Glial Sphingosine-Mediated Epigenetic Regulation Stabilizes Synaptic Function in Drosophila Models of Alzheimer's Disease.

Destabilization of neural activity caused by failures of homeostatic regulation has been hypothesized to drive the progression of Alzheimer's Disease (AD). However, the underpinning mechanisms that connect synaptic homeostasis and the disease etiology are yet to be fully understood. Here, we demonstrated that neuronal overexpression of amyloid ß (Aß) causes abnormal histone acetylation in peripheral glia and completely blocks presynaptic homeostatic potentiation (PHP) at the neuromuscular junction in Drosophila The synaptic deficits caused by Aß overexpression in motoneurons are associated with motor function impairment at the adult stage. Moreover, we found that a sphingosine analog drug, Fingolimod, ameliorates synaptic homeostatic plasticity impairment, abnormal glial histone acetylation, and motor behavior defects in the Aß models. We further demonstrated that perineurial glial sphingosine kinase 2 (Sk2) is not only required for PHP, but also plays a beneficial role in modulating PHP in the Aß models. Glial overexpression of Sk2 rescues PHP, glial histone acetylation, and motor function deficits that are associated with Aß in Drosophila Finally, we showed that glial overexpression of Sk2 restores PHP and glial histone acetylation in a genetic loss-of-function mutant of the Spt-Ada-Gcn5 Acetyltransferase complex, strongly suggesting that Sk2 modulates PHP through epigenetic regulation. Both male and female animals were used in the experiments and analyses in this study. Collectively, we provided genetic evidence demonstrating that abnormal glial epigenetic alterations in Aß models in Drosophila are associated with the impairment of PHP and that the sphingosine signaling pathway displays protective activities in stabilizing synaptic physiology.SIGNIFICANCE STATEMENT Fingolimod, an oral drug to treat multiple sclerosis, is phosphorylated by sphingosine kinases to generate its active form. It is known that Fingolimod enhances the cognitive function in mouse models of Alzheimer's disease (AD), but the role of sphingosine kinases in AD is not clear. We bridge this knowledge gap by demonstrating the relationship between impaired homeostatic plasticity and AD. We show that sphingosine kinase 2 (Sk2) in glial cells is necessary for homeostatic plasticity and that glial Sk2-mediated epigenetic signaling has a protective role in synapse stabilization. Our findings demonstrate the potential of the glial sphingosine signaling as a key player in glia-neuron interactions during homeostatic plasticity, suggesting it could be a promising target for sustaining synaptic function in AD.

Mepanipyrim induces visual developmental toxicity and vision-guided behavioral alteration in zebrafish larvae.

Mepanipyrim, an anilinopyrimidine fungicide, has been extensively used to prevent fungal diseases in fruit culture. Currently, research on mepanipyrim-induced toxicity in organisms is still very scarce, especially visual developmental toxicity. Here, zebrafish larvae were employed to investigate mepanipyrim-induced visual developmental toxicity. Intense light and monochromatic light stimuli-evoked escape experiments were used to investigate vision-guided behaviors. Meanwhile, transcriptomic sequencing and real-time quantitative PCR assays were applied to assess the potential mechanisms of mepanipyrim-induced visual developmental toxicity and vision-guided behavioral alteration. Our results showed that mepanipyrim exposure could induce retinal impairment and vision-guided behavioral alteration in larval zebrafish. In addition, the grk1b gene of the phototransduction signaling pathway was found to be a potential aryl hydrocarbon receptor (AhR)-regulated gene. Mepanipyrim-induced visual developmental toxicity was potentially related to the AhR signaling pathway. Furthermore, mepanipyrim-induced behavioral alteration was guided by the visual function, and the effects of mepanipyrim on long and middle wavelength light-sensitive opsins may be the main cause of vision-guided behavioral alteration. Our results provide insights into understanding the relationship between visual development and vision-guided behaviors induced by mepanipyrim exposure.

Application of Sigma metrics in the quality control strategies of immunology and protein analytes.

