TRIM21 promotes ubiquitination of SARS-CoV-2 nucleocapsid protein to regulate innate immunity.

The innate immune response is the first line of host defense against viral infections, but its role in immunity against SARS-CoV-2 remains unclear. By using immunoprecipitation coupled with mass spectroscopy, we observed that the E3 ubiquitin ligase TRIM21 interacted with the SARS-CoV-2 nucleocapsid (N) protein and ubiquitinated it at Lys375 . Upon determining the topology of the TRIM21-mediated polyubiquitination chain on N protein, we then found that polyubiquitination led to tagging of the N protein for degradation by the host cell proteasome. Furthermore, TRIM21 also ubiquitinated the N proteins of SARS-CoV-2 variants of concern, including Alpha, Beta, Gamma, Delta, and Omicron together with SARS-CoV and MERS-CoV variants. Herein, we propose that ubiquitylation and degradation of the SARS-CoV-2 N protein inhibited SARS-CoV-2 viral particle assembly, by which it probably involved in preventing cytokine storm. Eventually, our study has fully revealed the association between the host innate immune system and SARS-CoV-2 N protein, which may aid in developing novel SARS-CoV-2 treatment strategies.

Genetic and enzymatic characterization of two novel blaNDM-36, -37 variants in Escherichia coli strains.

The widespread of different NDM variants in clinical Enterobacterales isolates poses a serious public health concern, which requires continuous monitoring. In this study, three E. coli strains carrying two novel blaNDM variants of blaNDM-36, -37 were identified from a patient with refractory urinary tract infection (UTI) in China. We conducted antimicrobial susceptibility testing (AST), enzyme kinetics analysis, conjugation experiment, whole-genome sequencing (WGS), and bioinformatics analysis to characterize the blaNDM-36, -37 enzymes and their carrying strains. The blaNDM-36, -37 harboring E. coli isolates belonged to ST227, O9:H10 serotype and exhibited intermediate or resistance to all ß-lactams tested except aztreonam and aztreonam/avibactam. The genes of blaNDM-36, -37 were located on a conjugative IncHI2-type plasmid. NDM-37 differed from NDM-5 by a single amino acid substitution (His261Tyr). NDM-36 differed from NDM-37 by an additional missense mutation (Ala233Val). NDM-36 had increased hydrolytic activity toward ampicillin and cefotaxime relative to NDM-37 and NDM-5, while NDM-37 and NDM-36 had lower catalytic activity toward imipenem but higher activity against meropenem in comparison to NDM-5. This is the first report of co-occurrence of two novel blaNDM variants in E. coli isolated from the same patient. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of NDM enzymes.

Avibactam potentiated the activity of both ceftazidime and aztreonam against S. maltophilia clinical isolates in vitro.

BACKGROUND: Treatment options for Stenotrophomonas maltophilia (S. maltophilia) infections were limited. We assessed the efficacy of ceftazidime (CAZ), ceftazidime-avibactam (CAZ-AVI), aztreonam (ATM), and aztreonam-avibactam (ATM-AVI) against a selection of 76 S. maltophilia out of the 1179 strains isolated from the First Affiliated Hospital of Chongqing Medical University during 2011-2018. METHODS: We investigated the antimicrobial resistance profiles of the 1179 S. maltophilia clinical isolates from the first affiliated hospital of Chongqing Medical University during 2011-2018, a collection of 76 isolates were selected for further study of microbiological characterization. Minimum inhibitory concentrations (MICs) of CAZ, CAZ-AVI, ATM and ATM-AVI were determined via the broth microdilution method. We deemed that CAZ-AVI or ATM-AVI was more active in vitro than CAZ or ATM alone when CAZ-AVI or ATM-AVI led to a category change from "Resistant" or "Intermediate" with CAZ or ATM alone to "Susceptible" with CAZ-AVI or ATM-AVI, or if the MIC of CAZ-AVI or ATM-AVI was at least 4-fold lower than the MIC of CAZ or ATM alone. RESULTS: For the 76 clinical isolates included in the study, MICs of CAZ, ATM, CAZ-AVI and ATM-AVI ranged from 0.03-64, 1-1024, 0.016-64, and 0.06-64 µg/mL, respectively. In combined therapy, AVI was active at restoring the activity of 48.48% (16/33) and 89.71% (61/68) of S. maltophilia to CAZ and ATM, respectively. Furthermore, CAZ-AVI showed better results in terms of the proportion of susceptible isolates (77.63% vs. 56.58%, P < 0.001), and MIC50 (2 µg/mL vs. 8 µg/mL, P < 0.05) when compared to CAZ. According to our definition, CAZ-AVI was more active in vitro than CAZ alone for 81.58% (62/76) of the isolates. Similarly, ATM-AVI also showed better results in terms of the proportion of susceptible isolates (90.79% vs.10.53%, P < 0.001) and MIC50 (2 µg/mL vs. 64 µg/mL, P < 0.001) when compared to ATM. According to our definition, ATM-AVI was also more active in vitro than ATM alone for 94.74% (72/76) of the isolates. CONCLUSIONS: AVI potentiated the activity of both CAZ and ATM against S. maltophilia clinical isolates in vitro. We demonstrated that CAZ-AVI and ATM-AVI are both useful therapeutic options to treat infections caused by S. maltophilia.

