Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5.

PURPOSE: The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments. METHODS: The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics. RESULTS: Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including blaNDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, blaTEM-1B, aac(3)-lld, and blaCTX-M-261, a novel blaCTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The blaCTX-M-261 was derived from blaCTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder. CONCLUSION: Plasmid-mediated horizontal transfer plays an important role in blaNDM-5 and blaCTX-M-261 dissemination, which increases the threat to public health due to the resistance to most ß-lactam antibiotics. This is the first report of blaCTX-M-261 and blaNDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -.

Cellular Id1 inhibits hepatitis B virus transcription by interacting with the novel covalently closed circular DNA-binding protein E2F4.

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), which required developing novel therapies targeting the inhibition of HBV transcription and replication due to current limited treatment options. We explored novel target for the development of novel therapies targeting the inhibition of HBV replication and transcription. The expression of Id1 and E2F4 in HCC cells and tissues was detected by qRT-PCR and western blot. We investigated the Id1 and E2F4-mediated transcription of HBV infection by using HepG2.2.15, HepAD38, HepG2-NTCP cell lines and AAV/HBV-infected mice. Interactions between the two host proteins and viral covalently closed circular DNA (cccDNA) were assessed using subcellular localization, protein-protein interaction, chromatin immunoprecipitation, and luciferase assays. Ectopic Id1 significantly reduced HBV transcription and replication in both HBV-expressing cells and AAV/HBV-infected mice. Id1 and E2F4 could form a heterodimer to prevent E2F4 from promoting HBV transcription and replication. E2F4 could directly bind to cccDNA and activate the HBV core promoter in cell lines. Furthermore, in vitro binding experiments confirmed that the sequence 1758'-TTAAAGGTC-1766', which is highly conserved among HBV genotypes, is the target site of the E2F4 homodimer. The findings suggest that E2F4 function as novel cccDNA-binding protein to directly activate HBV transcription by binding to Cp promoter region. Our results highlight the ability that E2F4 represent a pan-potential therapeutic target against HBV transcription and provide more clues to better understand the life cycle of HBV.

In Vitro Activity of Auranofin in Combination With Aztreonam-Avibactam Against Metallo-ß-lactamase (MBL)-Producing Enterobacterales.

Objectives: To assess the efficacy of aztreonam-avibactam-auranofin (ATM-AVI-AUR) against a collection of 88 carbapenemase-producing Enterobacterales (CPE) clinical isolates and 6 in vitro selected ATM-AVI-resistant CPE with CMY-16 Tyr150Ser and Asn346His mutants or transformants. Methods: MICs of imipenem, ceftazidime-avibact8am (CAZ-AVI), ATM-AVI, CAZ-AVI-AUR and ATM-AVI-AUR were determined via the broth microdilution method. Genetic background and carbapenemase genes were determined by PCR and Sanger sequencing. Results: AUR alone showed little antibacterial activity with AUR MICs were greater than 64 µg/mL for all the 88 clinical CPE isolates. The addition of AUR (16 µg/mL) resulted in an 3-folding dilutions MIC reduction of ATM-AVI MIC50 (0.5 to 0.0625 µg/mL) and a 2-folding dilutions MIC reduction of MIC90 (1 to 0.25 µg/mL) against all 88 clinical CPE isolates, respectively. Notably, the reduced ATM-AVI MIC values were mainly found in MBL-producers, and the MIC50 and MIC90 reduced by 2-folding dilutions (0.25 to 0.0625 µg/mL) and 3-folding dilutions (2 to 0.25 µg/mL) respectively by AUR among the 51 MBL-producers. By contrast, the addition of AUR did not showed significant effects on ATM-AVI MIC50 (0.0625 µg/mL) and MIC90 (0.125 µg/mL) among single KPC-producers. Interestingly, the addition of AUR restored the ATM-AVI susceptibility against the 6 in vitro selected ATM-AVI-resistant CMY-16 Tyr150Ser and Asn346His mutants or transfromants, with the MICs reduced from ≥32 µg/mL (32->256 µg/mL) to ≤8 µg/mL (0.0625-8 µg/mL). Conclusions: Our results demonstrated that AUR potentiated the activities of CAZ-AVI and ATM-AVI against MBL-producing isolates in vitro. Importantly, AUR restored the ATM-AVI activity against ATM-AVI resistant mutant strains. As a clinically approved drug, AUR might be repurposed in combination with ATM-AVI to treat infections caused by highly resistant MBL-producing Enterobacterales.

Identification of a Novel blaNDM Variant, blaNDM-33, in an Escherichia coli Isolate from Hospital Wastewater in China.

