Klebsiella pneumoniae carbapenemase variants: the new threat to global public health.

Klebsiella pneumoniae carbapenemase (KPC) variants, which refer to the substitution, insertion, or deletion of amino acid sequence compared to wild blaKPC type, have reduced utility of ceftazidime-avibactam (CZA), a pioneer antimicrobial agent in treating carbapenem-resistant Enterobacterales infections. So far, more than 150 blaKPC variants have been reported worldwide, and most of the new variants were discovered in the past 3 years, which calls for public alarm. The KPC variant protein enhances the affinity to ceftazidime and weakens the affinity to avibactam by changing the KPC structure, thereby mediating bacterial resistance to CZA. At present, there are still no guidelines or expert consensus to make recommendations for the diagnosis and treatment of infections caused by KPC variants. In addition, meropenem-vaborbactam, imipenem-relebactam, and other new ß-lactam-ß-lactamase inhibitor combinations have little discussion on KPC variants. This review aims to discuss the clinical characteristics, risk factors, epidemiological characteristics, antimicrobial susceptibility profiles, methods for detecting blaKPC variants, treatment options, and future perspectives of blaKPC variants worldwide to alert this new great public health threat.

Comparison of three colloidal gold immunoassays and GeneXpert Carba-R for the detection of Klebsiella pneumoniae blaKPC-2 variants.

The increasing use of ceftazidime-avibactam has led to the emergence of a wide range of ceftazidime-avibactam-resistant blaKPC-2 variants. Particularly, the conventional carbapenemase phenotypic assay exhibited a high false-negative rate for KPC-2 variants. In this study, three colloidal gold immunoassays, including the Gold Mountainriver CGI test, Dynamiker CGI test and NG-Test CARBA5, and GeneXpert Carba-R, were used to detect the presence of KPC-2 carbapenemase and its various variants in 42 Klebsiella pneumoniae strains. These strains covered blaKPC-2 (13/42) and 16 other blaKPC-2 variants including blaKPC-12 (1/42), blaKPC-23 (1/42), blaKPC-25 (1/42), blaKPC-33 (6/42), blaKPC-35 (1/42), blaKPC-44 (1/42), blaKPC-71 (1/42), blaKPC-76 (8/42), blaKPC-78 (1/42), blaKPC-79 (1/42), blaKPC-100 (1/42), blaKPC-127 (1/42), blaKPC-128 (1/42), blaKPC-144 (1/42), blaKPC-157 (2/42), and blaKPC-180 (1/42). For KPC-2 strains, all four assays showed 100% negative percentage agreement (NPA) and 100% positive percentage agreement (PPA) with sequencing results. For all 16 KPC-2 variants, GeneXpert Carba-R showed 100% NPA and 100% PPA, and the three colloidal gold immunoassays showed 100% NPA, while the PPAs of the Gold Mountainriver CGI test, Dynamiker CGI test, and NG-Test CARBA5 were 87.5%, 87.5%, and 68.8%, respectively. We also found a correlation between the mutation site in the amino acid of the variants and false-negative results by colloidal gold immunoassays. In conclusion, the GeneXpert Carba-R has been proven to be a reliable method in detecting KPC-2 and its variants, and the colloidal gold immunoassay tests offer a practical and cost-effective approach for their detection. For the sample with a negative result by a colloidal gold immunoassay test but not matching the drug-resistant phenotype, it is recommended to retest using another type of kit or the GeneXpert Carba-R assay, which can significantly improve the accuracy of detection.

Antibiotic resistance and resistance mechanism of Corynebacterium kroppenstedtii isolated from patients with mastadenitis.