Low-dose antiplatelet therapy survey after intracerebral hemorrhage in China: a retrospective hospital-based study.

Restarting of antiplatelet therapy (AT) for patients with a history of intracerebral hemorrhage (ICH) is still a clinical dilemma in China. We aimed to investigate the association between low-dose AT and the long-term clinical outcome in Chinese ICH patients. A total of 312 patients with a history of ICH were retrospectively enrolled and followed. The ischemic vascular events, recurrent ICH, and all-cause death were reviewed retrospectively. We explored the predictors of ischemic vascular events and recurrent ICH from all patients using Cox proportional hazard regression model. One hundred fifty-one (48.4%) patients were treated with low-dose AT, and the median duration of follow-up was 4.0 years (interquartile range, 2.5-5 years). Compared to 30 (19.8%) of 151 participants who restarted low-dose AT had ischemic vascular events, 51 (31.7%) of 161 participants who did not receive AT showed ischemic vascular events (p=0.025). Eighteen (11.9%) of 151 participants treated with low-dose AT had recurrent ICH and 21 (13.0%) of 161 in non-AT participants (p=0.830). Cox regression analysis also showed that diabetes mellitus was an independent risk factor for ischemic vascular events (p=0.029). Uncontrolled blood pressure (BP) was independently associated with the risk for both ischemic vascular events (p=0.025) and recurrent ICH (p=0.001). Atrial fibrillation (AF) was an independent risk factor for recurrent ICH among patients with a history of ICH (p=0.018). In a Chinese population of patients with predominantly deep, mild to moderate severity ICH, restarting of low-dose AT at a median of 6.2 months was associated with a lower risk of ischemic vascular events without increased risk of recurrent ICH.

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.

Characteristics of Clostridium difficile isolates and the burden of hospital-acquired Clostridium difficile infection in a tertiary teaching hospital in Chongqing, Southwest China.

BACKGROUND: Clostridium difficile infection (CDI), especially hospital-acquired Clostridium difficile infection (HA-CDI), continues to be a public health problem and has aroused great concern worldwide for years. This study aimed to elucidate the clinical and epidemiological features of HA-CDI and the characteristics of C.difficile isolates in Chongqing, Southwest China. METHODS: A case-control study was performed to identify the clinical incidence and risk factors of HA-CDI. C. difficile isolates were characterised by polymerase chain reaction (PCR) ribotyping, multilocus sequence typing (MLST), toxin gene detection and antimicrobial susceptibility testing. RESULTS: Of the 175 suspicious patients, a total of 122 patients with antibiotic-associated diarrhea (AAD) were included in the study; among them, 38 had HA-CDI. The incidence of AAD and HA-CDI was 0.58 and 0.18 per 1000 patient admissions, respectively. Chronic renal disease and cephalosporin use were independent risk factors for HA-CDI. Fifty-five strains were assigned into 16 sequence types (STs) and 15 ribotypes (RTs). ST2/RT449 (8, 14.5%) was the predominant genotype. Of the 38 toxigenic isolates, A + B + CDT- isolates accounted for most (34, 89.5%) and 1 A + B + CDT+ isolate emerged. No isolate was resistant to vancomycin, metronidazole or tigecycline, with A-B-CDT- being more resistant than A + B + CDT-. CONCLUSIONS: Different genotypes of C. difficile strains were witnessed in Chongqing, which hinted at the necessary surveillance of HA-CDI. Adequate awareness of patients at high risk of HA-CDI acquisition is advocated and cautious adoption of cephalosporins should be highlighted.