BACKGROUND: Six Sigma (6σ) is an efficient laboratory management method. We aimed to analyze the performance of immunology and protein analytes in terms of Six Sigma. METHODS: Assays were evaluated for these 10 immunology and protein analytes: Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM), Complement 3 (C3), Complement 4 (C4), Prealbumin (PA), Rheumatoid factor (RF), Anti streptolysin O (ASO), C-reactive protein (CRP), and Cystatin C (Cys C). The Sigma values were evaluated based on bias, four different allowable total error (TEa) and coefficient of variation (CV) at QC materials levels 1 and 2 in 2020. Sigma Method Decision Charts were established. Improvement measures of analytes with poor performance were recommended according to the quality goal index (QGI), and appropriate quality control rules were given according to the Sigma values. RESULTS: While using the TEaNCCL , 90% analytes had a world-class performance with σ>6, Cys C showed marginal performance with σ<4. While using minimum, desirable, and optimal biological variation of TEa, only three (IgG, IgM, and CRP), one (CRP), and one (CRP) analytes reached 6σ level, respectively. Based on σNCCL that is calculated from TEaNCCL , Sigma Method Decision Charts were constructed. For Cys C, five multi-rules (13s /22s /R4s /41s /6X , N = 6, R = 1, Batch length: 45) were adopted for QC management. The remaining analytes required only one QC rule (13s , N = 2, R = 1, Batch length: 1000). Cys C need to improve precision (QGI = 0.12). CONCLUSIONS: The laboratories should choose appropriate TEa goals and make judicious use of Sigma metrics as a quality improvement tool.

Vogesella urethralis sp. nov., isolated from human urine, and emended descriptions of Vogesella perlucida and Vogesella mureinivorans.

A novel Vogesella strain, YM-1T, was recovered from human urine in PR China in 2017. Cells of strain YM-1T were Gram-stain-negative, rod-shaped, aerobic, motile, non-spore-forming and poly-ß-hydroxybutyrate-accumulating. The strain contained C16:1ω6c/C 16:1ω7c, C16:0 and C18:0ω7c as major fatty acids; phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospholipid as major polar lipids; and ubiquinone-8 as the predominant respiratory quinone. Comparison of 16S rRNA gene sequences indicated that this strain had highest similarities to Vogesella perlucida DS-28T (98.8 %) and Vogesella mureinivorans 389T (98.1 %). The results of phylogenetic analysis based on the 16S rRNA gene sequences revealed that the novel strain was clustered and well separated with V. perlucida DS-28T and V. mureinivorans 389T within the genus Vogesella. The average nucleotide identity (ANI) and amino acid identity (AAI) analyses showed that this strain was not identified as V. perlucida DS-28T or V. mureinivorans 389T, with values well below the threshold limit for species demarcation (ANI <88.1 %, AAI <88.6 %). Based on the above results, strain YM-1T is proposed to be a novel species of the genus Vogesella with the name Vogesella urethralis sp. nov. (YM-1T=NBRC 113779=CGMCC 1.17135).

Inhibition of apoptosis signal-regulating kinase by paeoniflorin attenuates neuroinflammation and ameliorates neuropathic pain.

BACKGROUND: Neuropathic pain is a serious clinical problem that needs to be solved urgently. ASK1 is an upstream protein of p38 and JNK which plays important roles in neuroinflammation during the induction and maintenance of chronic pain. Therefore, inhibition of ASK1 may be a novel therapeutic approach for neuropathic pain. Here, we aim to investigate the effects of paeoniflorin on ASK1 and neuropathic pain. METHODS: The mechanical and thermal thresholds of rats were measured using the Von Frey test. Cell signaling was assayed using western blotting and immunohistochemistry. RESULTS: Chronic constrictive injury (CCI) surgery successfully decreased the mechanical and thermal thresholds of rats and decreased the phosphorylation of ASK1 in the rat spinal cord. ASK1 inhibitor NQDI1 attenuated neuropathic pain and decreased the expression of p-p38 and p-JNK. Paeoniflorin mimicked ASK1 inhibitor NQDI1 and inhibited ASK1 phosphorylation. Paeoniflorin decreased the expression of p-p38 and p-JNK, delayed the progress of neuropathic pain, and attenuated neuropathic pain. Paeoniflorin reduced the response of astrocytes and microglia to injury, decreased the expression of IL-1ß and TNF-α, and downregulated the expression of CGRP induced by CCI. CONCLUSIONS: Paeoniflorin is an effective drug for the treatment of neuropathic pain in rats via inhibiting the phosphorylation of ASK1, suggesting it may be effective in patients with neuropathic pain.