To investigate the antibiotic resistance and resistance mechanism of Corynebacterium kroppenstedtii (C. kroppenstedtii) isolated from patients with mastadenitis. Ninety C. kroppenstedtii clinical isolates were obtained from clinical specimens in 2018-2019. Species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed by the broth microdilution method. The resistance genes were detected using PCR and DNA sequencing. The results of antimicrobial susceptibility testing indicated that the resistance rates of C. kroppenstedtii to erythromycin and clindamycin, ciprofloxacin, tetracycline, and trimethoprim-sulfamethoxazole were 88.9%, 88.9%, 67.8%, 62.2%, and 46.6%, respectively. None of the C. kroppenstedtii isolates was resistant to rifampicin, linezolid, vancomycin, or gentamicin. The gene of erm(X) was detected in all clindamycin and erythromycin-resistant strains. The gene of sul(1) and tet(W) were detected among all trimethoprim sulfamethoxazole-resistant strains and tetracycline-resistant strains, respectively. Furthermore, 1 or 2 amino acid mutations (mainly single mutation) were observed in the gyrA gene among ciprofloxacin-resistant strains.

Emergence and Recovery of Ceftazidime-avibactam Resistance in blaKPC-33-Harboring Klebsiella pneumoniae Sequence Type 11 Isolates in China.

This is the first report of ceftazidime-avibactam resistance caused by the blaKPC-33 mutation through the D179Y variant during the treatment of blaKPC-2-positive Klebsiella pneumoniae-related infections in China. The blaKPC-33-containing K. pneumoniae was susceptible to meropenem-vaborbactam, cefepime-zidebactam, tigecycline, and polymyxin B. The blaKPC-33 gene was located on a 77 551-bp transformable plasmid harboring qnrS1 and blaLAP-2. Detecting blaKPC-33-positive K. pneumoniae clinical strains is important for infection control.

Identification of Biomarkers for Osteosarcoma Based on Integration Strategy.

BACKGROUND Osteosarcoma (OS) is the most common primary malignant tumor of bone. The identification of novel biomarkers is necessary for the diagnosis and treatment of osteosarcoma. MATERIAL AND METHODS We obtained 11 paired fresh-frozen OS samples and normal controls from patients between September 2015 and February 2017. We used an integration strategy that analyzes next-generation sequencing data by bioinformatics methods based on the pathogenesis of osteosarcoma. RESULTS One susceptibility lncRNA and 7 susceptibility genes regulated by the lncRNA for osteosarcoma were effectively identified, and real-time PCR and clinical index ALP data were used to test their effectiveness. CONCLUSIONS The results showed that the expression levels of the 7 genes were highly consistent in the training and test sample sets, especially between the expression value of the gene ALPL and the plasma detection value of its encoded protein ALP. In particular, both the expression of gene ALPL and the plasma detection values of protein ALP encoded by gene ALPL showed a high degree of consistency among different data types. The identified lncRNA and genes effectively classified the samples proved so that they could be used as potential biomarkers of osteosarcoma. Our strategy may also be helpful for the identification of biomarkers for other diseases.

Results from the China Antimicrobial Surveillance Network (CHINET) in 2017 of the In Vitro Activities of Ceftazidime-Avibactam and Ceftolozane-Tazobactam against Clinical Isolates of Enterobacteriaceae and Pseudomonas aeruginosa.