In Vitro Activity of Ceftazidime-Avibactam against Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae Isolates.

Carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKp) strains have emerged while antimicrobial treatment options remain limited. Herein, we tested the in vitro activity of ceftazidime-avibactam and other comparator antibiotics against 65 CR-hvKp isolates. Ceftazidime-avibactam, colistin, and tigecycline are highly active in vitro against CR-hvKp isolates (MIC90, ≤1 µg/ml), including K. pneumoniae carbapenemase 2 (KPC-2)-producing ST11 CR-hvKp. On the basis of previous clinical experience and the in vitro data presented herein, we posit that ceftazidime-avibactam is a therapeutic option against CR-hvKp infections.

Multiplex PCR Analysis for Rapid Detection of Klebsiella pneumoniae Carbapenem-Resistant (Sequence Type 258 [ST258] and ST11) and Hypervirulent (ST23, ST65, ST86, and ST375) Strains.

Carbapenem-resistant and hypervirulent Klebsiella pneumoniae strains have emerged recently. These strains are both hypervirulent and multidrug resistant and may also be highly transmissible and able to cause severe infections in both the hospital and the community. Clinical and public health needs require a rapid and comprehensive molecular detection assay to identify and track the spread of these strains and provide timely infection control information. Here, we develop a rapid multiplex PCR assay capable of distinguishing K. pneumoniae carbapenem-resistant isolates of sequence type 258 (ST258) and ST11, and hypervirulent ST23, ST65/ST375, and ST86 clones, as well as capsular types K1, K2, K locus type 47 (KL47), and KL64, and virulence genes rmpA, rmpA2, iutA, and iroN The assay demonstrated 100% concordance with 118 previously genotyped K. pneumoniae isolates and revealed different populations of carbapenem-resistant and hypervirulent strains in two collections in China and the United States. The results showed that carbapenem-resistant and hypervirulent K. pneumoniae strains are still rare in the United States, whereas in China, ∼50% of carbapenem-resistant strains carry rmpA/rmpA2 and iutA virulence genes, which are largely associated with the epidemic ST11 strains. Similarly, a high prevalence of hypervirulent strains was found in carbapenem-susceptible isolates in two Chinese hospitals, but these primarily belong to ST23, ST65/ST375, and ST86, which are distinct from the carbapenem-resistant strains. Taken together, our results demonstrated that this PCR assay can be a useful tool for molecular surveillance of carbapenem-resistant and hypervirulent K. pneumoniae strains.

Antimicrobial susceptibilities and clinical characterization of Pseudomonas aeruginosa isolates from urinary tract infections.

INTRODUCTION: Pseudomonas aeruginosa is a uropathogen that is mainly involved in nosocomial infection. The aim of this study was to analyze the antimicrobial susceptibilities and clinical characterization of P. aeruginosa isolates from urinary tract infections (UTIs). MATERIALS AND METHODS: The study collected all P. aeruginosa UTI strains from a hospital in Chongqing, China, from January 1st, 2010 to December 31st, 2011. The antibiotic susceptibilities of the P. aeruginosa isolates were analyzed using the agar dilution method and the genotypes were assessed using random amplification of polymorphic DNA-PCR (RAPD-PCR). The clinical characteristics of the patients with UTIs were collected from the hospital information systems, and significance was analyzed using the proportion test. RESULTS: A total of 2,778 episodes of culture-proven UTIs were used in the study. There were 198 infections (7.1%) caused by P. aeruginosa. P. aeruginosa strains were highly resistant to most drugs tested. The RAPD-PCR data revealed that the 198 P. aeruginosa infections had 82 different genotypes. Antibacterial use, previous UTI, urinary tract catheter and urinary tract operation were found to be risk factors for the development of UTIs. CONCLUSIONS: P. aeruginosa is the second most common UTI pathogen in our hospital. We should closely monitor patients with risk factors for P. aeruginosa infection.

Crystallization and preliminary X-ray crystallographic studies of DnaJ from Streptococcus pneumoniae.

