Molecular Mechanisms Responsible KPC-135-Mediated Resistance to Ceftazidime-Avibactam in ST11-K47 Hypervirulent Klebsiella pneumoniae.

AbstractCeftazidime-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 harbored 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 harbored 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.

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.

Improved Analytical Model for Thermal Softening in Aluminum Alloys Form Room Temperature to Solidus.

In advanced solid-state manufacturing processes such as friction stir welding, the metal's temperature ranges from room temperature to the solidus temperature. The material strength in the temperature range is generally required for investigating the mechanical behaviors. In this communication paper, an analytical model is proposed for describing the thermal softening of aluminum alloys for room temperature to solidus temperature, in which the concept of temperature-dependent transition between two thermal softening regimes is implemented. It is demonstrated that the proposed model compares favorably to the well-known Sellars-Tegart model and Johnson-Cook model. The constants of the proposed model for nine typical engineering commercial aluminum alloys are documented.

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.

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.

Sexually dimorphic morphology, feeding behavior and gene expression profiles in cotton aphid Aphis gossypii.

BACKGROUND: Sexual dimorphism exists in most insects; however, less is known about sexual dimorphism in aphids. In this study, we identified sexually dimorphic differences in morphology, feeding behavior and gene expression between sexual females and males of the cotton aphid through electron microscopy, electrical penetration graph techniques and RNA sequencing. RESULTS: All males were alate with a slender reddish-yellow body and abdominal yellow-black stripes, whereas all sexual females were apterous with a pudgy green body. Sensillum types on the antennae were identical between the two sexes, although males had more sensilla, possibly because the antennae are significantly longer in males compared with sexual females. In terms of feeding behavior, males spent more time probing mesophyll cells and the phloem sieve, and salivating into the phloem sieve. By contrast, sexual females spent more time ingesting xylem sap. In total, 510 and 724 genes were specifically expressed in sexual females and males, respectively, and were significantly enriched in signaling pathways related to reproduction for sexual females (e.g. ovarian steroidogenesis, oxytocin signaling pathway) and energy and flight for males (e.g. thermogenesis, insulin signaling pathway). Moreover, 8551 differentially expressed genes were identified between the two sexes, of which the 3720 upregulated genes in sexual females were mostly enriched in signaling pathways of metabolism and energy, such as thermogenesis and the citrate cycle. CONCLUSION: This study provides insight into sexual dimorphism in aphids and lays a foundation for revealing the molecular mechanism underlying differences between the two sexes in cotton aphid. © 2023 Society of Chemical Industry.

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.

Performance of Ceftazidime-Avibactam 30/20-µg and 10/4-µg Disks for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

Ceftazidime-avibactam, a new ß-lactam-ß-lactamase inhibitor combination, is active against multidrug-resistant Enterobacterales and Pseudomonas aeruginosa isolates and has became available for clinical use in China in the latter half of 2019. In this study, we evaluated the performance of the disk diffusion test with ceftazidime-avibactam 10/4-µg and 30/20-µg disks, compared with the reference broth microdilution method, with a collection of 467 Enterobacterales and 182 P. aeruginosa nonduplicate clinical isolates. The results of antimicrobial susceptibility testing indicated that the categorical agreement (CA) of ceftazidime-avibactam 10/4-µg disk testing for all tested Enterobacterales isolates was 99.8%, with 0.5% very major errors (VMEs) and no major error (ME). The CA of ceftazidime-avibactam 10/4-µg disk testing for all tested P. aeruginosa isolates was 87.9%, with 15.5% MEs and no VME. The CA of ceftazidime-avibactam 30/20-µg disk testing for all tested Enterobacterales isolates was 99.4%, with 1.5% VMEs and no ME. The CA of ceftazidime-avibactam 30/20-µg disk testing for all tested P. aeruginosa isolates was 91.8%, with 2.5% VMEs and 9.9% MEs. Overall, ceftazidime-avibactam 10/4-µg disk testing showed superior performance and was more suitable for assessment of the susceptibility of Enterobacterales and P. aeruginosa isolates. IMPORTANCE Multidrug-resistant Enterobacterales and P. aeruginosa strains have become a global public threat, with the emergence and prevalence of plasmid-mediated extended-spectrum ß-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases disseminated worldwide. Ceftazidime-avibactam, which is commercially available, has shown excellent in vitro activity against multidrug-resistant and carbapenem-resistant Enterobacterales and P. aeruginosa isolates. Moreover, ceftazidime-avibactam has shown promise in treating infections caused by multidrug-resistant and carbapenem-resistant isolates. The disk diffusion test for ceftazidime-avibactam is the most common antimicrobial susceptibility testing method in most laboratories in China. The accurate detection of ceftazidime-avibactam susceptibility is of great significance for the rational clinical application of drugs. Here, we evaluated the performance of the ceftazidime-avibactam 10/4-µg and 30/20-µg disk diffusion tests, compared with the reference broth microdilution method, with clinical Enterobacterales and P. aeruginosa isolates.