Genome-wide identification and characterization of Eutrema salsugineum microRNAs for salt tolerance.

Eutrema salsugineum, a close relative of Arabidopsis thaliana, is a valuable halophytic model plant that has extreme tolerance to salinity. As posttranscriptional gene regulators, microRNAs (miRNAs) control gene expression and a variety of biological processes, including plant-stress responses. To identify salt-stress responsive miRNAs in E. salsugineum and reveal their possible roles in the adaptive response to salt stress, we chose the Solexa sequencing platform to screen the miRNAs in 4-week-old E. salsugineum seedlings under salt treatment. A total of 82 conserved miRNAs belonging to 27 miRNA families and 17 novel miRNAs were identified and 11 conserved miRNA families and 4 novel miRNAs showed a significant response to salt stress. To investigate the possible biological roles of miRNAs, 1060 potential targets were predicted. Moreover, 35 gene ontology (GO) categories and 1 pathway, including a few terms that were directly and indirectly related to salt stress, were significantly enriched in the salt-stress-responsive miRNAs targets. The relative expression analysis of six target genes was analyzed using quantitative real-time polymerase chain reaction (PCR) and showed a negative correlation with their corresponding miRNAs. Many stress regulatory and phytohormone regulatory cis-regulatory elements were widely present in the promoter region of the salt-responsive miRNA precursors. This study describes the large-scale characterization of E. salsugineum miRNAs and provides a useful resource for further understanding of miRNA functions in the regulation of the E. salsugineum salt-stress response.

MicroRNA expression profiling of the fifth-instar posterior silk gland of Bombyx mori.

BACKGROUND: The growth and development of the posterior silk gland and the biosynthesis of the silk core protein at the fifth larval instar stage of Bombyx mori are of paramount importance for silk production. RESULTS: Here, aided by next-generation sequencing and microarry assay, we profile 1,229 microRNAs (miRNAs), including 728 novel miRNAs and 110 miRNA/miRNA* duplexes, of the posterior silk gland at the fifth larval instar. Target gene prediction yields 14,222 unique target genes from 1,195 miRNAs. Functional categorization classifies the targets into complex pathways that include both cellular and metabolic processes, especially protein synthesis and processing. CONCLUSION: The enrichment of target genes in the ribosome-related pathway indicates that miRNAs may directly regulate translation. Our findings pave a way for further functional elucidation of these miRNAs and their targets in silk production.

Porin deficiency or plasmid copy number increase mediated carbapenem-resistant Escherichia coli resistance evolution.

This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 µg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 µg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

Dysregulated glial genes in Alzheimer's disease are essential for homeostatic plasticity: Evidence from integrative epigenetic and single cell analyses.

Synaptic homeostatic plasticity is a foundational regulatory mechanism that maintains the stability of synaptic and neural functions within the nervous system. Impairment of homeostatic regulation has been linked to synapse destabilization during the progression of Alzheimer's disease (AD). Recent epigenetic and transcriptomic characterizations of the nervous system have revealed intricate molecular details about the aging brain and the pathogenesis of neurodegenerative diseases. Yet, how abnormal epigenetic and transcriptomic alterations in different cell types in AD affect synaptic homeostatic plasticity remains to be elucidated. Various glial cell types play critical roles in modulating synaptic functions both during the aging process and in the context of AD. Here, we investigated the impact of glial dysregulation of histone acetylation and transcriptome in AD on synaptic homeostatic plasticity, using computational analysis combined with electrophysiological methods in Drosophila. By integrating snRNA-seq and H3K9ac ChIP-seq data from the same AD patient cohort, we pinpointed cell type-specific signature genes that were transcriptionally altered by histone acetylation. We subsequently investigated the role of these glial genes in regulating presynaptic homeostatic potentiation in Drosophila. Remarkably, nine glial-specific genes, which were identified through our computational method as targets of H3K9ac and transcriptional dysregulation, were found to be crucial for the regulation of synaptic homeostatic plasticity in Drosophila. Our genetic evidence connects abnormal glial transcriptomic changes in AD with the impairment of homeostatic plasticity in the nervous system. In summary, our integrative computational and genetic studies highlight specific glial genes as potential key players in the homeostatic imbalance observed in AD.