DnaJ, cooperating with DnaK and GrpE, promotes the folding of unfolded hydrophobic polypeptides, dissociates protein complexes and translocates protein across membranes. Additionally, DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. The crystals belong to space groups I222 or I212121 and the diffraction resolution is 3.0 Šwith unit-cell parameters a=47.68, b=104.45, c=234.57 Å. The crystal most likely contains one molecule in the asymmetric unit, with a VM value of 3.24 Å3 Da(-1) and a solvent content of 62.1%.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter strains in China: a multicenter genomic study.

Epidemiological characteristics and molecular features of carbapenem-resistant Enterobacter (CR-Ent) species remain unclear in China. In this study, we performed a genomic study on 92 isolates from Enterobacter-caused infections from a multicenter study in China. Whole genome sequencing (WGS) was used to determine the genome sequence of 92 non-duplicated CR-Ent strains collected from multiple tertiary health centres. The precise species of Enterobacter strains were identified by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH). Molecular features of high-risk CR-Ent sequence type (ST) lineages and carbapenemase-encoding plasmids were determined. The result revealed that the most common human-source CR-Ent species in China was E. xiangfangensis (66/92, 71.93%), and the proportion of carbapenemase-producing Enterobacter (CP-Ent) in CR-Ent was high (72/92, 78.26%) in comparison to other global regions. Furthermore, ST171 and ST116 E. xiangfangensis were the major lineages of CP-Ent strains, and ST171 E. xiangfangensis was more likely to cause infections in older patients. Genomic analysis also highlighted the likelihood of intra-hospital/inter-hospital clonal transmission of ST171 and ST116 E. xiangfangensis. In addition, the blaNDM-harbouring IncX3-type plasmid was identified as the prevalent carbapenemase-encoding plasmid carried by CR-Ent strains, and was experimentally confirmed to be able to self-transfer with high frequency. This study detailed the genomic and clinical characteristics of CR-Ent in China in the form of multicenter for the first time. The high risk of carbapenemase-producing ST171 and ST116 E. xiangfangensis, and the blaNDM-harbouring IncX3-type plasmid were detected and emphasized.

[Cloning and characterization of a new antibacterial target, 3,4-dihydroxy-2-butanone-4-phosphate synthase].

OBJECTIVE: Riboflavin, called generally vitamin B12, is the precursor of cofactor flavin adenine dinucleotide (FAD) and flavin mononucleotide, which plays crucial roles in the biosynthesis of organisms. Bacteria need to synthesize riboflavin to maintain their survival and proliferation if they cannot obtain flavin from the surroundings. 3,4-Dihydroxy-2-butanone4-phosphate synthase (DHBPs) is one of the key enzymes in biosynthesis of riboflavin. In the presence of Mg2+, DHBPs can degrade ribulose-5-phosphate (Ru5P) into formate and 3,4-dihydroxy-2-butanone-4-phosphate (DHBP). Potentially, these enzymes related to biosynthesis and metabolism of riboflavin, including DHBPs, may serve as the target for new antibacterial drug. In order to determine the three-dimensional structure and screen small molecule of inhibitor of DHBPs, we expressed and purified DHBPs from Streptococcus pneumonia (S. pn), and characterized the activity of this enzyme. METHOD: DHBPs gene was amplified by PCR, and over-expression plasmid pW28-DHBPs was constructed. pW28-DHBPs was transformed into Escherichia coli BL21 (DE3) to express DHBPs; the recombinant protein was purified by nickel column and ion-exchange column. The enzymatic activity was tested using spectroscopy. RESULTS: The plasmid of pW28-DHBPs was verified by restrictive enzyme digestion and sequencing. Soluble DHBPs was expressed in E. coli BL21, and purified with 95% purity. The result of size exclusion chromatography indicates that DHBPs was dimer in solution. Additionally, the recombinant protein has activity to hydrolyze Ru5P into formate and DHBP in the conditions of pH 7.5 and 25 degrees C and in the presence of Mg2+. CONCLUSIONS: DHBPs could be highly expressed in soluble form in E. coli BL21 strain, and the recombinant protein has activity to hydrolyze Ru5P.

Metagenomic next-generation sequencing clinches the diagnosis of Legionella pneumonia in a patient with acute myeloid leukemia: A case report and literature review.