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.

The application of Tong-fu therapeutic method on ulcerative colitis: A systematic review and meta-analysis for efficacy and safety of rhubarb-based therapy.

Background: Tong-fu therapeutic method (TFTM) is a traditional Chinese medicine treatment method for ulcerative colitis, which is a novel treatment strategies and have purgative effect. As the most representative medicinal of TFTM, Rhubarb has been reported to have a therapeutic impact on ulcerative colitis by regulating intestinal flora, anti-inflammation, and improving intestinal microcirculation. Although rhubarb has been widely used in Chinese medicine for the treatment of ulcerative colitis, the appropriate protocol is still demanded to its rational use in clinic, which promoted to evaluate the efficacy and safety for rhubarb-based therapy on ulcerative colitis. Method: Clinical trials were searched through PubMed, Cochrane Library, Web of Science, Excerpta Medica Database, Chinese National Knowledge Infrastructure, WAN FANG Database, Chinese Scientific Journal Database, and Chinese Biomedical Literature Database. The subgroup analyses were performed with three groups: medication, course of treatment, and route of administration. The statistical analyses were performed on Review Manager software (version 5.4.1). Results: A total of 2, 475 patients in 30 original studies were analyzed in this article. It was found that rhubarb-based therapy could increase clinical efficacy and reduce the recurrence rate. Subgroup analyses showed that rhubarb-based therapy was more effective than 5-aminosalicylic acid or sulfasalazine alone. In addition, the hypercoagulable state of ulcerative colitis could be ameliorated by decreasing platelet (PLT) and fibrinogen (FIB), and increasing prothrombin time (PT) significantly. Moreover, C-reaction protein (CRP), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and IL-1ß expression were significantly reduced, while IL-10 production was increased, which mediated the alleviation of intestinal inflammation stress. Conclusion: Rhubarb-based therapy could effectively improve ulcerative colitis. Of note, the rhubarb-based medicinal formulas combined with 5-ASA or SASP are more effective than the 5-ASA or SASP alone. In addition, although rhubarb has side effect, the results of our analysis showed that rhubarb-based therapy did not exhibit significant side effects. This means it has a high safety profile in clinical use. Moreover, the use of rhubarb-based therapy is recommend to use within 1-13 weeks or 3 months via administered orally or by enema, which is contributes to ensure the curative effect and avoid its toxic and side effects. As an important case of TFTM, rhubarb-based therapy provides evidence for the practical application of TFTM.

Effects of antioxidant intervention in patients with polycystic ovarian syndrome: A systematic review and meta-analysis.

BACKGROUND: The role of antioxidant intervention in polycystic ovary syndrome (PCOS) patients has been increasingly investigated in recent years. In order to further clarify whether antioxidant therapy is beneficial for PCOS patients and the emphasis of its effects, this study provides a systematic review and meta-analysis of randomized controlled trials examining the effect of antioxidant intervention on PCOS. METHODS: Enrolled study designs related to antioxidant interventions and PCOS, published from 1999 to 2020, were searched from EMBASE, PubMed, and Web of Science databases to sort out proven studies on antioxidant interventions and PCOS. Data were reported as weighted mean difference (WMD) or standard mean difference with associated confidence intervals of 95%. The analysis was conducted using Stata version 16.0. RESULTS: Twenty-three studies were included in total. Antioxidant intervention had a positive impact on homeostasis model assessment of insulin resistance (WMD = -0.37, P = .011) and Triglycerides (WMD = -25.51, P < .001). And antioxidant intervention did not improve testosterone levels significantly (WMD = -0.20, P = .2611). Subgroup analysis showed that except for the D-chiro-inosito subgroup, no difference in body mass index was observed between the intervention group and the control group. CONCLUSIONS: This meta-analysis demonstrates the efficacy of antioxidant intervention in patients with PCOS, demonstrating that antioxidant intervention has a significant effect on insulin resistance and lipid metabolism improvement. However, antioxidant intervention therapy has no discernible impact on testosterone levels or body mass index. Omega-3 may be a more effective antioxidant intervention for PCOS. In addition, this meta-analysis provides important reference opinions and treatment recommendations for PCOS.

High Prevalence and Overexpression of Fosfomycin-Resistant Gene fosX in Enterococcus faecium From China.