Since the discovery of NDM-1 and the worldwide reporting of different variants have raised alarms concerning global health, the problem of carbapenem-resistant Enterobacterales (CRE) has become increasingly serious. Therefore, research on the hydrolytic activity and molecular structure of NDM variants is beneficial to the development of antibacterial drugs. NDM has been evolving into variants that possess different hydrolysis activities toward ß-lactam antibiotics. Here, we characterized a novel blaNDM variant, named blaNDM-33, identified from a multidrug-resistant Escherichia coli strain from hospital sewage. NDM-33 differed from NDM-5 with a single-amino-acid substitution (A72T). blaNDM-5 was located in the Tn125-related blaNDM-33 region from an IncX3-type plasmid, pHD6415-NDM, that can be transferred horizontally. The genetic construct of blaNDM-33 showed higher MICs of carbapenems than a blaNDM-5 construct. Enzyme kinetics showed that NDM-33 had higher enzymatic activity for meropenem and cefazolin than NDM-5. The emergence of this novel NDM variant could pose a threat to public health because of its transferability and enhanced carbapenem activity. IMPORTANCE Our study described a novel NDM-33 variant from an E. coli strain isolated from hospital sewage, where it was associated with human disease and antibiotic exposure. Importantly, hospital sewage was increasingly considered to be related to CRE hosts. Pathogens were transmitted from reservoirs through direct and indirect contact, ingestion, and inhalation of contaminated water or aerosols. In addition, under the selective pressure of antibiotics, NDM variants will become the main strain in the hospital water system and evolve into high virulence and high resistance. The monitoring of NDM mutants is of great significance for preventing and controlling the evolution of superbugs.

E2F4 Promotes the Proliferation of Hepatocellular Carcinoma Cells through Upregulation of CDCA3.

Liver cancer, the second most commonly diagnosed cancer, is associated with high mortality rates. E2F4 is a member of the E2F transcription factor family. There are limited studies on the role of E2F4 in hepatocellular carcinoma (HCC). In this study, the expression of E2F4 in HCC tissue samples and cell lines was analyzed using quantitative real-time polymerase chain reaction. E2F4 expression positively correlated with tumor size in patients with HCC. Additionally, E2F4 expression was greater in HCC cells than in normal LO2 cells. Furthermore, overexpression of E2F4 significantly enhanced the proliferation, migration, and invasion of HCC cells. The results of a luciferase assay revealed that E2F4 upregulated the expression of CDCA3 by binding to its promoter region (1863'-ACGCGCGAGAATG-1875') and consequently promoted proliferation and cell cycle progression of HCC cells. Taken together, these results demonstrated that E2F4 might play a vital role in HCC progression and could serve as a potential biomarker for the diagnosis and as a therapeutic target of HCC.

In vitro Activity of Ceftazidime-Avibactam and Aztreonam-Avibactam Against Carbapenem-resistant Enterobacteriaceae Isolates Collected from Three Secondary Hospitals in Southwest China Between 2018 and 2019.

PURPOSE: To assess the antimicrobial activities of ceftazidime/avibactam (CAZ/AVI) and aztreonam/avibactam (ATM/AVI) against carbapenem-resistant Enterobacteriaceae (CRE) isolates collected from three secondary hospitals in Southwest China between 2018 and 2019. MATERIALS AND METHODS: A total of 120 unique CRE clinical isolates were collected and carbapenemase genes were detected using PCR. Antimicrobial susceptibility was determined using standard broth microdilution method and the results were interpreted according to CLSI breakpoints. RESULTS: The 120 carbapenem-resistant strains included 92 Klebsiella pneumoniae, 10 Escherichia coli, 10 Enterobacter cloacae, five Klebsiella aerogenes, and three Klebsiella oxytoca isolates. Seventy-four percent of these 120 CRE isolates were collected from patients located in non-ICUs; 65.0% of these CRE isolates were collected from male patients; and 34.2% of these isolates were isolated from respiratory tracts. Four different carbapenemase genes were identified among 103 carbapenemase-producing Enterobacteriaceae (CPE) isolates, including bla KPC-2 (n=77), bla NDM-1 (n=16), bla NDM-5 (n=12) and bla IMP-4 (n=2). Overall, 21.7%, 37.5%, 40.8%, 75.0%, and 100% of the CRE strains were susceptible to levofloxacin, trimethoprim/sulfamethoxazole, amikacin, CAZ/AVI, and ATM/AVI, respectively. In addition, antimicrobial susceptibility testing showed that 96.7% isolates (n=116) were resistant to aztreonam, and the addition of avibactam (4 mg/L) significantly reduced the MICs of those aztreonam-resistant isolates by more than 128-fold (range: ≤0.125-4 mg/L), and 90.0% (n=108) of total 120 isolates were inhibited at ATM/AVI concentration ≤1 mg/L. CONCLUSION: Our study revealed significant antimicrobial resistance among the CRE isolates against some commonly used antibiotics in three secondary Chinese hospitals. ATM/AVI exhibited potent activity against CRE isolates, including double carbapenemase-producing isolates, whereas CAZ/AVI was active against all KPC producers.

A Ceftazidime-Avibactam-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Strain Harboring blaKPC-14 Isolated in New York City.