Role of α2δ-3 in regulating calcium channel localization at presynaptic active zones during homeostatic plasticity.

The homeostatic modulation of synaptic transmission is an evolutionarily conserved mechanism that is critical for stabilizing the nervous system. At the Drosophila neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) compensates for impairments in postsynaptic glutamate receptors due to pharmacological blockade or genetic deletion. During PHP, there is an increase in presynaptic neurotransmitter release, counteracting postsynaptic changes and restoring excitation to baseline levels. Previous studies have shown that α2δ-3, an auxiliary subunit of voltage-gated calcium channels (VGCCs), is essential for both the rapid induction and sustained expression of PHP at the Drosophila NMJ. However, the molecular mechanisms by which α2δ-3 regulates neurotransmitter release during PHP remain to be elucidated. In this study, we utilized electrophysiological, confocal imaging, and super-resolution imaging approaches to explore how α2δ-3 regulates synaptic transmission during PHP. Our findings suggest that α2δ-3 governs PHP by controlling the localization of the calcium channel pore-forming α1 subunit at presynaptic release sites, or active zones. Moreover, we examined the role of two structural domains within α2δ-3 in regulating neurotransmitter release and calcium channel localization. Our results highlight that these domains in α2δ-3 serve distinct functions in controlling synaptic transmission and presynaptic calcium channel abundance, at baseline in the absence of perturbations and during PHP. In summary, our research offers compelling evidence that α2δ-3 is an indispensable signaling component for controlling calcium channel trafficking and stabilization in homeostatic plasticity.

Corrigendum: Role of α2δ-3 in regulating calcium channel localization at presynaptic active zones during homeostatic plasticity.

[This corrects the article DOI: 10.3389/fnmol.2023.1253669.].

Evaluation of the Zybio EXS3000 mass spectrometry in routine identification of Clinical isolates.

The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied in routine clinical microbiology laboratories as an efficient and reliable technique for diagnostic purpose. In this work, we evaluated the performance of the newly developed Zybio EXS3000 (Zybio Inc., China) in microbial identification and compared it with VITEK MS (bioMérieux, France). For this study, a total of 1340 isolates from various clinical specimens were collected. These isolates were analyzed simultaneously on both EXS3000 and VITEK MS. The inconsistent or unidentifiable data were further identified using the help of either 16S rRNA gene or ITS region sequencing. During the study, we observed that EXS3000 and VITEK MS provided positive confirmatory diagnostics for 95.0% and 96.5% of the isolates, respectively, which were consistent with the sequencing results. However, it is worth noting that the EXS3000 system needs to improve the identification performance of Candida albicans in the follow-up. There are no significant differences between the two devices in terms of microbial identification performance. The advantage of EXS3000 over VITEK MS is in its ability to perform in significantly lesser time period. In conclusion, the results of this investigation showed that EXS3000 can be used to identify microorganisms in clinical microbiology laboratories.

Molecular epidemiology and genomic dynamics of Pseudomonas aeruginosa isolates causing relapse infections.

