Coexistence of virulent and multidrug-resistant plasmids in an uropathogenic Escherichia coli.

OBJECTIVES: The emergence of pathogens co-harbouring multiple mobile resistance and virulence elements is of great concern in clinical settings. Herein, we report an O101: H10-ST167 Escherichia coli Hu106 strain isolated from the urinary tract of a female in China. METHODS: Antibiotic susceptibility testing was used to present the antimicrobial resistance spectrum. Whole-genome sequencing (WGS) and bioinformatic analysis were used to clarify the virulent and resistance mechanisms. Furthermore, the virulence of this strain was tested by the Greater wax moth larvae and siderophore production experiment. RESULTS: The strain E. coli Hu106 was resistant to almost all antimicrobials tested, and only susceptible to aztreonam, amikacin, and tigecycline. WGS analysis revealed that the strain Hu106 co-harboured blaNDM-9 and mcr-1 on p2-Hu106, belonging to IncHI2/IncHI2A (256,000 bp). The co-existence of both resistance genes, blaNDM-9 and mcr-1, on the plasmid p2-Hu106 was mainly acquired by transposition recombination of mobile antibiotic elements mediated by IS26 and/or IS1 on IncHI2/IncHI2A type plasmid. In addition, the virulence clusters aerobactin (iutA-iucABCD) and salmochelin (iroBCDEN) were identified on an IncFIB/IncFIC(IncFII) type plasmid p1-Hu106, flanked by small mobile elements such as IS1A, ISkpn28, and IS3, respectively. After performing genomic comparison of p1-Hu106 with the WGS in NCBI, we identified that the virulent plasmid p1-Hu106-like could spread in different clones of E. coli and Klebsiella pneumoniae, revealing its underlying dissemination mechanism between Enterobacterales. Furthermore, the strain caused a decreased survival rate of larvae and produced high siderophore units (62.33%), similar to hypervirulent K. pneumoniae NTUH-K2044. CONCLUSIONS: The strains co-carrying the multidrug-resistant plasmid p2-Hu106 and virulent plasmid p1-Hu106 should be closely monitored to prevent its further spreading.

Within-host acquisition of colistin-resistance of an NDM-producing Klebsiella quasipneumoniae subsp. similipneumoniae strain through the insertion sequence-903B-mediated inactivation of mgrB gene in a lung transplant child in China.

Background: Colistin, as the antibiotic of "last resort" for carbapenem-resistant Klebsiella, develop resistance during administration of this antimicrobial agent. We identified an NDM-1-producing Klebsiella quasipneumonuae subsp. similipneumoniae (KQSS) strain KQ20605 recovered from a child, which developed resistance to colistin (KQ20786) through acquiring an IS903B element between the -27th and -26th bp of mgrB promoter region after 6-day colistin usage. Objectives: The aim of this study is to explore the source of IS903B in the disruptive mgrB gene and its underlying mechanisms. Materials and methods: Antibiotics susceptibility testing was conducted via microbroth dilution method. The in vitro colistin-induced experiment of KQ20605 was performed to mimic the in vivo transition from colistin-sensitive to resistant. Whole-genome sequencing was used to molecular identification of colistin resistance mechanism. Results: The IS903B element integrated into mgrB gene of KQ20786 had a 100% nucleotide identity and coverage match with one IS903B on plasmid IncR, and only 95.1% (1005/1057) identity to those on chromosome. In vitro, upon the pressure of colistin, KQ20605 could also switch its phenotype from colistin-sensitive to resistant with IS elements (e.g., IS903B and IS26) frequently inserted into mgrB gene at "hotspots", with the insertion site of IS903B nearly identical to that of KQ20786. Furthermore, IS26 elements in this isolate were only encoded by plasmids, including IncR and conjugative plasmid IncN harboring bla NDM. Conclusion: Mobilizable IS elements on plasmids tend to be activated and integrated into mgrB gene at "hotspots" in this KQSS, thereby causing the colistin resistance emergence and further dissemination.

Carbapenem-resistant Citrobacter freundii harboring blaKPC-2 and blaNDM-1: a study on their transferability and potential dissemination via generating a transferrable hybrid plasmid mediated by IS6100.