The in vitro activities of ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C-T), and comparators were determined for 1,774 isolates of Enterobacteriaceae and 524 isolates of Pseudomonas aeruginosa collected by 30 medical centers from the China Antimicrobial Surveillance Network (CHINET) in 2017. Antimicrobial susceptibility testing was performed by the CLSI broth microdilution method, and blaKPC and blaNDM were detected by PCR for all carbapenem-resistant Enterobacteriaceae (CRE). Ceftazidime-avibactam demonstrated potent activity against almost all Enterobacteriaceae (94.6% susceptibility; MIC50, ≤0.25 mg/liter; MIC90, ≤0.25 to >32 mg/liter) and good activity against P. aeruginosa (86.5% susceptibility; MIC50/90, 2/16 mg/liter). Among the CRE, 50.8% (189/372 isolates) were positive for blaKPC-2, which mainly existed in ceftazidime-avibactam-susceptible Klebsiella pneumoniae isolates (92.1%, 174/189). Among the CRE, 17.7% (66/372 isolates) were positive for blaNDM, which mainly existed in strains resistant to ceftazidime-avibactam (71.7%, 66/92). Ceftolozane-tazobactam showed good in vitro activity against Escherichia coli and Proteus mirabilis (MIC50/90, ≤0.5/2 mg/liter; 90.5 and 93.8% susceptibility, respectively), and the rates of susceptibility of K. pneumoniae (MIC50/90, 2/>64 mg/liter) and P. aeruginosa (MIC50/90, 1/8 mg/liter) were 52.7% and 88.5%, respectively. Among the CRE strains, 28.6% of E. coli isolates and 85% of K. pneumoniae isolates were still susceptible to ceftazidime-avibactam, but only 7.1% and 1.9% of them, respectively, were susceptible to ceftolozane-tazobactam. The rates of susceptibility of the carbapenem-resistant P. aeruginosa isolates to ceftazidime-avibactam (65.7%) and ceftolozane-tazobactam (68%) were similar. Overall, both ceftazidime-avibactam and ceftolozane-tazobactam were highly active against clinical isolates of Enterobacteriaceae and P. aeruginosa recently collected across China, and ceftazidime-avibactam showed activity superior to that of ceftolozane-tazobactam against Enterobacteriaceae, whereas ceftolozane-tazobactam showed a better effect against P. aeruginosa.

Decreased Wnt5a Expression is a Poor Prognostic Factor in Triple-Negative Breast Cancer.

BACKGROUND: Wingless-type MMTV integration site family member 5A (Wnt5a) has been documented to either overexpress or be lost in several malignancies. Our study aimed to investigate the expression and clinical significance of Wnt5a protein in triple-negative breast cancer (TNBC). MATERIAL/METHODS: By using immunohistochemistry, Wnt5a expression was evaluated in 90 TNBC specimens. The association between Wnt5a expression and clinic-pathological factors was assessed by using the chi-square test. The survival analysis of patients was conducted by using the Kaplan-Meier and log-rank tests. Cox regression was utilized for the univariate and multivariate analyses of prognostic factors. RESULTS: Results showed that negative Wnt5a expression correlated with positive lymph node metastasis (LNM) (P=0.007) and Ki67 proliferation (P=0.002). Patients with negative Wnt5a expression had significantly poorer recurrence-free survival (RFS) than patients with positive Wnt5a expression (P=0.008). Multivariate Cox regression analysis revealed that decreased Wnt5a expression was an independent prognostic factor for RFS (P=0.014). CONCLUSIONS: Negative Wnt5a protein expression might contribute to the tumor progression and poor prognosis of TNBC and might be a new therapy target in TNBC.

Elevated expression of HABP1 is a novel prognostic indicator in triple-negative breast cancers.

Hyaluronan-binding protein 1 (HABP1) has been documented to overexpress in several malignancies. The aim of our study is to investigate the expression of HABP1 protein in triple-negative breast cancer (TNBC) and its clinical significance. Using immunohistochemistry, HABP1 expression was evaluated in 139 TNBC specimens. The association between HABP1 expression with clinicopathological parameters was assessed using chi-square test. The survival status of patients was analyzed using the Kaplan-Meier and log-rank tests. Cox regression was used for the multivariate analysis of prognostic factors. The results showed that HABP1 overexpression correlated with higher histological grade (P = 0.004), advanced TNM stage (P = 0.008), greater tumor size (P = 0.016), metastasis of lymph node (P < 0.001), and recurrence (P = 0.040). Furthermore, it indicated that patients with HABP1 overexpression had significantly poorer overall survival (OS) and disease-free survival (DFS) compared with patients (P < 0.001 for both). Multivariate Cox regression analysis revealed that elevated HABP1 expression was an independent prognostic factor for both OS (P = 0.035) and DFS (P = 0.013). In contrast to the effect of HABP1 in TNBCs with lymph node metastasis, HABP1 overexpression significantly affects prognosis (OS, P = 0.002; DFS, P = 0.003) in TNBCs without lymph node metastasis. In conclusion, HABP1 protein high expression may contribute to the tumor progression and poor prognosis of TNBC, especially in predicting prognosis in TNBCs without lymph node metastasis.