Pseudomonas aeruginosa (P. aeruginosa) is one of the leading causes of chronic infections, including reinfection, relapse, and persistent infection, especially in cystic fibrosis patients. Relapse P. aeruginosa infections are more harmful because of repeated hospitalization and undertreatment of antimicrobials. However, relapse P. aeruginosa infection in China remains largely unknown. Herein, we performed a 3-year retrospective study from 2019 to 2022 in a tertiary hospital, which included 442 P. aeruginosa isolates from 196 patients. Relapse infection was identified by screening clinical records and whole-genome sequencing (WGS). We found that 31.6% (62/196) of patients had relapsed infections. The relapse incidence of carbapenem-resistant P. aeruginosa infection (51.4%) is significantly higher than that of carbapenem-susceptible P. aeruginosa infection (20.2%, P < 0.0001). These isolates were assigned to 50 distinct sequence types and sporadically distributed in phylogeny, indicating that relapsed infections were not caused by certain lineages. Fast adaptation and evolution of P. aeruginosa isolates were reflected by dynamic changes of antimicrobial resistance, gene loss and acquisition, and single-nucleotide polymorphisms during relapse episodes. Remarkably, a convergent non-synonymous mutation that occurs in a pyochelin-associated virulence gene fptA (T1056C, M252T) could be a considerable target for the diagnosis and treatment of relapse P. aeruginosa infection. These findings suggest that integrated utilization of WGS and medical records provides opportunities for improved diagnostics of relapsed infections. Continued surveillance of the genomic dynamics of relapse P. aeruginosa infection will generate further knowledge for optimizing treatment and prevention in the future.IMPORTANCEPseudomonas aeruginosa is a predominant pathogen that causes various chronic infections. Relapse infections promote the adaptation and evolution of antimicrobial resistance and virulence of P. aeruginosa, which obscure evolutionary trends and complicate infection management. We observed a high incidence of relapse P. aeruginosa infection in this study. Whole-genome sequencing (WGS) revealed that relapse infections were not caused by certain lineages of P. aeruginosa isolates. Genomic dynamics of relapse P. aeruginosa among early and later stages reflected a plasticity scattered through the entire genome and fast adaptation and genomic evolution in different ways. Remarkably, a convergent evolution was found in a significant virulence gene fptA, which could be a considerable target for diagnosis and treatment. Taken together, our findings highlight the importance of longitudinal surveillance of relapse P. aeruginosa infection in China since cystic fibrosis is rare in Chinese. Integrated utilization of WGS and medical records provides opportunities for improved diagnostics of relapse infections.

Analysis of CYP3A4 genetic polymorphisms in Han Chinese.

Our study aimed to comprehensively investigate the genetic polymorphisms of CYP3A4 in Han Chinese. We sequenced the gene regions of CYP3A4, including its promoter, exons, surrounding introns and 3' untranslated region (3'UTR), from 100 unrelated-healthy Han Chinese individuals. We detected 11 SNPs, three of which are novel. According to in silico functional prediction of novel variants, 20148 A>G in exon 10, resulting in substitution of Tyr319 with Cys (CYP3A4*21), may induce dramatic alteration of protein conformation, and 26908 G>A in 3'UTR may disrupt post-transcriptional regulation. We identified five alleles in Han Chinese, the allele frequencies of CYP3A4*1, *5, *6, *18 and *21 are 97, 0.5, 1, 1 and 0.5%, respectively. Haplotype inference revealed 14 haplotypes, of which the major haplotype CYP3A4*1A constitutes 59% of the total chromosomes. We also examined the possible role of natural selection in shaping the variation of CYP3A4 and confirmed a trend, consistent with the action of positive selection. We systematically screened the genetic polymorphisms of CYP3A4 in Han Chinese, highlighted possible functional impairment of the novel allele and summarized the distinct allele and haplotype frequency distribution, with an emphasis on detecting the footprint of recent positive selection on the CYP3A4 gene in Han Chinese.

Clinical Molecular and Genomic Epidemiology of Morganella morganii in China.

Objectives: Ongoing acquisition of antimicrobial resistance genes has made Morganella morganii a new clinical treatment challenge. Understanding the molecular epidemiology of M. morganii will contribute to clinical treatment and prevention. Methods: We undertook a 6-year clinical molecular epidemiological investigation of M. morganii from three tertiary hospitals in China since 2014. Antimicrobial susceptibility testing was performed using a VITEK-2 system. All isolates were screened for ß-lactam and plasmid-mediated quinolone resistance genes by PCR. Isolates carrying carbapenem-resistant genes were subjected to whole-genome sequencing (WGS). The variation and evolution of these mobile genetic elements (MGEs) were then systematically analyzed. Results: Among all M. morganii isolates (n = 335), forty (11.9%) were recognized as multidrug resistant strains. qnrD1, aac(6')-Ib-cr, bla TEM-104, and bla CTX-M-162 were the top four most prevalent resistance genes. Notably, phylogenomic and population structure analysis suggested clade 1 (rhierBAPS SC3 and SC5) associated with multiple resistance genes seemed to be widely spread. WGS showed a bla OXA-181-carrying IncX3 plasmid and a Proteus genomic island 2 variant carrying bla CTX-M-3, aac(6')-Ib-cr coexisted in the same multidrug resistant strain zy_m28. Additionally, a bla IMP-1-carrying IncP-1ß type plasmid was found in the strain nx_m63. Conclusion: This study indicates a clade of M. morganii is prone to acquire resistance genes, and multidrug resistant M. morganii are increasing by harboring a variety of MGEs including two newly discovered ones in the species. We should be vigilant that M. morganii may bring more extensive and challenging antimicrobial resistance issue.