Introduction: The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of blaKPC-2- and blaNDM-1-coharboring carbapenem-resistant Citrobacter freundii (CRCF). Methods: Four blaKPC-2- and blaNDM-1-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination. Results: Three ST523 blaKPC-2- and blaNDM-1-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the blaKPC-2 and blaNDM-1 genes were coharbored on an IncR plasmid, probably generated by a blaKPC-2-harboring plasmid acquiring blaNDM-1, in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the blaKPC-2 and blaNDM-1 of the fourth CRCF strain are located on two different non-transferable plasmids lacking complete transfer elements. Additionally, throughout the course of the 10-day continuous passage, the genetic surroundings of blaNDM-1 in four CRCF strains were gradually excised from their plasmids after the 8th day, whereas they maintained 100% retention for blaKPC-2. Genome and plasmidome analyses revealed that blaKPC-2- or blaNDM-1-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae. Conclusion: Clonal dissemination of ST523 blaKPC-2- and blaNDM-1-coharboring CRCF strains was detected, and we first reported blaKPC-2 and blaNDM-1 concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented.

Evaluation of BACTEC MGIT 960 system for recovery of Nocardia from clinical specimens.

Nocardia spp. is an aerobic Gram-positive bacillus responsible for nocardiosis. Herein, we performed a retrospective study to evaluate the performance of BACTEC MGIT 960 system, in comparison with smear microscopy and blood agar plate (BAP) culture, to recover Nocardia from different clinical specimens. Furthermore, the inhibitory effect of antibiotics contained in MGIT 960 tube on Nocardia was also evaluated. The sensitivities for Nocardia recovery using smear microscopy, BAP culture, and MGIT 960 were 39.4% (54/137), 46.1% (99/215), and 81.3% (156/192), respectively. N. farcinica was the most detected species (60.4%, 136/225). In MGIT 960-recovered Nocardia strains, N. farcinica accounted for 76.9%. Furthermore, trimethoprim in MGIT 960 tube inhibited less N. farcinica growth than that of other Nocardia species, partially explaining why MGIT 960 recovered more N. farcinica from sputa. The current study demonstrated that MGIT 960 could recover Nocardia strains from heavily-contaminated samples if its components and antibiotics are redesigned.

Genetic Diversity of Polymyxin-Resistance Mechanisms in Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae: a Multicenter Study in China.

Polymyxin has been the last resort to treat multidrug-resistant Klebsiella pneumonia. However, recent studies have revealed that polymyxin-resistant carbapenem-resistant Klebsiella pneumonia (PR-CRKP) emerged due to the mutations in chromosomal genes or the plasmid-harboring mcr gene, leading to lipopolysaccharide modification or efflux of polymyxin through pumps. Further surveillance was required. In the present study we collected PR-CRKP strains from 8 hospitals in 6 provinces/cities across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features by whole-genome sequencing (WGS). The broth microdilution method (BMD) was performed to determine the MIC of polymyxin. Of 662 nonduplicate CRKP strains, 15.26% (101/662) were defined as PR-CRKP; 10 (9.90%) were confirmed as Klebsiella quasipneumoniae by WGS. The strains were further classified into 21 individual sequence types (STs) by using multilocus sequence typing (MLST), with ST11 being prevalent (68/101, 67.33%). Five carbapenemase types were identified among 92 CR-PRKP, blaKPC-2 (66.67%), blaNDM-1 (16.83%), blaNDM-5 (0.99%), blaIMP-4 (4.95%), and blaIMP-38 (0.99%). Notably, 2 PR-CRKP strains harbored both blaKPC-2 and blaNDM-1. The inactivation of mgrB, associated significantly with high-level polymyxin resistance, was mainly caused by the insertion sequence (IS) insertion (62.96%, 17/27). Furthermore, acrR was inserted coincidently by ISkpn26 (67/101, 66.33%). The deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47 (capsule locus types), and diverse mutations of the ramR gene were identified. Only one strain carried the mcr gene. In summary, the high IS-inserted mgrB inactivation, the close relationship between ST11 and the deletion or splicing mutations of the crrCAB, and the specific features of PR-K. quasipneumoniae constituted notable features of our PR-CRKP strains in China. IMPORTANCE Polymyxin-resistant CRKP is a serious public health threat whose resistance mechanisms should be under continuous surveillance. Here, we collected 662 nonduplicate CRKP strains across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features. Polymyxin resistance mechanism in 101 PR-CRKP strains in China were also investigated, 9.8% of which (10/101) were K. quasipneumoniae, as determined via WGS, and inactivation of mgrB remained the most crucial polymyxin resistance mechanism, significantly related to high-level resistance. Deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47. Diverse mutations of the ramR gene were identified. The plasmid complementation experiment and mRNA expression analysis further confirmed that the mgrB promoter and ramR played a critical role in polymyxin resistance. This multicenter study contributed to the understanding of antibiotic resistance forms in China.