Legionella pneumonia caused by Legionella pneumophila is a multi-system disease that is a life-threatening, acute, and severe form of pneumonia. L. pneumophila is widespread and the clinical manifestations of Legionella pneumonia are similar to those of typical and atypical pneumonia. Current diagnostic scores and radiologic evidence have limited diagnostic value. Thus, it is likely that many cases of Legionella pneumonia remain unreported. We describe a woman with a medical history of acute myeloid leukemia who suffered from repeated fever, and no relief following initial empirical antibiotic treatment. Ultimately, she was diagnosed with Legionella pneumonia based on metagenomic next-generation sequencing (mNGS). We also performed a systematic review of the literature and identified 5 other patients who were diagnosed with Legionella pneumonia using mNGS, and reviewed their clinical characteristics, biological characteristics, epidemiological features, laboratory results, clinical findings, and treatments. This literature review showed that accurate etiological diagnosis is becoming increasingly essential for a definitive diagnosis and treatment strategies. The clinical manifestations of Legionella pneumonia are non-specific, and many routine laboratory diagnostic tests cannot identify Legionella. mNGS, an indispensable approach for identifying microorganisms, can provide a promising tool for the rapid and accurate etiological diagnosis methods contributing to early diagnosis, early treatment, and improved prognosis, especially for uncommon species such as L. pneumophila.

Genetic Diversity and in vitro Activity of Aztreonam/Avibactam and Ceftazidime/Avibactam Against Carbapenem-Resistant Enterobacterales: A Multi-Center Study in Southwest China.

Purpose: This study aimed to understand the distribution characteristics of carbapenemase genes and assess the antimicrobial activities of aztreonam/avibactam (ATM/AVI) and ceftazidime/avibactam (CAZ/AVI) against carbapenem-resistant Enterobacterales (CRE) isolates in Chongqing, Southwest China. Methods: CRE isolates and their clinical information were collected from 22 hospitals covering all the five regions across Chongqing between January 1, 2016 and December 31, 2017. PCR was used to screen for common carbapenemase genes. And minimum inhibitory concentrations (MICs) were determined by broth microdilution method. Results: A total of 312 unduplicated CRE isolates (eg, 206 Klebsiella pneumoniae, 43 Escherichia coli, and 42 Enterobacter cloacae) were collected during the two-year study period. Among these CRE isolates, 92.3% carried carbapenemase genes, with a majority of isolates carrying single bla KPC-2 (47.1%) or single bla NDM/IPM (36.2%) and 8.9% of isolates carrying two or three carbapenemase genes. Notably, 95.6% (197/206) K. pneumoniae, 86.0% (37/43) E. coli and 88.1% (37/42) E. cloacae harbored carbapenemase genes. In addition, bla KPC-2 was prevalent in K. pneumoniae (70.4%), while bla NDM was predominant in E. coli (83.7%) and E. cloacae (78.6%). Besides, only metallo-ß-lactamase (MBL) genes were detected in the CRE isolates from children. Overall, 0.0%, 48.1%, 59.0%, 61.5% and 63.1% of the CRE isolates were resistant to ATM/AVI, CAZ/AVI, nitrofurantoin, amikacin and trimethoprim/sulfamethoxazole, respectively. 99.7% of the total 312 isolates could be killed by ATM/AVI with the MIC 1 µg/mL, whereas CAZ/AVI showed good antibacterial activity (98.0% susceptible) against the bla KPC-2-carriers with the MIC50/90 values of 1/4 µg/mL. Conclusion: The distribution features of carbapenemase genes in Chongqing were comprehensively illustrated in terms of species and sources of CRE for the first time in this multi-center study that covered all the geographical locations across Chongqing. ATM/AVI showed superior activity against all CRE isolates regardless of their genotype, whereas CAZ/AVI was active against almost all KPC-producers.

Differences in the Distribution of Species, Carbapenemases, Sequence Types, Antimicrobial Heteroresistance and Mortality Rates Between Pediatric and Adult Carbapenemase-Producing Enterobacterales in Bloodstream Infections.