Ceftazidime-avibactam is a potent antibiotic combination against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae Here, we describe a unique ceftazidime-avibactam-resistant and carbapenem-susceptible K. pneumoniae strain harboring a novel blaKPC-14 variant. This strain was isolated from a New York City patient in 2003, which predates the introduction of avibactam. Despite resistance to ceftazidime-avibactam, the strain was susceptible to imipenem-relebactam and meropenem-vaborbactam. Comprehensive genomic sequencing revealed that blaKPC-14 is harbored on an ST6 IncN plasmid associated with the early spread of blaKPCIMPORTANCE KPC is currently the most common carbapenemase identified in the United States. More than 40 KPC variants have been described, of which KPC-2 and KPC-3 are the most frequent clinical variants. However, our understanding of the genetic structures and ß-lactam resistance profiles of other novel KPC variants remains incomplete. Here, we report a novel blaKPC variant (blaKPC-14) and the complete genome sequence of blaKPC-14-harboring K. pneumoniae strain BK13048, which is susceptible to carbapenems but resistant to ceftazidime-avibactam. To the best of our knowledge, this is one of the earliest KPC-producing K. pneumoniae strains exhibiting resistance to ceftazidime-avibactam.

Genetic diversity and in vitro activity of ceftazidime/avibactam and aztreonam/avibactam against imipenem-resistant Enterobacteriaceae isolates in Southwest China: A single-centre study.

OBJECTIVES: The aim of this study was to investigate the molecular mechanisms of imipenem resistance in Enterobacteriaceae and to assess the antimicrobial activities of ceftazidime/avibactam (CAZ/AVI) and aztreonam/avibactam (ATM/AVI) against imipenem-resistant clinical isolates in a tertiary hospital in China. METHODS: A total of 91 imipenem-resistant Enterobacteriaceae were collected and genes encoding carbapenemases, ESBLs, AmpC ß-lactamases and porins were detected using PCR. MICs and susceptibility were determined using in-house-prepared broth microdilution panels and were interpreted according to CLSI breakpoints. RESULTS: Imipenem-resistant isolates comprising 54 Klebsiella pneumoniae, 18 Escherichia coli, 8 Enterobacter cloacae, 6 Serratia marcescens, 3 Klebsiella oxytoca and 2 Klebsiella aerogenes were collected independently. Five different carbapenemase genes were identified, namely blaKPC-2 (n = 60), blaNDM-5 (n = 14), blaNDM-1 (n = 11), blaKPC-3 (n = 4) and blaIMP-4 (n = 1). Among the 91 carbapenem-resistant Enterobacteriaceae (CRE), 85 isolates harboured at least one ESBL and/or AmpC gene, including 5 strains without carbapenemase-encoding genes. Moreover, 31 K. pneumoniae carried ompK35 and/or ompK36 mutations. MLST results showed that the K. pneumoniae belonged to 12 different STs, with ST11 being predominant (29/54; 53.7%). Overall, 17.6%, 25.3%, 41.8%, 65.9% and 100% of the CRE strains were susceptible to amikacin, trimethoprim/sulfamethoxazole, tetracycline, CAZ/AVI and ATM/AVI, respectively. CONCLUSION: This study revealed that CRE isolates differ significantly in their species, STs, porins and carbapenemase genes in a single Chinese hospital. ATM/AVI exhibited potent activity against CRE isolates, even for the most notorious double-carbapenemase-producers with porin defects, whereas CAZ/AVI was active against all the non-metallo-ß-lactamase-producing isolates.

The HBx and HBc of hepatitis B virus can influence Id1 and Id3 by reducing their transcription and stability.

Hepatitis B virus (HBV) infection is closely related with the occurrence and development of hepatocellular carcinoma (HCC), in which Hepatitis B virus x protein (HBx) and core protein (HBc) play crucial roles. Additionally, inhibitors of differentiation (Id) proteins exhibited significant correlation with liver cancer development. Here, we identified that HBV dramatically inhibited the expression of Id1 and Id3 in both protein and transcriptional levels for the first time, whereas there was little effect of the virus on Id2. Additionally, two HBV coded protein, HBc and HBx, could reduce the expression of Id1 and Id3 distinctly, whereas the other two viral proteins, HBs and HBp were unable to affect Id1 and Id3 proteins. Both the activity inhibitors and activators further confirmed that HBc inhibited the expression of Id1 and Id3 by BMP/Smad signaling pathway. HBx could interact with both Id1 and Id3 at residues 112-136 of HBx protein, and it could inhibit the two Id proteins by accelerating their degradation. This is the first report about HBc and HBx regulating Id1 and Id3, whereas the detailed mechanism associated with above needed further experiments to clarify.

In vitro selection of aztreonam/avibactam resistance in dual-carbapenemase-producing Klebsiella pneumoniae.

OBJECTIVES: To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. METHODS: The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. RESULTS: We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. CONCLUSIONS: Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.