Enterococci are one of the main causes of gastrointestinal tract infections in the healthcare system and can develop resistance to fosfomycin through plasmid or chromosomally encoded fosfomycin resistance genes. To investigate the mechanisms of fosfomycin resistance, a total of 4,414 clinical isolates of non-replicated clinical enterococci collected from 62 hospitals in 26 provinces or cities in China were tested. Antibiotic susceptibility testing, detection of fosfomycin resistance genes, and cloning of the fosX gene were done. The PFGE, MLST, qRT-PCR, and next genome sequencing were carried out. The results revealed that the fosfomycin-resistant rate of enterococci was 3.5% (153/4,414), and the major resistance mechanism was fosX (101/153) and fosB (52/153) genes. The fosX gene could increase 4- fold fosfomycin MIC in Enterococcus faecium BM4105RF transformants, and the results of PFGE showed the 101 E. faecium carrying fosX were grouped into 48 pulse types. The multilocus sequence typing identified ST555 as the vast majority of STs, mostly distributed in Shanghai, China. Furthermore, the fosX gene expression was strongly related to the fosfomycin-resistant levels of enterococci. The present study was the first to describe the high prevalence presence of the fosX gene in E. faecium from China.

Characterization of the First Carbapenem-Resistant Pseudocitrobacter faecalis Harboring blaOXA-181 in China.

With the wide use of carbapenems, carbapenem-resistant Enterobacterales have been increasingly reported worldwide. In this study, one blaOXA-181-positive Pseudocitrobacter faecalis strain was isolated from the blood culture of a patient with a bloodstream infection in China, which was its first clinical report outside Pakistan. Species identification of P. faecalis was initially performed using MALDI-TOF/MS and further confirmed by 16S rRNA gene and housekeeping gene sequencing. The antimicrobial susceptibility testing was determined through the broth microdilution method, and their clonal relationship was analyzed by pulsed-field gel electrophoresis. To study the transmission and genetic structure of the blaOXA-181 gene, a transformation test and whole-genome sequencing (WGS) were performed. The results of the antimicrobial susceptibility testing indicated this P. faecalis was resistant to carbapenems, quinolones, and commonly used ß-lactam/ß-lactamase inhibitor combinations. Through WGS and transformation experiments, blaOXA-181 and qnrS1 genes causing antibiotic resistance were located on a 55,148-bp length IncX3 type plasmid with a truncated ColKp3 replicon gene. As a rare species of Enterobacterales, P. faecalis was clinically reported in China for the first time, and the blaOXA-181 gene it carried was located on a globally disseminated IncX3 plasmid. The spread of such bacteria and antibiotic resistance requires more clinical attention.

Multiple Novel Ceftazidime-Avibactam-Resistant Variants of blaKPC-2-Positive Klebsiella pneumoniae in Two Patients.

As the first-line antimicrobial agent for the infection caused by carbapenem-resistant Enterobacterales, ceftazidime-avibactam develops drug resistance during its ever-growing clinical use. In this study, we report multiple novel variants in blaKPC-2-positive Klebsiella pneumoniae from two separate patients during their exposure to ceftazidime-avibactam. For one patient, the blaKPC-2 gene carried by K. pneumoniae mutated into blaKPC-35, blaKPC-78, and blaKPC-33 over the same period, while that for the other patient mutated into blaKPC-79 and further evolved into blaKPC-76 to enhance resistance level, among which blaKPC-76 and blaKPC-79 were reported for the first time. In contrast with blaKPC-2, the emergent mutations within the Ω-loop conferred high-level resistance to ceftazidime-avibactam with a sharp reduction of carbapenemase activity. These blaKPC-positive K. pneumoniae isolated from sputum (both patients) and cerebrospinal fluid (patient 2) belonged to ST11 and ST859, respectively. All strains located blaKPC alleles on IncFII/IncR plasmids, except one on an IncFII plasmid. Such blaKPC-2 variants first appeared after 9 to 18 days of ceftazidime-avibactam usage, but the lack of its feasible detection method often led to the assumption of ceftazidime-avibactam sensitivity resulting in clinical incorrect usage. Subsequent substitution of ceftazidime-avibactam with carbapenems also failed, because the blaKPC-2-containing K. pneumoniae dominated again. Ultimately, treatment failed even with the therapeutic regimen of ceftazidime-avibactam combined with carbapenems, because of the inadequate concentration of avibactam in infection sites and decreased drug sensitivity of strains caused by increased expression of blaKPC and point mutation of ompK35 and ompK36. As novel KPC variants conferring resistance to ceftazidime-avibactam are constantly emerging worldwide, quick and efficient laboratory detection and surveillance are urgently needed for infection control. IMPORTANCE Carbapenem-resistant K. pneumoniae which was classified as the most urgent threat by World Health Organization, is the most critical public health concern due to its high mortality rate. Recently, the rapid mutation of blaKPC has occurred during anti-infective therapy, which posed an unexpected challenge for both the diagnostic laboratory and clinical practice.