The dissemination of carbapenemase-producing Enterobacterales (CPE) is worrisome given their scarce treatment options. CPE bloodstream infections (BSIs) had a high mortality rate in adults, and there was little data on pediatric CPE-BSIs around the world. We comprehensively explored the differences in the clinical and microbiological characteristics between pediatric and adult CPE-BSIs. Forty-eight pediatric and 78 adult CPE-BSIs cases were collected. All-cause 30 day-mortality in children with CPE-BSIs (14.6%, 7/48) was significantly lower than that in adult patients (42.3%, 33/78, p = 0.001). The subgroup in adults empirically treated with tigecycline as an active drug displayed a significantly higher 30-days crude mortality (63.3%, 19/30) than the subgroup treated without tigecycline (29.2%, 14/48, p = 0.003). K. pneumoniae was the most prevalent species in both the pediatric (45.8%, 22/48) and adult populations (64.1%, 50/78), with discrepant carbapenemase genes in each population: 95.4% (21/22) of the pediatric K. pneumoniae isolates carried bla NDM, while 82.0% (41/50) of the adult strains harbored bla KPC. The ratio of E. coli in children (37.5%) was significantly higher than that in adults (12.8%, p = 0.002). In both populations, the majority of E. coli expressed bla NDM, particularly bla NDM-5. With statistical significance, bla NDM was much more common in children (95.8%, 46/48) than in adults (34.6%, 27/78). The rate of multiple-heteroresistance phenotypes in children was as high as 87.5%, which was much lower in adults (57.1%). Agar dilution checkboard experiment against one pediatric carbapenemase-producing E. coli isolates showed that the combination of amikacin and fosfomycin yielded an additive effect. Overall, K. pneumoniae was the most common CPE-BSIs pathogen in both populations, with NDM-producing K. pneumoniae and KPC-producing ST11 K. pneumoniae being the most prevalent species in children and adults, respectively. E. coli was more prevalent in children than in adults, yet bla NDM-5 was the most common carbapenem-resistant mechanism in E. coli in both populations. The wide range of multiple-heteroresistance combination traits found in different pathogen species from different host populations should provide a good foundation for future combination therapy design. Further investigations from more CPE isolates of various species are needed to evaluate the possible in vitro partial synergy of the amikacin and fosfomycin combination.

Bone marrow mesenchymal stromal cells with support of bispecific antibody and ultrasound-mediated microbubbles prevent myocardial fibrosis via the signal transducer and activators of transcription signaling pathway.

BACKGROUND AIMS: This study was initiated to investigate the efficacy of myocardial fibrosis intervention via signal transducer and activators of transcription (STAT) signaling using bone marrow (BM) mesenchymal stromal cells (MSC) with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). METHODS: BiAb (anti-CD29 × anti-myosin light chain antibody; AMLCA) was prepared and combined with isolated MSC from male mice and transfused into female mice with isoproterenol-induced myocardial fibrosis via the tail vein, followed by MB (MSC + BiAb + MB). This study included seven groups: MSC + BiAb + MB; MSC; BiAb; MB; MSC + BiAb; untreated; and control. Five weeks after treatment, expression levels of the sex-determining region of Y-chromosome (SRY), matrix metalloproteinases (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent quantitative real-time polymerase chain reaction (qRT-PCR). Collagen distribution was observed using Sirius Red staining. The protein expression of signal transducer and activators of transcription (STAT)1 and STAT3 was detected by Western blot. RESULTS: The highest homing number of MSC was in the MSC + BiAb + MB group, second highest in the MSC + BiAb group, and lowest in MSC alone. Compared with the untreated group, MSC + BiAb + MB, MSC + BiAb and MSC groups had decreased levels of MMP-9, TIMP-1, STAT1 and collagen deposition, and increased levels of STAT3. Upregulated STAT3 and downregulated TIMP-1 were significantly different in MSC + BiAb + MB compared with MSC alone or MSC + BiAb. CONCLUSIONS: The homing rate and repairing efficacy of MSC improved with treatment utilizing a combination of BiAb and MB. MSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway.

Resistance Trends of Klebsiella pneumoniae Causing Urinary Tract Infections in Chongqing, 2011-2019.