Novel microRNAs in silkworm (Bombyx mori).

We acquired more than 4 million useful sequences using a high-throughput method from a library for miRNA identification, which is constructed from a mixture of 14 RNA samples from different developmental stages. We mapped 247,410 reads to known silkworm miRNAs in miRBase (13.0), 701,913 reads to other RNA molecules based on sequence homology, and 3,219,395 reads to the silkworm genome. Our analysis identified 54 silkworm known miRNAs. A striking strand bias between miRNAs and their corresponding miRNA*s was found, and was speculated to reflect that transcripts from the passenger strand of pre-miRNAs may have important biological roles. Using an elaborate screening protocol, we predicted 287 candidate novel miRNAs (represent 116,494 short reads), and 59 of them have both miRNA and miRNA* sequences. Most of the previously identified silkworm miRNAs are cross-species conserved with a high abundance, while those predicted candidates tend to be species-specific miRNAs. Our discovery of SNPs among miRNAs implied within-species functional diversity. Target prediction uncovers that considerable silkworm miRNAs may aim at modulating more than one hormone signaling pathway components and/or hormone biosynthesis-related proteins implying their important roles in silkworm development.

A discovery of novel microRNAs in the silkworm (Bombyx mori) genome.

MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

Impact of an Intervention to Control Imipenem-Resistant Acinetobacter baumannii and Its Resistance Mechanisms: An 8-Year Survey.

BACKGROUND: This study aimed to examine the impact of an intervention carried out in 2011 to combat multi-drug resistance and outbreaks of imipenem-resistant Acinetobacter baumannii (IRAB), and to explore its resistance mechanism. METHODS: A total of 2572 isolates of A. baumannii, including 1673 IRAB isolates, were collected between 2007 and 2014. An intervention was implemented to control A. baumannii resistance and outbreaks. Antimicrobial susceptibility was tested by calculating minimal inhibitory concentrations (MICs), and outbreaks were typed using pulsed-field gel electrophoresis (PFGE). Resistance mechanisms were explored by polymerase chain reaction (PCR) and whole genome sequencing (WGS). RESULTS: Following the intervention in 2011, the resistance rates of A. baumannii to almost all tested antibiotics decreased, from 85.3 to 72.6% for imipenem, 100 to 80.8% for ceftriaxone, and 45.0 to 6.9% for tigecycline. The intervention resulted in a decrease in the number (seven to five), duration (8-3 months), and departments (five to three) affected by outbreaks; no outbreaks occurred in 2011. After the intervention, only bla AMPC (76.47 to 100%) and bla TEM- 1 (75.74 to 96.92%) increased (P < 0.0001); whereas bla GES- 1 (32.35 to 3.07%), bla PER- 1 (21.32 to 1.54%), bla OXA- 58 (60.29 to 1.54%), carO (37.50 to 7.69%), and adeB (9.56 to 3.08%) decreased (P < 0.0001). Interestingly, the frequency of class B ß-lactamase genes decreased from 91.18% (bla SPM- 1) and 61.03% (bla IMP- 1) to 0%, while that of class D bla OXA- 23 increased to 96.92% (P < 0.0001). WGS showed that the major PFGE types causing outbreaks each year (type 01, 11, 18, 23, 26, and 31) carried the same resistance genes (bla KPC- 1, bla ADC- 25, bla OXA- 66, and adeABC), AdeR-S mutations (G186V and A136V), and a partially blocked porin channel CarO. Meanwhile, plasmids harboring bla OXA- 23 were found after the intervention. CONCLUSION: The intervention was highly effective in reducing multi-drug resistance of A. baumannii and IRAB outbreaks in the long term. The resistance mechanisms of IRAB may involve genes encoding ß-lactamases, efflux pump overexpression, outer membrane porin blockade, and plasmids; in particular, clonal spread of bla OXA- 23 was the major cause of outbreaks. Similar interventions may also help reduce bacterial resistance rates and outbreaks in other hospitals.