Downregulation of the long noncoding RNA EGOT correlates with malignant status and poor prognosis in breast cancer.

Eosinophil granule ontogeny transcript (EGOT) is a long noncoding RNA involved in the regulation of eosinophil granule protein transcript expression. However, little is known about the role of EGOT in malignant disease. This study aimed to assess the potential role of EGOT in the pathogenesis of breast cancer. Quantitative real-time polymerase chain reaction was performed to detect the expression levels of EGOT in 250 breast cancerous tissues and 50 adjacent noncancerous tissues. The correlation of EGOT expression with clinicopathological features and prognosis was also analyzed. EGOT expression was lower in breast cancer compared with the adjacent noncancerous tissues (P < 0.001), and low levels of EGOT expression were significantly correlated with larger tumor size (P = 0.022), more lymph node metastasis (P = 0.020), and higher Ki-67 expression (P = 0.017). Moreover, patients with low levels of EGOT expression showed significantly worse prognosis for overall survival (P = 0.040), and this result was further validated in a larger cohort from a public database. Multivariate analysis suggested that low levels of EGOT were a poor independent prognostic predictor for breast cancer patients (HR = 1.857, 95 % CI = 1.032-3.340, P = 0.039). In conclusion, EGOT may play an important role in breast cancer progression and prognosis and may serve as a new potential prognostic target in breast cancer patients.

Study of circulating antibodies against CD25 and FOXP3 in breast cancer.

Our recent work suggests that circulating levels of anti-CD25 and anti-FOXP3 antibodies were significantly increased in patients with either lung cancer or esophageal cancer. To confirm if these two autoantibodies are specific for certain types of malignant tumors, the present work was thus undertaken to examine an alteration of anti-CD25 and anti-FOXP3 IgG levels in breast cancer. A total of 152 patients with breast cancer and 112 control subjects were recruited in this study. The levels of circulating anti-CD25 and anti-FOXP3 IgG antibodies were tested using an in-house enzyme-linked immunosorbent assay (ELISA). Student's t test showed no significant differences in the levels of either anti-CD25 IgG or anti-FOXP3 IgG between patients with breast cancer and control subjects, although patients at stage I had increased levels of anti-CD25 IgG compared with control subjects (t = 2.11, P = 0.037); there was no significant association of the anti-FOXP3 IgG levels with stages of breast cancer. In conclusion, circulating IgG autoantibody to CD25 instead of FOXP3 may be a potential biomarker for early diagnosis of breast cancer but further investigation remains needed to replicate this initial finding.

Anti-arthritic effects of FasL gene transferred intra-articularly by an inducible lentiviral vector containing improved tet-on system.

The objective of this study is to construct and identify an inducible lentiviral vector containing improved tet-on system and FasL gene and observe its effects on pristane-induced arthritis (PIA). FasL gene was amplified from the spleen of Lewis rats by RT-PCR. The tet-on system was improved with insertion of a chicken chromatin insulator (cHS4) element and an rtTA-dependent, tet-responsive element containing modifications of the tetO sequence (TRE-tight1). Pro-apoptosis effect of the vector pTREFasLcHS4V16 on synovial cells was evaluated by flow cytometer in vitro. Anti-arthritis effects of the vector on PIA after intra-articular injection were observed by clinical evaluation and joint histology. Cytokines in synovial tissue were measured by ELISA. The recombinant inducible lentiviral vector pTREFasLcHS4V16 was successfully constructed. The expression response and the pro-apoptosis effects of the vector were doxycycline dose-dependent. The vector injected intra-articularly attenuated the severity of PIA and decreased the level of cytokines in inflamed joints. pTREFasLcHS4V16 with an improved tet-on system can precisely regulate the expression of FasL gene and apoptosis. Anti-arthritis effects were observed after intra-articular injection of the inducible vector.