PURPOSE: To analyze the characteristics and trends of drug resistance for Klebsiella pneumoniae (K. pneumoniae), isolated from urinary tract infections (UTIs), to common antibiotics used in clinics. METHODS: This retrospective study was conducted in a teaching hospital in Chongqing from 2011 to 2019. Laboratory data of isolated bacteria were collected and analyzed. RESULTS: Among the 17,966 non-repetitive strains isolated from the urine sample, a total of 1543 K. pneumoniae isolates were identified, with an isolation frequency secondary only to Escherichia coli (E. coli) and there was a peak in the K. pneumoniae isolates in the year 2013. During the period, the rate of extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae fell from 48.4% in 2011 to 32.9% in 2019, and a marked jump of resistance was seen in carbapenems from 2.2% to 18.0%. The peak of carbapenem resistance rate (22.6%) to K. pneumoniae was observed in 2017 along with a low ESBL-producing rate (30.9%). Piperacillin/tazobactam and cefepime resistance levels went up from 4.4% to 25.7% and from 18.2% to 30.5%, respectively. Moreover, the K. pneumoniae isolates resistance rate to carbapenems and amikacin gradually grew up, showing their peaks in 2017, and then dropped year by year. However, ceftazidime and aztreonam resistance levels were relatively stable, fluctuating between 21.8% and 35.6%, 32.2% and 39.4%, respectively. CONCLUSION: There is a significant upward tendency in carbapenem resistance rate and a downward tendency in ESBL-production rate in K. pneumoniae isolates from UTIs, and continuous surveillance is necessary in the future.

Design of Rapid Detection System for Five Major Carbapenemase Families (bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

PURPOSE: Carbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (bla KPC, bla NDM, bla VIM, bla IMP, and bla OXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed. MATERIALS AND METHODS: Five sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-"standard strains", including 1 bla NDM-1, 1 bla NDM-5, 1 bla NDM-6, 1 bla NDM-7, 2 bla IMP-4, 1 bla IMP-8, 2 bla KPC-2, 1 bla KPC-3, 1 bla KPC-4, 1 bla KPC-5, 1 bla KPC-6, 1 bla KPC-7, 1 bla OXA-48 and 1 bla OXA-181 carrier, and 1 bla VIM and bla OXA-244, 1 bla KPC-2 and bla IMP-4, 1 bla KPC-2 and bla VIM-1 and 1 bla KPC-2 and bla NDM-1-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 bla NDM, 2 bla OXA-48-like, 1 bla KPC and bla VIM, 2 bla IMP, and 37 bla KPC carriers) and 61 non-CPE, were also detected. RESULTS: With the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 103 CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates. CONCLUSION: Therefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.

MBLs, Rather Than Efflux Pumps, Led to Carbapenem Resistance in Fosfomycin and Aztreonam/Avibactam Resistant Elizabethkingia anophelis.

OBJECTIVE: To assess the risk factors associated with infections and in-hospital mortality, antimicrobial susceptibility patterns and carbapenem resistance mechanisms in E. anophelis. METHODS: This retrospective case-control study was conducted to reveal the risk factors associated with Elizabethkingia anophelis (E. anophelis) infection and in-hospital mortality in a university tertiary hospital in southwest China, using multivariable logistic-regression analyses. Complete 16S rRNA gene sequencing was used to reconfirm the identity of all isolates. We employed the broth microdilution method to investigate the antimicrobial susceptibility profiles. The presence of resistance genes was confirmed by polymerase chain reaction and DNA sequencing. Full-length resistance genes were cloned into the pET-28a vector for further functional studies. RESULTS: Our multivariate analysis indicated that coronary artery disease, chronic obstructive pulmonary disease, surgery in the past 6 months, anemia and systemic steroid use were independent risk factors for the acquisition of E. anophelis. Additionally, anemia was the only independent risk factor associated with in-hospital mortality in patients with E. anophelis infections. E. anophelis isolates showed high in-vitro susceptibility towards minocycline (100%) and piperacillin/tazobactam (71.8%), but were resistant to colistin, fosfomycin, ceftazidime/avibactam and aztreonam/avibactam. The PCR revealed the presence of blaGOB and blaBlaB in 37 isolates, and blaCME ß-lactamase genes in 36 isolates out of 39 E. anophelis isolates. Additionally, we showed that two metallo-ß-lactamases (MBLs) BlaB and GOB, were responsible for carbapenem resistance and the serine-ß-lactamase, CME, was functionally involved in resistance to cephalosporins and monobactams. Interestingly, the various putative efflux pumps in E. anophelis were not responsible for resistance. CONCLUSION: Our findings will help clinicians to identify high-risk patients and suggests that minocycline should be considered as a therapeutic option for E. anophelis infections. Additionally, carbapenem resistance in E. anophelis is mainly associated with the MBLs, BlaB and GOB, rather than various putative efflux pumps.

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.