Ceftazidime/avibactam Improves the Antibacterial Efficacy of Polymyxin B Against Polymyxin B Heteroresistant KPC-2-Producing Klebsiella pneumoniae and Hinders Emergence of Resistant Subpopulation in vitro.

Due to the increasing multidrug resistance and limited antibiotics, polymyxin B revived as the last resort for the treatment of carbapenemase-producing Klebsiella pneumoniae (CRKP). Unfortunately, the heteroresistance hampers polymyxin B monotherapy treatment via the amplification of resistant subpopulation. Reliable polymyxin B based combinations are demanded. Ceftazidime/avibactam has been regarded as a new salvage therapy against CRKP. The occurrence of heteroresistance was confirmed by population analysis profiling (PAP). Our study demonstrated that polymyxin B and ceftazidime/avibactam combinations improved the in vitro antimicrobial activity of polymyxin B and delayed or suppressed the regrowth of resistant subpopulation by time-kill studies. Ceftazidime/avibactam at around MIC values (0.5-1 × MIC) plus clinically achievable concentrations of polymyxin B (0.5-2 mg/L) resulted in sustained killing against polymyxin B-heteroresistant isolates. Active PmrAB and PhoPQ systems and a pmrA mutation (G53R) in resistant subpopulation might associate with heteroresistance, but further investigation was required. Our findings suggested that the heteroresistance represented barriers to polymyxin B efficacy, and the combination of polymyxin B with ceftazidime/avibactam could be potentially valuable for the treatment of heteroresistant CRKP. Further, in vivo studies need to be performed to evaluate the efficacy of this combination against heteroresistant strains.

High Prevalence of Metallo-ß-Lactamase-Producing Enterobacter cloacae From Three Tertiary Hospitals in China.

Enterobacter cloacae has recently emerged as one of the most common carbapenem-resistant Enterobacteriaceae. The emergence and spread of metallo-ß-lactamase-producing E. cloacae have posed an immediate threat globally. Here, we investigated the molecular characteristics of 84 carbapenem-resistant Enterobacter cloacae (CREL) collected from three tertiary hospitals in China between 2012 and 2016. Species identification and antimicrobial susceptibility testing were performed using a VITEK-2 system. Carbapenems, polymyxins B, and tigecycline were tested by broth microdilution method. The carbapenem in activation method (CIM) and cefoxitin three-dimensional test were used to detect carbapenemase and AmpC ß-lactamase, respectively. Isolates were screened for ß-lactam resistance genes by PCR, and expression of ompC and ompF was determined by qRT-PCR. Genetic relatedness was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), while selected isolates were subjected to whole-genome sequencing. Among the 84 CREL isolates, 50 (59.5%) were detected as carbapenemase producers. NDM-1 was the dominant carbapenemase (80.0%), followed by IMP-26 (8.0%) and IMP-4 (6.0%). Notably, we identified the first NDM-1 and IMP-1 co-producing E. cloacae, carrying plasmids of several incompatibility (Inc) groups, including IncHI2, IncHI2A, and IncN. Most isolates showed decreased expression of ompC and/or ompF, and contained a broad distribution of ESBLs and AmpC ß-lactamases. These findings suggested that different molecular mechanisms, including carbapenemase, ESBL and/or AmpC plus loss of porins, have contributed to carbapenem resistance. The bla NDM-1-harboring plasmids contained highly conserved gene environment around bla NDM-1 (bla NDM-1-ble MBL-trpF-dsbD-cutA1-groES-groEL), which could be associated with the potential dissemination of bla NDM-1. IMP-type MBL was located within a variety of integrons and usually contained various gene cassettes encoding multidrug resistance. These isolates produced 54 different pulsotypes, and were classified into 42 STs by MLST. Nineteen bla NDM-1-positive E. cloacae isolates obtained from Ningxia had the same pulsotype (PFGE type 1), belonging to ST78 within clonal complex 74 (CC74). The plasmid-based replicon typing indicated that IncX3 plasmids mediated the dissemination of bla NDM-1 among these homologous strains. This is the first report on the outbreak of NDM-1-producing E. cloacae ST78 with contribution of IncX3 plasmids in Northwestern China. There's an immediate need to intensify surveillance attentively to prevent and control the further spread of NDM-1 in China.