Metabolic regulation of mRNA splicing.

Alternative mRNA splicing enables the diversification of the proteome from a static genome and confers plasticity and adaptiveness on cells. Although this is often explored in development, where hard-wired programs drive the differentiation and specialization, alternative mRNA splicing also offers a way for cells to react to sudden changes in outside stimuli such as small-molecule metabolites. Fluctuations in metabolite levels and availability in particular convey crucial information to which cells react and adapt. We summarize and highlight findings surrounding the metabolic regulation of mRNA splicing. We discuss the principles underlying the biochemistry and biophysical properties of mRNA splicing, and propose how these could intersect with metabolite levels. Further, we present examples in which metabolites directly influence RNA-binding proteins and splicing factors. We also discuss the interplay between alternative mRNA splicing and metabolite-responsive signaling pathways. We hope to inspire future research to obtain a holistic picture of alternative mRNA splicing in response to metabolic cues.

Spread and evolution of blaKPC-plasmid between Serratia marcescens and Klebsiella pneumoniae.

OBJECTIVES: blaKPC-carrying Enterobacterales have post great challenges to global healthcare systems. In this study, we reported the evolution and spread of blaKPC between Serratia marcescens and Klebsiella pneumoniae. METHODS: Four S. marcescens and one K. pneumoniae strains were isolated from the sputum samples of the patient. Antimicrobial susceptibility tests and whole genome sequencing were performed to investigate the phenotype & genotype of strains. Conjugation assays, cloning experiment and kinetic parameters measuring were performed to explore the spread and antimicrobial resistance mechanisms. RESULTS: The evolution and transmission of blaKPC-2 occurred during the treatment of ceftazidime-avibactam and trimethoprim-sulfamethoxazole. Analysis of the antimicrobial susceptibility and genetic profiles of the clinical strains showed that blaKPC-2 evolved into blaKPC-71 and blaKPC-44, together with resistance to ceftazidime-avibactam and carbapenems susceptibility recovery under antimicrobial pressure. Cloning and expression of blaKPC-44 & blaKPC-71 in E. coli DH5α showed that KPC-44 and KPC-71 resulted in a 64∼128-fold increase in the MIC value for ceftazidime-avibactam. Meanwhile, the kinetic assays also showed that the enzyme activity of KPC-44 and KPC-71 towards carbapenems was destroyed and couldn't be inhibited by avibactam. Based on the conjugation assay and whole genome sequence analyses, we provided evolutionary insights into the transmission pathway trace of blaKPC-bearing plasmids between S. marcescens and K. pneumoniae. CONCLUSIONS: Mixed-species co-infection is one of the risk factors leading to the spread of plasmids carrying carbapenem-resistant genes, and increased surveillance of multidrug-resistant Enterobacterales is urgently needed.

Dynamic evolution of ceftazidime-avibactam resistance from a single patient through the IncX3_NDM-5 plasmid transfer and blaKPC mutation.

