recycle and re-use for Pb-Zn contaminated soil remediation.