Comparison of Four Carbapenemase Detection Methods for blaKPC-2 Variants.

Recently, various blaKPC-2 variants resistant to ceftazidime-avibactam have begun to emerge in clinical settings, but it is unclear which testing method is most appropriate for detecting these variants. Strains were subjected to antimicrobial susceptibility testing using the broth microdilution method. Four carbapenemase detection methods, modified carbapenem inactivation method (mCIM) and EDTA carbapenem inactivation method (eCIM), APB/EDTA (carbapenemase inhibitor APB [3-aminophenylboronic acid] and EDTA enhancement method), NG-test Carba 5, and GeneXpert Carba-R were used to try to detect KPC-2 variants in 19 Klebsiella pneumoniae isolates. Among those blaKPC-2 variants, blaKPC-33-, blaKPC-35-, blaKPC-71-, blaKPC-76-, blaKPC-78-, and blaKPC-79-positive isolates accounted for 26.3% (5/19), 15.8% (3/19), 5.3% (1/19), % 42.1% (8/19), 5.3% (1/19), and 5.3% (1/19), respectively. All 19 K. pneumoniae carrying blaKPC-2 variants showed resistance to ceftazidime-avibactam (MICs:16 to >64 mg/L), and 14 strains were susceptible to imipenem (MICs: 0.25 to 1 mg/L). None of the blaKPC-2 variants could be detected using either the mCIM or the APB/EDTA method, while five strains carrying blaKPC-2 variants (blaKPC-35, blaKPC-78, and blaKPC-79) tested KPC positive when using NG-test Carba 5. However, GeneXpert Carba-R was able to detect blaKPC-2 variants (harboring blaKPC-33, blaKPC-35, blaKPC-71, blaKPC-76, blaKPC-78, and blaKPC-79) carried by all 19 K. pneumoniae. The emergence of new KPC variants poses an increased challenge for carbapenemase detection methods, and laboratories should use the appropriate assays to accurately detect these variants. IMPORTANCE Carbapenemase detection is essential for the appropriate treatment of CRE infections. Several clinical laboratories have begun using relevant carbapenemase assays such as mCIM and eCIM, the APB/EDTA method, NG-test Carba 5, and GeneXpert Carba-R to detect carbapenemases. Nevertheless, some of these methods may have limitations for detecting blaKPC-2 variants. Additionally, there has been little relevant research on evaluate the differences between these standard methods for detecting blaKPC-2 variants. Therefore, we investigated the reliability of these classic methods for assessing 19 K. pneumoniae with blaKPC-2 variants. Our results showed that none of the blaKPC-2 variants could be detected using either the mCIM or APB/EDTA method, while five strains (harboring blaKPC-35, blaKPC-78,and blaKPC-79) tested KPC positive when using NG-test Carba 5. GeneXpert Carba-R could detect six blaKPC-2 variants carried by all 19 K. pneumoniae. This study may be valuable for clinical laboratories in their efforts to test for various blaKPC-2 variants.

First Report of bla IMP-4 and bla SRT-2 Coproducing Serratia marcescens Clinical Isolate in China.

Carbapenem-resistant Enterobacterales (CRE) has become a major therapeutic concern in clinical settings, and carbapenemase genes have been widely reported in various bacteria. In Serratia marcescens, class A group carbapenemases including SME and KPC were mostly identified. However, there are few reports of metallo-ß-lactamase-producing S. marcescens. Here, we isolated a carbapenem-resistant S. marcescens (S378) from a patient with asymptomatic urinary tract infection which was then identified as an IMP-4-producing S. marcescens at a tertiary hospital in Sichuan Province in southwest of China. The species were identified using MALDI-TOF MS, and carbapenemase-encoding genes were detected using PCR and DNA sequencing. The results of antimicrobial susceptibility testing by broth microdilution method indicated that the isolate S. marcescens S378 was resistant to meropenem (MIC = 32 µg/ml) and imipenem (MIC = 64 µg/ml) and intermediate to aztreonam (MIC = 8 µg/ml). The complete genomic sequence of S. marcescens was identified using Illumina (Illumina, San Diego, CA, United States) short-read sequencing (150 bp paired-end reads); five resistance genes had been identified, including bla IMP-4, bla SRT-2, aac(6')-Ic, qnrS1, and tet(41). Conjugation experiments indicated that the bla IMP-4-carrying plasmid pS378P was conjugative. Complete sequence analysis of the plasmid pS378P bearing bla IMP-4 revealed that it was a 48,780-bp IncN-type plasmid with an average GC content of 50% and was nearly identical to pP378-IMP (99% nucleotide identity and query coverage).