The rapid dissemination of carbapenem-resistant Enterobacterales (CRE) especially carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a great threat to global public health. Ceftazidime-avibactam, a novel ß-lactam/ß-lactamase inhibitor combination, has been widely used due to its excellent antibacterial activity against KPC-producing K. pneumoniae. However, several resistance mechanisms have been reported since its use. Here, we conducted a series of in vitro experiments to reveal and demonstrate the dynamic evolution of ceftazidime-avibactam resistance including interspecies IncX3_NDM-5 plasmid transfer between Enterobacter cloacae and K. pneumoniae and blaKPC mutation from blaKPC-2 to blaKPC-33. Through the analysis of conjugation frequency and fitness cost, the IncX3_NDM-5 plasmid in this study showed strong transmissibility and stability in E. coli EC600 and clinical strain K. pneumoniae 5298 as recipient strain. With increasing ceftazidime-avibactam concentration, the conjugation frequency remained at 10-3-10-5, while the mutation frequency of K. pneumoniae 5298 was 10-6-10-8 at the same concentration. Further plasmid analysis (the IncX3_NDM plasmid from this study and other 658 plasmids from the NCBI database) revealed the diverse origin and genetic structure of blaNDM-5 carrying plasmids. E. coli (42.9%), China (43.9%), IncX3 (66.6%) are the most common strains, regions, and Inc types respectively. By analysing of genetic environment detected in IncX3 plasmids, the dominant structures (168/258, 65.1%) were identified: ISKox3-IS26-blaNDM-5-IS5-ISAba125-Tn3000-Tn3. In additon, several structural variations were found in the core gene structure. In conclusion, the high fitness and transmissibility of the IncX3_NDM-5 plasmids were noteworthy. More importantly, the diverse ceftazidime-avibactam resistance mechanisms including blaNDM-5 tranfer and blaKPC-2 mutation highlighted the importance of the continuous monitoring of antimicrobial susceptibility and carbapenemases subtype during ceftazidime-avibactam treatment.

Molecular mechanisms responsible KPC-135-mediated resistance to ceftazidime-avibactam in ST11-K47 hypervirulent Klebsiella pneumoniae.

Ceftazidime-avibactam resistance attributable to the blaKPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the blaKPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The blaKPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the blaKPC-2-positive cloned strain, only the MIC of ceftazidime increased against blaKPC-135-positive K. pneumoniae and wasn't inhibited by avibactam (MIC 32 µg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

In vitro mimicry of in vivo KPC mutations by ceftazidime-avibactam: phenotypes, mechanisms, genetic structure and kinetics of enzymatic hydrolysis.

Ceftazidime-avibactam (CZA) is employed for the treatment of infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP). Resistance to CZA is frequently linked to point mutations in the blaKPC. We conducted in vitro simulations of in vivo blaKPC mutations using CZA. Four pre-therapy KPC-KP isolates (K1, K2, K3, and K4) were evaluated, all initially exhibited susceptibility to CZA and produced KPC-2. The crucial distinction was that following CZA treatment, the blaKPC-2 mutated in K1, K2, and K3, rendering them resistant to CZA, while K4 achieved microbiological clearance, and blaKPC-2 remained unaltered. The induction assay identified various blaKPC-2 variants, including blaKPC-25, blaKPC-127, blaKPC-100, blaKPC-128, blaKPC-137, blaKPC-138, blaKPC-144 and blaKPC-180. Our findings suggest that the resistance of KPC-KP to CZA primarily results from the emergence of KPC variants, complemented by increased blaKPC expression. A close correlation exists between avibactam concentration and the rate of increased CZA minimum Inhibitory concentration, as well as blaKPC mutation. Inadequate avibactam concentration is more likely to induce resistance in strains against CZA, there is also a higher likelihood of mutation in the blaKPC-2 and the optimal avibactam ratio remains to be determined. Simultaneously, we selected a blaKPC-33-producing K. pneumoniae strain (mutated from blaKPC-2) and induced it with imipenem and meropenem, respectively. The blaKPC-2 was detected during the process, indicating that the mutation is reversible. Clinical use of carbapenems to treat KPC variant strains increases the risk of infection, as the gene can mutate back to blaKPC-2, rendering the strain even more cross-resistant to carbapenems and CZA.

Complex evolutionary trajectories in vivo of two novel KPC variants conferring ceftazidime-avibactam resistance.