Molecular Characteristics of an NDM-4 and OXA-181 Co-Producing K51-ST16 Carbapenem-Resistant Klebsiella pneumoniae: Study of Its Potential Dissemination Mediated by Conjugative Plasmids and Insertion Sequences.

The carbapenem-resistant Klebsiella pneumoniae (CRKP) strain GX34 was recovered from the respiratory tract of an elderly male with severe pneumonia, and only susceptible to amikacin, tigecycline, and colistin. Complete genome suggested that it belonged to K51-ST16 and harbored plasmid-encoded NDM-4 and OXA-181, located on IncFIB plasmid GX34p1_NDM-4 and ColKP3/IncX3 plasmid GX34p4_OXA-181, respectively. A series of transconjugants generated in the plasmid conjugation assays, including Escherichia coli J53-N1 (harboring a self-transmissible and blaNDM-1-producing plasmid Eco-N-1-p), J53-N2 (harboring a blaNDM-4-producing plasmid and a helper plasmid GX34p5), and J53-O (harboring a blaOXA-181-producing plasmid), could be stably inherited after 10 days of serial passage and no significant biological fitness costs were detected. Furthermore, we first reported the blaNDM-1 gene, derived from blaNDM-4 mutation (460C>A) under meropenem pressure, could be in vitro transferred into a self-conjugative, recombined plasmid Eco-N-1-p of J53-N1. Eco-N-1-p was mainly recombined by GX34p4_OXA-181 (40,449 bp, 75.16%) and GX34p1_NDM-4 (8,553 bp, 15.89%), in which IS26 and IS5-like probably played a major role. Eco-N-1-p could be transferred into the conjugation recipient K. pneumoniae KP54 and make the latter sacrifice fitness. The retention rates of blaNDM-1 remained high stability (>80% after 200 generations). The comparative genomic analysis of GX34 and those carrying blaNDM-4 or blaOXA-181 genes retrieved from the NCBI RefSeq database showed all blaNDM-4 (26/26, 100.00%) and blaOXA-181 (13/13, 100.00%) were surrounded by IS26. The immediate environment of blaNDM-4 and blaOXA-181 in GX34 and some retrieved strains shared identical features, hinting at their possible dissemination. Effective measures should be taken to monitor the spread of this clone.

Intranasal Transplantation of Human Neural Stem Cells Ameliorates Alzheimer's Disease-Like Pathology in a Mouse Model.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by memory impairments, which has no effective therapy. Stem cell transplantation shows great potential in the therapy of various disease. However, the application of stem cell therapy in neurological disorders, especially the ones with a long-term disease course such as AD, is limited by the delivery approach due to the presence of the brain blood barrier. So far, the most commonly used delivery approach in the therapy of neurological disorders with stem cells in preclinical and clinical studies are intracranial injection and intrathecal injection, both of which are invasive. In the present study, we use repetitive intranasal delivery of human neural stem cells (hNSCs) to the brains of APP/PS1 transgenic mice to investigate the effect of hNSCs on the pathology of AD. The results indicate that the intranasally transplanted hNSCs survive and exhibit extensive migration and higher neuronal differentiation, with a relatively limited glial differentiation. A proportion of intranasally transplanted hNSCs differentiate to cholinergic neurons, which rescue cholinergic dysfunction in APP/PS1 mice. In addition, intranasal transplantation of hNSCs attenuates ß-amyloid accumulation by upregulating the expression of ß-amyloid degrading enzymes, insulin-degrading enzymes, and neprilysin. Moreover, intranasal transplantation of hNSCs ameliorates other AD-like pathology including neuroinflammation, cholinergic dysfunction, and pericytic and synaptic loss, while enhancing adult hippocampal neurogenesis, eventually rescuing the cognitive deficits of APP/PS1 transgenic mice. Thus, our findings highlight that intranasal transplantation of hNSCs benefits cognition through multiple mechanisms, and exhibit the great potential of intranasal administration of stem cells as a non-invasive therapeutic strategy for AD.

MicroRNA-34a-mediated death of acute myeloid leukemia stem cells through apoptosis induction and exosome shedding inhibition via histone deacetylase 2 targeting.