More and more ceftazidime-avibactam resistant KPC-producing K. pneumoniae have been reported with its widespread use, and the detection rate of KPC variants has increased dramatically. However, the evolutionary mechanism and fitness effects during KPC mutation remained unknown. Here, we report the complex in vivo evolutionary trajectories of two novel KPC variants, KPC-155 (L169P/GT242A) and KPC-185 (D179Y/GT242A), from Klebsiella pneumoniae in the same patient. The novel variants were shown to confer ceftazidime-avibactam resistance but restore carbapenem susceptibility based on the results of plasmid transformation assays, cloning experiments, and enzyme kinetic measurements. In vitro competition experiments highlighted the adaptive advantage conferred by strains carrying these KPC variants, which could lead to the rapid spread of these ceftazidime-avibactam resistant strains. The growth curve indicated that blaKPC-185 had better growth conditions at lower avibactam concentration compared to blaKPC-155, which was consistent with ceftazidime-avibactam use in vivo. In addition, replicative transposition of the IS26-flanked translocatable unit (IS26-ISKpn6-blaKPC-ISKpn27-IS26) also contributes to the blaKPC amplification and formation of two copies (blaKPC-2 and blaKPC-185), conferring both carbapenem and ceftazidime-avibactam resistance. However, strains with double copies showed reduced competitive advantage and configuration stability. The comparative plasmid analysis of IS26 group (IS26-blaKPC-IS26) and Tn1721 group (Tn1721-blaKPC-IS26) revealed that IS26-insertion could influence the distribution of resistance genes and ability of self-conjugation. The dynamic changes in blaKPC configuration highlight the need for consistent monitoring including antimicrobial susceptibility testing and determination of blaKPC subtypes-during clinical treatment, especially when ceftazidime-avibactam is administered.

Development of Computational Approach for Analyzing In-Process Thermal-Mechanical Condition during Friction Stir Welding for Prediction of Material Bonding Defect.

Unlike the conventional fusion welding process, friction stir welding (FSW) relies on solid-state bonding (SSB) to join metal surfaces. In this study, a straightforward computational methodology is proposed for predicting the material bonding defects during FSW using quantitative evaluation of the in-process thermal-mechanical condition. Several key modeling methods are integrated for predicting the material bonding defects. FSW of AA2024 is taken as an example to demonstrate the performance of the computational analysis. The dynamic sticking (DS) model is shown to be able to predict the geometry of the rotating flow zone near the welding tool. Butting interface tracking (BIT) analysis shows a significant orientation change occurring to the original butting interface, owing to the material flow in FSW, which has a major impact on the bonding pressure at the butting interface. The evolution of the interfacial temperature and the interfacial pressure at the butting interface was obtained to analyze their roles in the formation of material bonding. Four bonding-quality indexes for quantifying the thermal-mechanical condition are tested to show their performance in characterizing the bonding quality during FSW. When the BQI is below a critical value, a bonding defect will be generated. The paper indicates that the simulation-based prediction of a material bonding defect is highly feasible if the developed methodology is extended to quantitatively determine the critical value of the bonding quality index for successful SSB for various alloys.

Towards an Optimized Artificial Neural Network for Predicting Flow Stress of In718 Alloys at High Temperatures.

Artificial neural networks (ANNs) have been an important approach for predicting the value of flow stress, which is dependent on temperature, strain, and strain rate. However, there is still a lack of sufficient knowledge regarding what structure of ANN should be used for predicting metal flow stress. In this paper, we train an ANN for predicting flow stress of In718 alloys at high temperatures using our experimental data, and the structure of the ANN is optimized by comparing the performance of four ANNs in predicting the flow stress of In718 alloy. It is found that, as the size of the ANN increases, the ability of the ANN to retrieve the flow stress results from a training dataset is significantly enhanced; however, the ability to predict the flow stress results absent from the training does not monotonically increase with the size of the ANN. It is concluded that the ANN with one hidden layer and four nodes possesses optimized performance for predicting the flow stress of In718 alloys in this study. The reason why there exists an optimized ANN size is discussed. When the ANN size is less than the optimized size, the prediction, especially the strain dependency, falls into underfitting and fails to predict the curve. When the ANN size is less than the optimized size, the predicted flow stress curves with the temperature, strain, and strain rate will contain non-physical fluctuations, thus reducing their prediction accuracy of extrapolation. For metals similar to the In718 alloy, ANNs with very few nodes in the hidden layer are preferred rather than the large ANNs with tens or hundreds of nodes in the hidden layers.