We investigated the role of leukemia stem cells in chemoresistance and recurrence of acute myeloid leukemia. Total RNA was isolated from cells or tissues using TRIzol reagent. Cell viability was assessed with the tetrazolium assay. MicroRNA-34a (miR-34a), which acts on cell death regulation pathways, was noticeably downregulated in non-M3 acute myeloid leukemia stem cells compared with normal hematopoietic stem cells. Furthermore, inhibition of miR-34a-mediated suppression in leukemia stem cells was associated with poor clinical outcomes and impaired treatment efficacy in acute myeloid leukemia. Transfection with a miR-34a mimic triggered leukemia stem cell death and prevented leukemia. Bioinformatics analysis and a dual-luciferase reporter assay showed that miR-34a targeted the 3'-untranslated region of histone deacetylase 2, and the reinforced expression of miR-34a remarkably stimulated the expression of histone deacetylase 2 in leukemia stem cells. Ectopic miR-34a expression triggered death of leukemia stem cells via pathways involving the Janus kinase 1-signal transducer and activator of transcription 2-p53 axis. Targeting leukemia stem cells to trigger cell death through upregulation of miR-34a expression could be used to diagnose and treat acute myeloid leukemia.

[Effects of lentivirus-mediated heat shock protein 70 gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia].

OBJECTIVE: To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on calcium homeostasis in PC12 cells undergone ischemia and hypoxia, and the mechanism involved. METHODS: PC12 cells at logarithmic phase were collected, and were divided into recombination lentivirus infection group (infected by lentivirus containing HSP70 and fluorescent gene), lentivirus control group (infected by lentivirus containing fluorescin without HSP70 gene) and non-infection group. HSP70 gene and protein expressions in PC12 cells were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot. After being challenged with ischemia and hypoxia for 4 hours, the viability of cells was detected by methyl thiazolyl tetrazolium (MTT), the levels of lactic acid dehydrogenase (LDH) in cell supernatant were determined by LDH measurement test kit. The concentration of intracelluer calcium ([Ca²âº] i ) was assayed by flow cytometer. The activities of Na⁺ -K⁺ -ATPase, Ca⁺ -Mg⁺ -ATPase and total ATPase were measured by ATPase test kits. RESULTS: The expressions of exogenous HSP70 gene and protein were found by RT-PCR and Western Blot in the recombination lentivirus infection group. After being challenged with ischemia and hypoxia, the viability of cells in the recombination lentivirus infection group was increased significantly as compared with the lentivirus control group and non-infection group ( A value: 0.575 ± 0.020 vs. 0.395 ± 0.014, 0.363 ± 0.045, t1 = 17.996, t2= 10.600, both P < 0.001), the levels of LDH in culture medium and the concentration of [Ca⁺] i were decreased significantly [LDH ( U/L ): 743.46 ± 23.68 vs. 935.43±34.77, 962.89 ± 26.68, t1 = 11.179, t2 = 15.044, both P < 0.001; [ Ca²âº] i relative fluorescence: ( 31.60 ± 2.43 )% vs. (41.48 ± 3.33)%, ( 40.40 ± 3.05 )%, t1 = 5.853, t2= 5.502, both P < 0.001], and the activities of Na⁺ -K⁺ -ATPase, Ca²âº -Mg²âº -ATPase and total ATPase were increased significantly [ Na⁺ -K⁺ -ATPase (µmol × mg⁻¹× h⁻¹): 8.608 ± 0.307 vs. 6.728 ± 0.173, 6.450 ± 0.091, t1 = 9.237, P1= 0.001, t2 = 11.675, P2 < 0.001; Ca²âº -Mg²âº -ATPase (µmol×mg⁻¹ × h⁻¹): 10.523 ± 0.036 vs. 7.910 ± 0.209, 8.064 ± 0.195, t1 = 9.718, P1 = 0.001, t2= 11.535, P2 < 0.001; total ATPase (µmol×mg⁻¹× h⁻¹): 17.041 ± 0.324 vs. 14.150 ± 0.182, 13.983 ± 0.085, t1= 16.113, t2 = 17.602, both P < 0.001]. There was no statistical difference in above indexes between lentivirus control group and non-infection group. CONCLUSIONS: HSP70 can maintain the PC12 cells calcium homeostasis, which may be one of the important mechanisms of anti-apoptosis.